四碳醇的发酵生产 |
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申请号 | CN200680039955.1 | 申请日 | 2006-10-25 | 公开(公告)号 | CN101297042A | 公开(公告)日 | 2008-10-29 |
申请人 | 纳幕尔杜邦公司; | 发明人 | G·K·唐纳森; A·C·埃利奥特; D·弗林特; L·A·马吉奥-霍尔; V·纳加拉简; | ||||
摘要 | 提供了 发酵 生产四 碳 醇的方法。具体地,通过表达异丁醇 生物 合成途径的重组细菌的发酵生长来生产丁醇,优选异丁醇。 | ||||||
权利要求 | 1.重组微生物宿主细胞,其包括至少一个编码多肽的DNA分子, 该多肽催化选自以下的底物至产物的转化: |
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说明书全文 | 发明领域本发明涉及工业微生物学和醇类生产的领域。更具体地,通过重 组微生物的工业发酵来产生异丁醇。 发明背景丁醇是重要的工业化学物质,用作燃料添加剂,塑料工业中的原 料化学物质以及食品和香料工业中的食品级萃取剂。每年通过石油化 学方法产生100至120亿磅的丁醇,并且对这种日用化学物质的需求 很可能增加。 异丁醇化学合成的方法是已知的,如氧化合成,一氧化碳的催化 氢化(Ullmann’s Encyclopedia of Industrial Chemistry,第6版,2003, Wiley-VCHVerlag GmbH and Co.,Weinheim,Germany,Vol.5, pp.716-719)以及甲醇和n-丙醇的Guerbet缩合(Carlini等,J.Mol.Catal. A:Chem.220:215-220(2004))。这些方法使用源自石油化学,并且 通常是昂贵的,环境不友好的原料。从植物产生的原料生产异丁醇将 最小化温室气体释放并将表示本领域中的进展。 异丁醇是作为酵母发酵的副产物通过生物产生的。它是作为通过 该组真菌不完全代谢氨基酸的结果形成的“杂醇油”的组分。从L-缬 氨酸的分解代谢特异性地产生异丁醇。作为氮源收集L-缬氨酸的胺基 后,通过所谓的Ehrlich途径将所得到的α-酮酸脱羧基并还原成异丁醇 (Dickinson等,J.Biol.Chem.273(40):25752-25756(1998))。在饮料 发酵过程中获得的杂醇油和/或其组分的产量通常是低的。例如,报道 了啤酒发酵中产生的异丁醇浓度低于百万分之16(Garcia等,Process Biochemistry 29:303-309(1994))。将外源L-缬氨酸添加至发酵中提 高了异丁醇的产量,如Dickinson等所述的,上文,其中报道了通过在 发酵中提供20g/L浓度的L-缬氨酸获得了3g/L异丁醇的产量。然而, 将缬氨酸用作原料对于工业规模的异丁醇生产成本过高。直接从糖生 物合成异丁醇在经济上是可行的,并将表示本领域中的进展。没有设 计重组微生物来生产异丁醇的报道。 因此,存在对环境负责的,成本有效的作为单一产物的异丁醇生 产方法的需求。本发明通过提供表达异丁醇生物合成途径的重组微生 物生产宿主解决了这种需求。 发明概述 本发明提供了具有工程化异丁醇生物合成途径的重组微生物。该 工程化的微生物用于异丁醇的商业性生产。因此,在一个实施方案中, 本发明提供了重组微生物宿主细胞,其包括至少一个编码多肽的DNA 分子,该多肽催化选自以下的底物至产物的转化: i)丙酮酸至乙酰乳酸(途径步骤a) ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b) iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c) iv)α-酮基异戊酸至异丁醛(途径步骤d),和 v)异丁醛至异丁醇(途径步骤e) 其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且 其中所述微生物宿主细胞产生异丁醇。 在另一个实施方案中,本发明提供了重组微生物宿主细胞,其包 括至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物 的转化: i)丙酮酸至乙酰乳酸(途径步骤a) ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b) iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c) iv)α-酮基异戊酸至异丁酰-CoA(途径步骤f) v)异丁酰-CoA至异丁醛(途径步骤g),和 vi)异丁醛至异丁醇(途径步骤e) 其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且 其中所述微生物宿主细胞产生异丁醇。 在另一实施方案中,本发明提供了重组微生物宿主细胞,其包括 至少一个编码多肽的DNA分子,该多肽催化选自以下的底物至产物的 转化: i)丙酮酸至乙酰乳酸(途径步骤a) ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b) iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c) iv)α-酮基异戊酸至缬氨酸(途径步骤h) v)缬氨酸至异丁胺(途径步骤i), vi)异丁胺至异丁醛(途径步骤j),和 vii)异丁醛至异丁醇(途径步骤e) 其中至少一个DNA分子对于所述微生物宿主细胞是异源的,并且 其中所述微生物宿主细胞产生异丁醇。 在另一实施方案中,本发明提供了生产异丁醇的方法,其包括: 1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA 分子,该多肽催化选自以下的底物至产物的转化: i)丙酮酸至乙酰乳酸(途径步骤a) ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b) iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c) iv)α-酮基异戊酸至异丁醛(途径步骤d),和 v)异丁醛至异丁醇(途径步骤e) 其中至少一个DNA分子对所述微生物宿主细胞是异源的;和 2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养 基中的可发酵碳底物。 在另一实施方案中,本发明提供了生产异丁醇的方法,其包括: 1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA 分子,该多肽催化选自以下的底物至产物的转化: i)丙酮酸至乙酰乳酸(途径步骤a) ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b) iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c) iv)α-酮基异戊酸至异丁酰-CoA(途径步骤f) v)异丁酰-CoA至异丁醛(途径步骤g),和 vi)异丁醛至异丁醇(途径步骤e) 其中至少一个DNA分子对于所述微生物宿主细胞是异源的;和 2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养 基中的可发酵碳底物。 在另一实施方案中,本发明提供了生产异丁醇的方法,其包括: 1)提供重组微生物宿主细胞,其包括至少一个编码多肽的DNA 分子,该多肽催化选自以下的底物至产物的转化: i)丙酮酸至乙酰乳酸(途径步骤a) ii)乙酰乳酸至2,3-二羟基异戊酸(途径步骤b) iii)2,3-二羟基异戊酸至α-酮基异戊酸(途径步骤c) iv)α-酮基异戊酸至缬氨酸(途径步骤h) v)缬氨酸至异丁胺(途径步骤i), vi)异丁胺至异丁醛(途径步骤j),和 vii)异丁醛至异丁醇(途径步骤e) 其中至少一个DNA分子对于所述微生物宿主细胞是异源的;和 2)在由此产生异丁醇的条件下将(i)中的宿主细胞接触发酵培养 基中的可发酵碳底物。 在可替换的实施方案中,本发明提供了通过本发明的方法产生的 含有异丁醇的发酵培养基。 附图和序列表简述 从以下形成本申请一部分的详述,附图和附带的序列描述可以更 全面地了解本发明。 图1显示了四个不同的异丁醇生物合成途径。标记为“a”,“b”, “c”,“d”,“e”,“f”,“g”,“h”,“i”,“j”和“k”的步骤表示以下所 述的底物至产物的转化。 以下的序列符合37C.F.R.1.821-1.825(“含有核苷酸序列和/或氨基 酸序列公开内容的专利申请的要求-序列规定”)并符合世界知识产权组 织(WIPO)标准ST.25(1998)以及EPO和PCT的序列表要求(细则 5.2和49.5(a-bis),和行政指令的208条和附录C)。用于核苷酸和氨基 酸序列数据的符号和格式遵照37C.F.R.§1.822中列出的规定。 表1 基因和蛋白质SEQ ID编号的概括 描述 SEQ ID NO:核酸 SEQ ID NO:肽 肺炎克雷伯氏菌(Klebsiella pneumoniae)budB (乙酰乳酸合酶) 1 2 枯草芽孢杆菌(Bacillus subtillis)alsS (乙酰乳酸合酶) 78 178 乳酸乳球菌(Lactococcus lactis)als (乙酰乳酸合酶) 179 180 大肠杆菌(E.coli)ilvC (乙酰醇酸还原异构酶) 3 4 酿酒酵母(S.cerevisiae)ILV5 (乙酰醇酸还原异构酶) 80 181 海藻甲烷球菌(M.maripaludis)ilvC (酮醇酸还原异构酶) 182 183 枯草芽孢杆菌ilvC (乙酰醇酸还原异构酶) 184 185 大肠杆菌ilvD (乙酰醇酸脱水酶) 5 6 酿酒酵母ILV3 (二醇酸脱水酶) 83 186 海藻甲烷球菌ilvD (二醇酸脱水酶) 187 188 枯草芽孢杆菌ilvD (二醇酸脱水酶) 189 190 乳酸乳球菌kivD (支链α-酮酸脱羧酶),密码子最佳化 7 8 乳酸乳球菌kivD (支链α-酮酸脱羧酶) 191 8 乳酸乳球菌kdcA (支链α-酮酸脱羧酶) 192 193 鼠伤寒沙门氏菌(Salmonella typhimurium) (吲哚丙酮酸脱羧酶) 194 195 丙醇丁酮梭菌(Clostridium acetobutylicum)pdc (丙酮酸脱羧酶) 196 197 大肠杆菌yqhD (支链醇脱氢酶) 9 10 酿酒酵母YPR1 (2-甲基丁醛还原酶) 198 199 酿酒酵母ADH6 (NADPH-依赖性肉桂醇脱氢酶) 200 201 丙醇丁酮梭菌bdhA (NADH-依赖性丁醇脱氢酶A) 202 203 丙醇丁酮梭菌bdhB (丁醇脱氢酶) 158 204 枯草芽孢杆菌bkdAA (支链酮酸脱氢酶E1亚基) 205 206 枯草芽孢杆菌bkdAB (支链α-酮酸脱氢酶E1亚基) 207 208 枯草芽孢杆菌bkdB (支链α-酮酸脱氢酶E2亚基) 209 210 枯草芽孢杆菌lpdV (支链α-酮酸脱氢酶E3亚基) 211 212 恶臭假单胞菌(P.putida)bkdA1 (酮酸脱氢酶E1-α亚基) 213 214 恶臭假单胞菌bkdA2 (酮酸脱氢酶E1-β亚基) 215 216 恶臭假单胞菌bkdB (转酰酶) 217 218 恶臭假单胞菌lpdV (硫辛酰胺脱氢酶) 219 220 拜氏梭菌(C.beijerinckii)ald (辅酶A酰化醛脱氢酶) 221 222 丙醇丁酮梭菌adhe1 (醛脱氢酶) 223 224 丙醇丁酮梭菌adhe (醇-醛脱氢酶) 225 226 恶臭假单胞菌nahO (乙醛脱氢酶) 227 228 嗜热栖热菌(T.thermophilus) (乙醛脱氢酶) 229 230 大肠杆菌avtA (缬氨酸-丙酮酸转氨酶) 231 232 地衣芽孢杆菌(B.licheniformis)avtA (缬氨酸-丙酮酸转氨酶) 233 234 大肠杆菌ilvE (支链氨基酸转氨酶) 235 236 酿酒酵母BAT2 (支链氨基酸转氨酶) 237 238 嗜热碱甲烷杆菌(M.thermoautotrophicum) (支链氨基酸转氨酶) 239 240 天蓝色链霉菌(S.coelicolor) (缬氨酸脱氢酶) 241 242 枯草芽孢杆菌bcd (亮氨酸脱氢酶) 243 244 生绿链霉菌(S.viridifaciens) (缬氨酸脱氢酶) 245 246 反硝化产碱菌(A.denitrificans)aptA (ω-氨基酸:丙酮酸转氨酶) 247 248 真养雷氏菌(R.eutropha) (丙氨酸-丙酮酸转氨酶) 249 250 S.oneidensis (β丙氨酸-丙酮酸转氨酶) 251 252 恶臭假单胞菌 (β丙氨酸-丙酮酸转氨酶) 253 254 肉桂地链霉菌(S.cinnamonensis)icm (异丁酰-CoA变位酶) 255 256 肉桂地链霉菌icmB (异丁酰-CoA变位酶) 257 258 天蓝色链霉菌SCO5415 (异丁酰-CoA变位酶) 259 260 天蓝色链霉菌SCO4800 (异丁酰-CoA变位酶) 261 262 除虫链霉菌(S.avermitilis)icmA (异丁酰-CoA变位酶) 263 264 除虫链霉菌icmB (异丁酰-CoA变位酶) 265 266 SEQ ID NO:11-38,40-69,72-75,85-138,144,145,147-157, 159-176是本文所述实施例中使用的寡核苷酸克隆,筛选或测序引物的 核苷酸序列。 SEQ ID NO:39是实施例16中所述的cscBKA基因簇的核苷酸序 列。 SEQ ID NO:70是实施例13中所述的葡萄糖异构酶启动子1.6GI 的核苷酸序列。 SEQ ID NO:71是实施例13中所述的1.5GI启动子的核苷酸序列。 SEQ ID NO:76是实施例17中所述的GPD启动子的核苷酸序列。 SEQ ID NO:77是实施例17中所述的CYC1终止子的核苷酸序列。 SEQ ID NO:79是实施例17中所述的FBA启动子的核苷酸序列。 SEQ ID NO:81是实施例17中所述的ADH 1启动子的核苷酸序列。 SEQ ID NO:82是实施例17中所述的ADH1终止子的核苷酸序列。 SEQ ID NO:84是实施例17中所述的GMP启动子的核苷酸序列。 SEQ ID NO:139是蔗糖水解酶(CscA)的氨基酸序列。 SEQ ID NO:140是D-果糖激酶(CscK)的氨基酸序列。 SEQ ID NO:141是蔗糖透性酶(CscB)的氨基酸序列。 SEQ ID NO:142是实施例20中所述的质粒pPF988DssPspac的核 苷酸序列。 SEQ ID NO:143是实施例20中所述的质粒pPF988DssPgroE的核 苷酸序列。 SEQ ID NO:146是实施例20中所述的pPF988Dss载体片段的核 苷酸序列。 SEQ ID NO:177是实施例21中所述的质粒pPF988整合载体的核 苷酸序列。 SEQ ID NO:267是实施例21中所述的质粒pC194的核苷酸序列。 发明详述 本发明涉及使用重组微生物生产异丁醇的方法。本发明满足各种 商业和工业需求。丁醇是具有多种应用的重要工业日用化学品,其中 其作为燃料或燃料添加剂的潜能是特别重要的。尽管只是四碳醇,丁 醇具有与汽油相似的能含量并可以与任何矿物燃料混合。喜欢将丁醇 作为燃料或燃料添加剂,因为它在标准内燃机中燃烧时只产生CO2并 且几乎没有SOX或NOX。此外,丁醇比迄今为止最优选的燃料添加剂 乙醇的腐蚀性小。 除了作为生物燃料或燃料添加剂的用途,丁醇具有影响新兴燃料 电池工业中氢分配问题的潜能。目前燃料电池受到与氢运输和分配相 关的安全问题的困扰。对于氢含量可以将丁醇容易地重组,并可以通 过现有的加油站分配,以燃料电池或车辆需要的纯度。 最后,本发明从源自植物的碳源生产异丁醇,避免了与丁醇生产 的标准石化方法相关的负面环境影响。 以下的定义和缩写用于权利要求和说明书的解释。 如在此所用的术语“发明”和“本发明”是非限制性的术语,不 是用来指特定发明的任何单个实施方案,而是包括如说明书和权利要 求中所述的所有可能的实施方案。 术语“异丁醇生物合成途径”指的是产生异丁醇的酶途径。 在此可交替使用的术语“乙酰乳酸合酶”和“乙酰乳酸合成酶” 指的是催化丙酮酸转化成乙酰乳酸和CO2的酶。优选的乙酰乳酸合酶 已知为EC编号2.2.1.69(Enzyme Nomenclature(酶命名)1992,Academic Press,San Diego)。这些酶可从各种来源获得,包括但不限于,枯草芽 孢杆菌(GenBank No:CAB 15618(SEQ ID NO:178),Z99122(SEQ ID NO:78),各自为NCBI(国立生物技术信息中心)氨基酸序列, NCBI核苷酸序列),肺炎克雷伯氏菌(GenBank No:AAA25079(SEQ ID NO:2),M73842(SEQ ID NO:1)),和乳酸乳球菌(GenBank No: AAA25161(SEQ ID NO:180),L16975(SEQ ID NO:179))。 在此可交替使用的术语“乙酰醇酸异构还原酶”和“乙酰醇酸还 原异构酶”指的是使用NADPH(还原型烟酰胺腺嘌呤二核苷酸磷酸酯) 作为电子供体催化乙酰乳酸转化成2,3-二羟基异戊酸的酶。优选的乙酰 醇酸异构还原酶已知为EC编号1.1.1.86,并且可从大量的微生物阵列 中获得序列,这些微生物包括但不限于,大肠杆菌(GenBank No: NP_418222(SEQ ID NO:4),NC_000913(SEQ ID NO:3)),酿酒酵 母(GenBank No:NP_013459(SEQ ID NO:181),NC_001144(SEQ ID NO:80)),海藻甲烷球菌(GenBank No:CAF30210(SEQ ID NO: 183),BX957220(SEQ ID NO:182)),和枯草芽孢杆菌(GenBank No: CAB 14798(SEQ ID NO:158),Z99118(SEQ ID NO:184))。 术语“乙酰醇酸脱水酶”指的是催化2,3-二羟基异戊酸转化成α- 酮基异戊酸的酶。优选的乙酰醇酸脱水酶已知为EC编号4.2.1.9。这些 酶可从大量的微生物阵列中获得序列,这些微生物包括但不限于,大 肠杆菌(GenBank No:YP_026248(SEQ ID NO:6),NC_000913(SEQ ID NO:5)),酿酒酵母(GenBank No:NP_012550(SEQ ID NO:186), NC_001142(SEQ ID NO:83)),海藻甲烷球菌(GenBank No:CAF29874 (SEQ ID NO:188),BX957219(SEQ ID NO:187)),和枯草芽孢杆 菌(GenBank No:CAB14105(SEQ ID NO:190),Z99115(SEQ ID NO: 189))。 术语“支链α-酮酸脱羧酶”指的是催化α-酮基异戊酸转化成异丁 醛和CO2的酶。优选的支链α-酮酸脱羧酶已知为EC编号4.1.1.72并可 从各种来源获得,包括但不限于,乳酸乳球菌(GenBank No:AAS49166 (SEQ ID NO:193),AY548760(SEQ ID NO:192);CAG34226(SEQ ID NO:8),AJ746364(SEQ ID NO:191),鼠伤寒沙门氏菌(GenBank No:NP_461346(SEQ ID NO:195),NC_003197(SEQ ID NO:194)), 和丙醇丁酮梭菌(GenBank No:NP_149189(SEQ ID NO:197), NC_001988(SEQ ID NO:196))。 术语“支链醇脱氢酶”指的是催化异丁醛转化成异丁醇的酶。优 选的支链醇脱氢酶已知为EC编号1.1.1.265,但也可以归类于其他醇脱 氢酶(特别是,EC 1.1.1.1或1.1.1.2)。这些酶使用NADH(还原型烟 酰胺腺嘌呤二核苷酸)和/或NADPH作为电子供体,并从各种来源可 获得,包括但不限于,酿酒酵母(GenBank No:NP_010656(SEQ ID NO: 199),NC_001136(SEQ ID NO:198);NP_014051(SEQ ID NO:201), NC_001145(SEQ ID NO:200)),大肠杆菌(GenBank No:NP_417484 (SEQ ID NO:10),NC_000913(SEQ ID NO:9)),和丙醇丁酮梭菌 (GenBank No:NP_349892(SEQ ID NO:203),NC_003030(SEQ ID NO:202);NP_349891(SEQ ID NO:204),NC_003030(SEQ ID NO: 158))。 术语“支链酮酸脱氢酶”指的是催化α-酮基异戊酸转化成异丁酰 -CoA(异丁酰-辅酶A)的酶,使用NAD+(烟酰胺腺嘌呤二核苷酸) 作为电子受体。优选的支链酮酸脱氢酶已知为EC编号1.2.4.4。这些支 链酮酸脱氢酶由四个亚基构成,并且所有亚基的序列可从大量微生物 阵列中获得,这些维生物包括但不限于,枯草芽孢杆菌(GenBank No: CAB14336(SEQ ID NO:206),Z99116(SEQ ID NO:205);CAB14335 (SEQ ID NO:208),Z99116(SEQ ID NO:207);CAB14334(SEQ ID NO:210),Z99116(SEQ ID NO:209);和CAB14337(SEQ ID NO: 212),Z99116(SEQ ID NO:211))和恶臭假单胞菌(GenBank No: AAA65614(SEQ ID NO:214),M57613(SEQ ID NO:213);AAA65615 (SEQ ID NO:216)。M57613(SEQ ID NO:215);AAA65617(SEQ ID NO:218),M57613(SEQ ID NO:217);和AAA65618(SEQ ID NO: 220),M57613(SEQ ID NO:219))。 术语“酰化醛脱氢酶”指的是催化异丁酰-CoA转化成异丁醛的酶, 使用NADH或NADPH作为电子供体。优选的酰化醛脱氢酶已知为EC 编号1.2.1.10和1.2.1.57。这些酶可从多个来源获得,包括但不限于, 拜氏梭菌(GenBank No:AAD31841(SEQ ID NO:222),AF157306 (SEQ ID NO:221)),丙醇丁酮梭菌(GenBank No:NP_149325(SEQ ID NO:224),NC_001988(SEQ ID NO:223);NP_149199(SEQ ID NO:226),NC_001988(SEQ ID NO:225)),恶臭假单胞菌(GenBank No:AAA89106(SEQ ID NO:228),U13232(SEQ ID NO:227)), 和嗜热栖热菌(GenBank No:YP_145486(SEQ ID NO:230),NC_006461 (SEQ ID NO:229))。 术语“转氨酶”指的是催化α-酮基异戊酸转化成L-缬氨酸的酶, 使用丙氨酸或谷氨酸作为胺供体。优选的转氨酶已知为EC编号 2.6.1.42和2.6.1.66。这些酶可从多种来源获得。丙氨酸依赖性酶的来 源实例包括,但不限于,大肠杆菌(GenBank No:YP_026231(SEQ ID NO:232),NC_000913(SEQ ID NO:231))和地衣芽孢杆菌(GenBank No:YP_093743(SEQ ID NO:234),NC_006322(SEQ ID NO:233))。 谷氨酸依赖性酶的来源实例包括,但不限于,大肠杆菌(GenBank No: YP_026247(SEQ ID NO:236),NC_000913(SEQ ID NO:235)),酿 酒酵母(GenBank No:NP_012682(SEQ ID NO:238),NC_001142 (SEQ ID NO:237))和嗜热碱甲烷杆菌(GenBank No:NP_276546 (SEQ ID NO:240),NC_000916(SEQ ID NO:239))。 术语“缬氨酸脱氢酶”指的是催化α-酮基异戊酸转化成L-缬氨酸 的酶,使用NAD(P)H作为电子供体和氨作为胺供体。优选的缬氨酸脱 氢酶已知为EC编号1.4.1.8和1.4.1.9,并可从各种来源获得,包括但 不限于,天蓝色链霉菌(GenBank No:NP_628270(SEQ ID NO:242), NC_003888(SEQ ID NO:241))和枯草芽孢杆菌(GenBank No: CAB14339(SEQ ID NO:244),Z99116(SEQ ID NO:243))。 术语“缬氨酸脱羧酶”指的是催化L-缬氨酸转化成异丁胺和CO2 的酶。优选的缬氨酸脱羧酶已知为EC编号4.1.1.14。这些酶可在链霉 菌中找到,如例如,生绿链霉菌(GenBank No:AAN10242(SEQ ID NO: 246),AY116644(SEQ ID NO:245))。 术语“ω转氨酶”指的是催化异丁胺转化成异丁醛的酶,使用合适 的氨基酸作为胺供体。优选的ω转氨酶已知为EC编号2.6.1.18并可从 各种来源获得,包括但不限于,反硝化产碱菌(AAP92672(SEQ ID NO: 248),AY330220(SEQ ID NO:247)),真养雷氏菌(GenBank No: YP_294474)(SEQ ID NO:250),NC_007247(SEQ ID NO:249)), Shewanella oneidensis(GenBank No:NP_719046(SEQ ID NO:252), NC_004347(SEQ ID NO:251)),和恶臭假单胞菌(GenBank No: AAN66223(SEQ ID NO:254),AE016776(SEQ ID NO:253))。 术语“异丁酰-CoA突变酶”指的是催化丁酰-CoA转化成异丁酰 -CoA的酶。这种酶使用辅酶B12作为辅因子。优选的异丁酰-CoA突变 酶已知为EC编号5.4.99.13。在各种链霉菌中找到这种酶,包括但不限 于,肉桂地链霉菌(GenBank No:AAC08713(SEQ ID NO:256),U67612 (SEQ ID NO:255);CAB59633(SEQ ID NO:258),AJ246005(SEQ ID NO:257)),天蓝色链霉菌(GenBank No:CAB70645(SEQ ID NO: 260),AL939123(SEQ ID NO:269);CAB92663(SEQ ID NO:262); AL939121(SEQ ID NO:261));和除虫链霉菌(GenBank No:NP_824008 (SEQ ID NO:264),NC_003155(SEQ ID NO:263);NP_824637(SEQ ID NO:266),NC_003155(SEQ ID NO:265))。 术语“兼性厌氧菌”指的是在好氧和厌氧环境中都可以生长的微 生物。 术语“碳底物”或“可发酵碳底物”指的是能够被本发明的宿主 微生物代谢的碳源,并且是选自单糖,寡糖,多糖和单碳底物或其混 合物的碳源。 术语“基因”指的是能够表达为特定蛋白质的核酸片段,任选包 括编码序列之前(5’非-编码序列)和之后(3’非-编码序列)的调控序 列。“天然基因”指的是与其自身调控序列在自然状态发现的基因。 “嵌合基因”指的是不是天然基因的任何基因,包括不是在自然状态 一起发现的调控序列和编码序列。因此,嵌合基因可以包括来自不同 来源的调控序列和编码序列,或调控序列和编码序列源自相同的来源, 但以不同于自然状态发现的方式排列。“内源基因”指的是处于其在生 物体基因组中的天然位置的天然基因。“外源基因”或“异源基因”指 的是通常不是在宿主生物体中发现的而是通过基因转移引入宿主生物 体中的基因。外源基因可以包括插入非天然生物体中的天然基因,或 嵌合基因。“转基因”是已经通过转化程序引入基因组中的基因。 如在此所用的,术语“编码序列”指的是编码特定氨基酸序列的 DNA序列。“合适的调控序列”指的是位于编码序列上游(5’非-编码 序列),之内或下游(3’非-编码序列)的核苷酸序列,并且其影响相连 编码序列的转录,RNA加工或稳定性,或翻译。调控序列可以包括启 动子,翻译前导序列,内含子,多腺苷酸化识别序列,RNA加工位点, 效应子结合位点和茎-环结构。 术语“启动子”指的是能够控制编码序列或功能性RNA表达的 DNA序列。通常,编码序列位于启动子序列的3’端。启动子可以完全 源自天然基因,或由源自自然状态发现的不同启动子的不同序列构成, 或甚至包括合成的DNA片段。本领域技术人员应当理解不同的启动子 可以指导不同组织或细胞类型中的,或在不同的发育阶段的,或应答 不同的环境或生物条件的基因表达。使基因在大部分时间在大部分细 胞类型中得到表达的启动子通常称为“组成型启动子”。进一步认识到 因为在大部分情况中,调控序列的确切分界线还没有得到完全限定, 因此不同长度的DNA片段可能具有相同的启动子活性。 术语“可操纵地连接”指的是单个核酸片段上的核酸序列连接, 使得一个的功能受到另一个的影响。例如,在其能够影响编码序列的 表达时,启动子可操纵地连接编码序列(即,编码序列处于启动子的 转录控制下)。可以将编码序列以正义或反义方向可操纵地连接调控序 列。 如在此所用的术语“表达”指的是源自本发明核酸片段的正义 (mRNA)或反义RNA的转录和稳定累积。表达还可以指mRNA翻译 成多肽。 如在此所用的术语“转化”指的是核酸片段转移至宿主生物体中, 导致遗传上稳定的遗传。将含有转化的核酸片段的宿主生物体称为“转 基因”或“重组”或“转化的”生物体。 术语“质粒”,“载体”和“盒”指的是额外的染色体序列,其通 常携带不是细胞重要代谢的一部分的基因,且通常是环状双链DNA片 段的形式。这样的序列可以是源自任何来源的单链或双链DNA或RNA 的自主复制序列,基因组整合序列,噬菌体或核苷酸序列,线性或环 状的,其中各种核苷酸序列已经连接或重组成独特的构建体,其能够 将用于选定的基因产物的启动子片段和DNA序列以及合适的3’未翻译 序列引入细胞中。“转化盒”指的是含有外源基因并具有除了促进特定 宿主细胞转化的外源基因以外的序列的特定载体。“表达盒”指的是含 有外源基因并具有除了允许该基因在外源宿主中提高的表达的外源基 因以外的序列的特定载体。 如在此所用的术语“密码子简并性”指的是遗传密码中允许核苷 酸序列改变而没有影响所编码多肽的氨基酸序列的性质。本领域技术 人员公知核苷酸密码子使用中由特定宿主细胞呈现的“密码子偏好” 确定了给定的氨基酸。因此,合成用于在宿主细胞中提高表达的基因 时,理想的是设计基因使得密码子使用的频率接近宿主细胞的优选密 码子使用的频率。 术语“密码子最佳化”,当涉及用于各种宿主转化的基因和核酸分 子的编码片段时,指的是基因或核酸分子编码片段中的密码子改变, 这种改变反应出宿主生物体的典型密码子使用而没有改变由DNA编码 的多肽。 在此使用的标准重组DNA和分子克隆技术是本领域公知的,并描 述于Sambrook,J.,Fritsch,E.F.和Maniatis,T.,Molecular Cloning: ALaboratory Manual(分子克隆:实验室手册),第二版,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY(1989)(此后称为 “Maniatis”);和Silhavy,T.J.,Bennan,M.L.和Enquist,L.M., Experiments with Gene Fusions(使用基因融合的表达),Cold Spring Harbor Laboratory Press.Cold Spring Harbor,NY(1984);和Ausubel, F.M.等,Current Protocols in Molecular Biology(分子生物学中的通用 实验方案),Greene Publishing Assoc.and Wiley-Interscience出版 (1987)。 异丁醇生物合成途径 利用碳水化合物的微生物使用Embden-Meyerhof-Parnas(EMP)途 径,Entner-Doudoroff途径和戊糖磷酸酯循环作为主要的代谢途径来提 供用于生长和维持的能量和细胞前体。这些途径具有共同的中间产物 甘油醛-3-磷酸,并且最终直接或结合EMP途径形成丙酮酸。随后,丙 酮酸通过各种方式转化成乙酰-辅酶A(乙酰-CoA)。乙酰-CoA用作关 键的中间产物,例如,在产生脂肪酸,氨基酸和次级代谢产物中。糖 转化成丙酮酸的组合反应产生了能量(例如,腺苷-5’-三磷酸,ATP) 和还原等价物(例如,还原型烟酰胺腺嘌呤二核苷酸,NADH,和还原 型烟酰胺腺嘌呤二核苷酸磷酸酯,NADPH)。NADH和NADPH必须 再循环成其氧化形式(各自为NAD+和NAPD+)。在无机电子受体(例 如,O2,NO3-和SO42-)的存在下,还原等价物可以用来扩大能量池; 或者,可以形成还原碳副产物。 本发明能够通过提供四个完整的反应途径使用重组微生物从碳水 化合物来源产生异丁醇,如图1中所示的。其中三个途径包括丙酮酸 通过一系列的酶步骤转化成异丁醇。优选的异丁醇步骤(图1,步骤a 至e),包括以下的底物至产物的转化: a)丙酮酸至乙酰乳酸,例如由乙酰乳酸合酶催化的, b)乙酰乳酸至2,3-二羟基异戊酸,例如由乙酰醇酸异构还原酶催 化的, c)2,3-二羟基异戊酸至α-酮基异戊酸,例如由乙酰醇酸脱水酶催 化的, d)α-酮基异戊酸至异丁醛,例如由支链酮酸脱羧酶催化的, e)异丁醛至异丁醇,例如由支链醇脱氢酶催化的。 该途径结合了已知在公认的缬氨酸生物合成(丙酮酸至α-酮基异 戊酸)和缬氨酸分解代谢(α-酮基异戊酸至异丁醇)途径中涉及的酶。 因为许多缬氨酸生物合成酶还在异亮氨酸生物合成途径中催化类似的 反应,因此底物特异性在选择基因来源中是一个主要的考虑因素。为 此,对于乙酰乳酸合酶的主要目标基因是来自芽孢杆菌(alaS)和克雷 伯氏菌(budB)的那些。已知这些特定的乙酰乳酸合酶参与这些生物 体中的丁二醇发酵,并显示出对丙酮酸的亲和性高于酮丁酸(Gollop 等,J.Bacteriol.172(6):3444-3449(1990);Holtzclaw等,J.Bacteriol. 121(3):917-922(1975))。第二个和第三个途径步骤各自是由乙酰醇 酸还原异构酶和脱水酶催化的。已经从各种来源表征了这些酶,如例 如,大肠杆菌(Chunduru等,Biochemistry 28(2):486-493(1989);Flint 等,J.Biol.Chem.268(29):14732-14742(1993))。已知优选的异丁醇 途径的最后两个步骤在酵母中发生,酵母可以使用缬氨酸作为氮源, 并且在过程中分泌异丁醇。α-酮基异戊酸可以通过各种酮酸脱羧酶转化 成异丁醛,如例如丙酮酸脱羧酶。为了防止丙酮酸远离异丁醇产生的 方向错误,对丙酮酸的亲和性降低的脱羧酶是理想的。迄今为止,本 领域中已知存在两种这样的酶(Smit等,Appl.Environ.Microbiol.71(1): 303-311(2005);de la Plaza等,FEMS Microbiol.Lett.238(2):367-374 (2004))。两种酶都来自乳酸乳球菌的菌株并对酮异戊酸的偏好超过 丙酮酸50-200倍。最后,在酵母中已经鉴定了多种醛还原酶,许多具 有重叠的底物特异性。已知偏好支链底物胜于乙醛的那些包括但不限 于,醇脱氢酶VI(ADH6)和Ypr1p(Larroy等,Biochem.J.361(Pt1): 163-172(2002);Ford等,Yeast 19(12):1087-1096(2002)),两者都 使用了NADPH作为电子供体。最近已经在大肠杆菌中鉴定了用支链底 物激活的NADPH-依赖性还原酶,YqhD(Sulzenbacher等,J.Mol. Biol.342(2):489-502(2004))。 丙酮酸转化成异丁醇的另一个途径包括以下的底物至产物的转化 (图1,步骤a,b,c,f,g,e): a)丙酮酸至乙酰乳酸,例如由乙酰乳酸合酶催化的, b)乙酰乳酸至2,3-二羟基异戊酸,例如由乙酰醇酸异构还原酶催 化的, c)2,3-二羟基异戊酸至α-酮基异戊酸,例如由乙酰醇酸脱水酶催 化的, f)α-酮基异戊酸至异丁酰-CoA,例如由支链酮酸脱氢酶催化的, g)异丁酰-CoA至异丁醛,例如由酰化醛脱氢酶催化的, e)异丁醛至异丁醇,例如由支链醇脱氢酶催化的。 该途径中的头三个步骤(a,b,c)和上述的那些相同。通过支链 酮酸脱氢酶的作用将α-酮基异戊酸转化成异丁酰-CoA。虽然酵母只使 用缬氨酸作为氮源,但许多其他生物体(真核生物和原核生物)可以 也可以使用缬氨酸作为碳源。这些生物体具有支链酮酸脱氢酶(Sokatch 等,J.Bacteriol.148(2):647-652(1981)),其产生异丁酰-CoA。异丁 酰-CoA通过酰化醛脱氢酶可以转化成异丁醛。已经描述了明串珠菌 (Leuconostoc)和丙酸杆菌(Propionibacterium)中用支链底物激活的 脱氢酶,但还没有克隆(Kazahaya等,J.Gen.Appl.Microbiol.18:43-55 (1972);Hosol等,J.Ferment.Technol.57:418-427(1979))。然而, 还可能的是已知作用于直链酰基-CoA(即,丁酰-CoA)的酰化醛脱氢 酶也作用于异丁酰-CoA。然后异丁醛通过支链醇脱氢酶转化成异丁醇, 如以上对于第一个途径中所述的。 丙酮酸转化成异丁醇的另一个途径包括以下的底物至产物的转化 (图1,步骤a,b,c,h,i,j,e): a)丙酮酸至乙酰乳酸,例如由乙酰乳酸合酶催化的, b)乙酰乳酸至2,3-二羟基异戊酸,例如由乙酰醇酸异构还原酶催 化的, c)2,3-二羟基异戊酸至α-酮基异戊酸,例如由乙酰醇酸脱水酶催 化的, h)α-酮基异戊酸至缬氨酸,例如由缬氨酸脱氢酶或转氨酶催化的, i)缬氨酸至异丁胺,例如由缬氨酸脱羧酶催化的, j)异丁胺至异丁醛,例如由ω转氨酶催化的,和 e)异丁醛至异丁醇,例如由支链醇脱氢酶催化的。 该途径中的头三个步骤(a,b,c)和上述的那些相同。该途径需 要加入缬氨酸脱氢酶或合适的转氨酶。缬氨酸(和或亮氨酸)脱氢酶 催化还原性胺化并使用氨;氨的Km值在毫摩尔范围中(Priestly等, Biochem J.261(3):853-861(1989);Vancura等,J.Gen.Microbiol. 134(12):3213-3219(1988),Zink等,Arch.Biochem.Biophys.99:72-77 (1962);Sekimoto等,J.Biochem(日本)116(1):176-182(1994))。 转氨酶通常使用谷氨酸或丙氨酸作为氨基供体,并已经从各种生物体 得到了表征(Lee-Peng等,J.Bacteriol.139(2):399-345(1979);Berg 等,J.Bacteriol.155(3):1009-1014(1983))。丙氨酸-特异性酶是理想 的,因为除去胺基时,该步骤的丙酮酸产生可以结合途径中后来的丙 酮酸消耗(参见以下的)。下一个步骤是缬氨酸的脱羧作用,这是在链 霉菌中的valanimycin生物合成中发生的反应(Gang等,Mol.Microbiol. 46(2):505-517(2002))。所得到的异丁胺可以在吡哆醛5’-磷酸-依赖 性反应中转化成异丁醛,例如通过ω-转氨酶家族的酶。已经证明来自 河流弧菌(Vibrio fluvialis)这样的酶具有异丁胺活性(Shin等,Biotechnol. Bioeng.65(2):206-211(1999))。已经克隆了另一种来自反硝化产碱菌 的ω-转氨酶并且具有一定的丁胺活性(Yun等,Appl.Environ.Microbiol. 70(4):2529-2534(2004))。在这种方向中,这些酶使用丙酮酸作为氨 基受体,产生丙氨酸。如上所述,在途径的早期使用产生丙酮酸的转 氨酶可以抵消对丙酮酸池的不利影响。然后异丁醛通过支链醇脱氢酶 转化成异丁醇,如以上对于第一个途径所述的。 第四个异丁醇生物合成途径包括图1中步骤k,g,e所示的底物 至产物的转化。已知各种生物体通过丁酰-CoA中间产物产生丁酸和/ 或丁醇(Dürre等,FEMS Microbiol.Rev.17(3)251-262(1995); Abbad-Andaloussi等,Microbiology 142(5):1149-1158(1996))。通过 加入能够转化丁酰-CoA至异丁酰-CoA的突变酶在这些生物体中工程 化异丁醇产生(图1,步骤k)。已经从链霉菌克隆了异丁酰-CoA突变 酶(辅酶B12-依赖性酶)的两个亚基的基因(Ratnatilleke等,J.Biol. Chem.274(44):31679-31685(1999))。异丁酰-CoA转化成异丁醛(图 1中的步骤g),其转化成异丁醇(图1中的步骤e)。 因此,在提供丙酮酸至异丁醇的多个重组途径中,存在各种选择 来满足单个转化步骤,并且本领域技术人员将能够利用公众可获得的 序列来构建相关的途径。以下的表2中列出了本领域已知的且异丁醇 生物合成途径的构建中有用的代表性基因的列表。 表2 异丁醇生物合成途径基因的来源 基因 GenBank引用 乙酰乳酸合酶 Z99122,枯草芽孢杆菌完整基因组(21个片段中19个): 从3608981至3809670gi|3248830|emb|Z99122.2|BSUB0019[32468830] M73842,肺炎克雷伯氏菌乙酰乳酸合酶(iluk)基因,完整cds gi|1429210|gb|M73842.1|KPNILUK[149210] L16975,乳酸乳球菌α-乙酰乳酸合酶(als)基因,完整cds gi|473900|gb|L16975.1|LACALS[473900] 乙酰醇酸异构还 原酶 NC_000913,大肠杆菌K12,完整基因组 gi|49175990|ref|NC_000913.2|[49175990] NC_001144,酿酒酵母染色体XII,完整的染色体序列 gi|42742286|ref|NC_001144.3|[42742286] BX957220,海藻甲烷球菌S2完整基因组;片段2/5 gi|44920669|emb|BX957220.1|[44920669] Z99118,枯草芽孢杆菌完整基因组(21个片段中15个):从2812801 至3013507 gi|32468802|emb|Z99118.2|BSUB0015[32468802] 乙酰醇酸脱水酶 NC_000913,大肠杆菌K12,完整基因组 gi|49175990|ref|NC_000913.2|[49175990] NC_001142,酿酒酵母染色体X,完整染色体序列 gi|42742252|ref|NC_001142.5|[42742252] BX957219,海藻甲烷球菌S2完整基因组;片段1/5 gi|45047123|emb|BX957219.1|[45047123] Z99115,枯草芽孢杆菌完整基因组(21个片段中12个):从2207806 至2409180 gi|32468778|emb|Z99115.2|BSUB0012|[32468778] 支链α酮酸脱羧 酶 AY548760,乳酸乳球菌支链α酮酸脱羧酶(kdcA)基因,完整cds gi|44921616|gb|AY548760.1|[44921616] AJ746364,α酮异戊酸脱羧酶的乳酸乳球菌subsp.lactis kivd基因,菌 株IFPL730 gi|51870501|emb|AJ746364.1|[51870501] NC_003197,鼠伤寒沙门氏菌LT2,完整基因组 gi|16763390|ref|NC_003197.1|[16763390] NC_001988,丙醇丁酮梭菌ATCC 824质粒pSOL1,完整序列 gi|15004705|ref|NC_001988.2|[15004705] 支链醇脱氢酶 NC_001136,酿酒酵母染色体IV,完整染色体序列 gi|50593138|ref|NC_001136.6|[50593138] NC_001145,酿酒酵母染色体XIII,完整染色体序列 gi|44829554|ref|NC_001145.2|[44829554] NC_000913,大肠杆菌K12,完整基因组 gi|49175990|ref|NC_000913.2|[49175990] NC_003030,丙醇丁酮梭菌ATCC824,完整基因组 gi|15893298|ref|NC_003030.1|[15893298] 支链酮酸脱氢酶 Z99116,枯草芽孢杆菌完整基因组(21个片段中13个):从2409151 至2613687 gi|32468787|emb|Z99116.2|BSUB0013[32468787] M57613,恶臭假单胞菌支链酮酸脱氢酶操纵子(bkdA1,bkdA1和 bkdA2),转酰酶E2(bkdB),bkdR和硫辛酰胺脱氢酶(lpdV)基因, 完整cds gi|790512|gb|M57613.1|PSEBKDPPG2[790512] 酰化醛脱氢酶 AF157306,拜氏梭菌菌株NRRL B593假定的蛋白,辅酶A酰化醛脱 氢酶(ald),乙酰醋酸:丁酸/醋酸辅酶A转移酶(ctfA),乙酰醋酸:丁 酸/醋酸辅酶A转移酶(ctfB),和乙酰醋酸脱羧酶(adc)基因,完整 cds gi|47422980|gb|AF157306.2|[47422980] NC_001988,丙醇丁酮梭菌ATCC824质粒pSOL1,完整序列 gi|15004705|ref|NC_001988.2|[15004705] U13232,恶臭假单胞菌NCIB9816乙醛脱氢酶(nahO)和4-羟基-2- 氧戊酸醛缩酶(nahM)基因,完整cds,和4-草酰巴豆酯脱羧酶(nahK) 基因和2-氧代-4-戊酸甲酯水合酶(nahL)基因,部分cds gi|595671|gb|U13232.1|PPU13232[595671] 转氨酶 NC_000913,大肠杆菌K12,完整基因组 gi|49175990|ref|NC_000913.2|[49175990] NC_006322,地衣芽孢杆菌ATCC14580,完整基因组 gi|52783855|ref|NC_006322.1|[52783855] NC_001142,酿酒酵母染色体X,完整染色体序列 gi|42742252|ref|NC_001142.5|[42742252] NC_000916,嗜热碱甲烷杆菌str.Delta H,完整基因组 gi|15678031|ref|NC_000916.1|[15678031] 缬氨酸脱氢酶 NC_003888,天蓝色链霉菌A3(2),完整基因组 gi|32141095|ref|NC_003888.3|[321410945] Z99116,枯草芽孢杆菌完整基因组(21个片段中13个):从2409151 至2613687 gi|32468787|emb|Z99116.2|BSUB0013[32468787] 缬氨酸脱羧酶 AY116644,生绿链霉菌氨基酸转氨酶基因,部分cds;酮酸还原异构 酶,乙酰乳酸合酶小亚基,乙酰乳酸合酶大亚基,完整cds;氧化偶 氮基抗生素valanimycin基因簇,完整序列;以及公认的转移酶,和 公认的分泌的蛋白基因,完整cds gi|27777548|gb|AY116644.1|[27777548] ω转氨酶 AY330220,反硝化产碱菌ω氨基酸:丙酮酸转氨酶(aptA)基因,完 整cds gi|33086797|gb|AY330220.1|[33086797] NC_007347,真养雷氏菌JMP134染色体1,完整序列 gi|73539706|ref|NC_007347.1|[73539706] NC_004347,Shewanella oneidensis MR-1,完整基因组 gi|24371600|ref|NC_004347.1|[24371600] NZ_AAAG02000002,深红红螺菌(Rhodospirillum rubrum)Rrub02_2, 全基因组鸟枪序列 gi|48764549|ref|NZ_AAAG02000002.1|[48764549] AE016776,恶臭假单胞菌KT2440,完整基因组的21个片段中的3 个 gi|26557019|gb|AE016776.1|[26557019] 异丁酰-CoA突 变酶 U67612,肉桂地链霉菌辅酶B12-依赖性异丁酰CoA突变酶(icm)基 因,完整cds gi|3002491|gb|U67612.1|SCU67612[3002491] AJ246005,异丁酰-CoA突变酶的肉桂地链霉菌icmB基因,小亚基 gi|6137076|emb|AJ246005.1|SCI246005[6137076] AL939123,天蓝色链霉菌A3(2)完整基因组;片段20/29 gi|24430032|emb|AL939123.1|SCO939123[24430032] AL9939121,天蓝色链霉菌A3(2)完整基因组;片段18/29 gi|24429533|emb|AL939121.1|SCO939121[24429533] NC_003155,除虫链霉菌MA-4680,完整基因组 gi|57833846|ref|NC_003155.3|[57833846| 用于异丁醇生产的微生物宿主 用于异丁醇生产的微生物宿主可以选自细菌,蓝细菌,丝状真菌 和酵母。用于异丁醇生产的微生物宿主优选耐异丁醇,使得产量不受 到丁醇毒性的限制。在高异丁醇滴定水平具有代谢活性的微生物不是 本领域公知的。尽管已经从产溶剂(solventogenic)梭菌分离了耐丁醇的 突变体,但是关于其他潜在有用菌株的丁醇耐受性的信息获得得很少。 对于细菌醇耐受性比较的大部分研究表明丁醇比乙醇更毒(de Cavalho 等,Microsc.Res.Tech.64:215-22(2004)和Kabelitz等,FEMS Microbiol. Lett.220:223-227(2003))。Tomas等(J.Bacteriol.186:2006-2018 (2004))报道了丙醇丁酮梭菌发酵过程中的1-丁醇产量受到了1-丁醇 毒性的限制。1-丁醇对丙醇丁酮梭菌的主要影响是膜功能的破坏 (Hermann等,Appl.Environ.Microbiol.50:1238-1243(1985))。 选择用于异丁醇生产的微生物宿主优选耐异丁醇,并应当能够将 碳水化合物转化成异丁醇。选择合适微生物宿主的标准包括以下的: 内在的异丁醇耐受性,高葡萄糖利用率,用于基因操纵的遗传工具的 可用性和产生稳定的染色体改变的能力。 可以通过基于菌株内在耐受性的筛选来鉴定具有异丁醇耐受性的 合适宿主菌株。可以通过测定在最小培养基中生长时引起50%生长速 率抑制(IC50)的异丁醇浓度来测量微生物对异丁醇的内在耐受性。 可以使用本领域已知的方法来测定IC50值。例如,可以将目标微生物 在各种含量的异丁醇存在下生长并通过测量600纳米处的光密度来监 控生长速率。从生长曲线的对数部分计算倍增时间并用作生长速率的 测量。可以从生长的百分比抑制对异丁醇浓度的图中测定出产生50% 生长抑制的异丁醇浓度。优选,宿主菌株对于异丁醇应当具有高于约 0.5%的IC50。 用于异丁醇生产的微生物宿主还应当以高速率来利用葡萄糖。大 部分微生物能够利用碳水化合物。然而,特定的环境微生物不能高效 地利用碳水化合物,并因此不是合适的宿主。 在遗传上修饰宿主的能力对于任何重组微生物的产生是必须的。 基因转移技术的方式可以通过电穿孔,缀合,转导或天然转化。各种 宿主可缀合质粒和抗药性标记是可获得的。基于在该宿主中抗生素抗 性标记的性质,对宿主生物体定制克隆载体。 微生物宿主还必须得到操纵,以便通过删除各种基因使碳流的竞 争途径失活。这需要利用指导失活的转座子或染色体整合载体。此外, 生产宿主应当顺从化学诱变,使得可以获得提高内在异丁醇耐受性的 突变。 基于上述的标准,用于异丁醇生产的合适微生物宿主包括,但不 限于,梭菌属,发酵单胞菌属(Zymomonas),埃希氏菌属(Escherichia), 沙门氏菌属,红球菌属(Rhodococcus),假单胞菌属,芽孢杆菌属,乳 杆菌属(Lactobacillus),肠球菌属(Enterococcus),产碱杆菌属 (Alcaligenes),克雷伯氏菌属,类芽孢杆菌属(Paenibacillus),节杆 菌属(4rthrobacter),棒状杆菌属(Corynebacterium),短杆菌属 (Brevibacterium),毕赤氏酵母属(Pichia),假丝酵母属(Candida), 汉森酵母属(Hansenula)和酵母属(Saccharomyces)的成员。优选的 宿主包括:大肠杆菌(Escherichia coli),真养产碱杆菌(Alcaligenes eutrophus),地衣芽孢杆菌,浸麻类芽孢杆菌(Paenibacillus macerans), 红串红球菌(Rhodococcus erythropolis),恶臭假单胞菌,植物乳杆菌 (Lactobacillus plantarum),屎肠球菌(Enterococcus faecium),鹑鸡肠 球菌(Enterococcus gallinarium),粪肠球菌(Enterococcus faecalis), 枯草芽孢杆菌和酿酒酵母。 生产宿主的构建 可以使用本领域公知的技术来构建含有必需基因的重组生物体, 该基因编码可发酵碳底物转化成异丁醇的酶途径。在本发明中,可以 从各种来源分离编码本发明异丁醇生物合成途径之一的酶,例如,乙 酰乳酸合酶,乙酰醇酸异构还原酶,乙酰醇酸脱水酶,支链α-酮酸脱 羧酶,如上所述。 从细菌基因组获得所需基因的方法是分子生物学领域中常见的和 公知的。例如,如果基因的序列是已知的,可以通过限制性核酸内切 酶分泌合适的基因组文库,并可以使用与所需基因序列互补的探针来 筛选。一旦序列得到分离,使用标准引物定向扩增方法如聚合酶链式 反应(U.S.4,683,202)扩增DNA来获得适于使用合适载体转化的适量 DNA。用于在异源宿主中表达的密码子最佳化的工具是容易获得的。 基于宿主生物体的GC含量可以使用一些用于密码子最佳化的工具。表 3中给出了一些实例微生物宿主的GC含量。 表3 微生物宿主的GC含量 菌株 %GC 地衣芽孢杆菌 46 枯草芽孢杆菌 42 丙醇丁酮梭菌 37 大肠杆菌 50 恶臭假单胞菌 61 真养产碱杆菌 61 浸麻类芽孢杆菌 51 红串红球菌 62 短杆菌 50 多粘类芽孢杆菌(Paenibacillus polymyxa) 50 一旦鉴定并分离出相关的途径基因,可以通过本领域公知的方式 将它们转化值合适的表达载体中。用于转化各种宿主细胞的载体或盒 是常见的并从如下的公司可购得,(Madison,WI), Invitrogen Corp.(Carlsbad,CA),Stratagene(La Jolla,CA)和New England Biolabs,Inc.(Beverly,MA)。通常,载体和盒含有指导相关 基因的盒翻译的序列,选择标记和允许自主复制或染色体整合的序列。 合适的载体包括带有转录启动控制的基因5’端和控制转录终止的DNA 片段的3’端。两个控制片段可以源自与转化的宿主细胞同源的基因, 尽管可以理解这样的控制片段也可以源自不是选为生产宿主的特定物 种的天然基因。 用于驱动所需宿主细胞中相关途径编码片段的表达的启动控制片 段或启动子是为数众多的并是本领域技术人员熟知的。实际上,能够 驱动这些遗传序列的任何启动子都适用于本发明,包括但不限于, CYC1,HIS3,GAL1,GAL10,ADH1,PGK,PHO5,GAPDH,ADC1, TRP1,URA3,LEU2,ENO,TPI,CUP1,FBA,GPD和GPM(用于 在酵母中的表达);AOX1(用于在毕赤氏酵母中的表达);和lac,ara, tet,trp,IPL,IPR,T7,tac和trc(用于大肠杆菌,产碱杆菌和假单胞 菌中的表达);amy,apr,npr启动子和各种噬菌体启动子,用于枯草 芽孢杆菌,地衣芽孢杆菌和浸麻类芽孢杆菌中的表达;nisA(用于革兰 氏阳性细菌中的表达,Eichenbaum等,Appl.Environ.Microbiol.64(8): 2763-2769(1998));和合成的P11启动子(用于植物乳杆菌中的表达, Rud等,Microbiology 152:1011-1019(2006))。 终止控制片段也可以源自优选宿主的各种天然基因。任选地,终 止位点不是必需的,然而,如果包括,是最优选的。 特定的载体能够在大范围的宿主细菌中复制并可以通过缀合来转 移。pRK4的完整和注释序列以及三个相关的载体-pRK437,pRK442 和pRK442(H)是可获得的。已经证明这些衍生物对于革兰氏阴性细菌 中的遗传操纵是有价值的工具(Scott等,Plasmid50(1):74-79(2003))。 宽宿主范围的Inc P4质粒RSF1010的几个质粒衍生物也是可获得的, 具有可以在各种革兰氏阴性细菌中起作用的启动子。质粒pAYC36和 pAYC37,具有活性启动子和多个克隆位点,以允许异源基因在革兰氏 阴性细菌中的表达。 还广泛使用了染色体基因替代工具。例如,已经修改宽宿主范围 复制子pWV101的热敏感性变体来构建质粒pVE6002,其可以用于在 各种革兰氏阳性细菌中实现基因替代(Maguin等,J.Bacteriol.174(17): 5633-5638(1992))。此外,可利用体外transposome来形成来自商业来 源如的各种基因组中的随机突变。 以下更详细地描述了各种优选微生物宿主中异丁醇生物合成途径 的表达。 大肠杆菌中异丁醇生物合成途径的表达 用于大肠杆菌转化的载体或盒是常见的并从以上所列的公司可购 得。例如,可以从各种来源分离异丁醇生物合成途径的基因,克隆至 修饰的pUC9载体中并转化至大肠杆菌NM522中,如实施例6和7中 所述的。 红串红球菌中异丁醇生物合成途径的表达 可以将一系列大肠杆菌-红球菌穿梭载体用于红串红球菌中的表 达,包括但不限于,pRhBR17和pDA71(Kostichka等,Appl.Microbiol. Biotechnol.62:61-68(2003))。此外,可以将一系列启动子用于红串 红球菌中的异源基因表达(参见例如Nakashima等,Appl.Environ. Microbiol.70:5557-5568(2004),和Tao等,Appl.Microbiol.Biotechnol. 2005,DOI 10.1007/s00253-005-0064)。可以使用Tao等,上文和Brahs 等(Appl.Environ.Microbiol.66:2029-2036(2000))所述的方法形成 红串红球菌中染色体基因的靶向基因破坏。 如上所述,最初可以在pDA71或pRhBR71中克隆异丁醇生产需要 的异源基因并转化至大肠杆菌中。然后通过电穿孔将载体转化至红串 红球菌中,如Kostichka等所述的。可以将重组体生长于含有葡萄糖的 培养基中并使用本领域已知的方法进行异丁醇的生产。 枯草芽孢杆菌中异丁醇生物合成途径的表达 用于枯草芽孢杆菌中的基因表达和突变形成的方法也是本领域公 知的。例如,可以从各种来源分离异丁醇生物合成途径的基因,克隆 至修饰的pUC19载体中并转化至枯草芽孢杆菌BE1010中,如实施例8 中所述的。此外,可以将异丁醇生物合成途径的五个基因分成两个用 于表达的操纵子,如实施例20中所述的。将途径的三个基因(bubB, ilvD和kivD)整合至枯草芽孢杆菌BE1010的染色体中(Payne和 Jackson,J.Bacteriol.173:2278-2282(1991))。将剩余的两个基因(ilvD 和bdhB)克隆至表达载体中并转化至带有整合的异丁醇基因的芽孢杆 菌菌株中。 地衣芽孢杆菌中异丁醇生物合成途径的表达 在枯草芽孢杆菌中复制的大部分质粒和穿梭载体可以用于转化地 衣芽孢杆菌,通过原生质体转化或电穿孔。在质粒pBE20或pBE60衍 生物中克隆异丁醇生产需要的基因(Nagarajan等,Gene 114:121-126 (1992))。转化地衣芽孢杆菌的方法是本领域已知的(例如,参见, Fleming等,Appl.Environ.Microbiol.61(11):3775-3780(1995))。可 以将为了在枯草芽孢杆菌中表达而构建的质粒转化至地衣芽孢杆菌中 来产生生产异丁醇的重组微生物宿主。 浸麻类芽孢杆菌中异丁醇生物合成途径的表达 可以如以上对枯草芽孢杆菌中的表达所述的来构建质粒并用于转 化浸麻类芽孢杆菌,通过原生质体转化,以产生生产异丁醇的重组微 生物宿主。 真养产碱杆菌(雷氏菌)中异丁醇生物合成途径的表达 用于真养产碱杆菌中的基因表达和突变形成的方法是本领域已知 的(参见,例如,Taghavi等,Appl.Environ.Microbiol.,60(10):3585-3591 (1994))。可以在上述的任一种宽宿主范围载体中克隆用于异丁醇生 物合成途径的基因,并电穿孔来产生生产异丁醇的重组体。已经详细 描述了产碱杆菌中的多(羟基丁酸)途径,各种修饰真养产碱杆菌的 遗传技术是已知的,并且那些工具可以应用于工程化异丁醇生物合成 途径。 恶臭假单胞菌中异丁醇生物合成途径的表达 用于恶臭假单胞菌中的基因表达的方法是本领域已知的(参见, 例如,Ben-Bassat等,U.S.专利No.6,586,229,在此将其引入作为参考)。 可以将丁醇途径基因插入pPUC18中并将该连接的DNA电穿孔至电感 受态恶臭假单胞菌DOT-T1C5aAR1细胞中来产生生产异丁醇的重组 体。 酿酒酵母中异丁醇生物合成途径的表达 用于酿酒酵母中的基因表达的方法是本领于已知的(参见,例如, Methods in Enzymology(酶学方法),194卷,Guide to Yeast Genetics and Molecular and Cell Biology(酵母遗传学以及分子和细胞生物学指南) (A部分,2004,Christine Guthrie and Gerald R.Fink(编辑),Elsevier Academic Press,San Diego,CA)。酵母中基因的表达通常需要启动子, 接着为目标基因,以及转录终止子。在构建用于编码异丁醇生物合成 途径的基因的表达盒中可以使用各种酵母启动子,包括但不限于组成 型启动子FBA,GPD,ADH1和GPM,和诱导型启动子GAL1,GAL10 和CUP1。合适的转录终止子包括但不限于FBAt,GPDt,GMPt,ERG10t, GAL1t,CYC1和ADH1。例如,可以将异丁醇生物合成途径的合适启 动子,转录终止子和基因克隆至大肠杆菌-酵母穿梭载体中,如实施例 17中所述的。 植物乳杆菌中异丁醇生物合成途径的表达 乳杆菌属属于乳杆菌目(Lactobacillales)家族并且在枯草芽孢杆 菌和链球菌的转化中所用的许多质粒和载体可以用于乳杆菌。合适载 体的非限制性实例包括pAMβ1及其衍生物(Renault等,Gene 183: 175-182(1996);和O’Sullivan等,Gene 137:227-231(1993));pMBB1 和pHW800,pMBB1的衍生物(Wyckoff等,Appl.Environ.Microbiol.62: 1481-1486(1996));pMG1,一种可缀合质粒(Tanimoto等,J.Bacteriol. 184:5800-5804(2002));pNZ9520(Kleerebezem等,Appl.Environ. Microbiol.63:4581-4584(1997));pAM401(Fujimoto等,Appl.Environ. Microbiol.67:1262-1267(2001));和pAT392(Arthur等,Antimicrob. Agents Chemother.38:1899-1903(1994))。已经报道了几个来自植物 乳杆菌的质粒(例如,van Kranenburg R,Golic N,Bongers R,Leer RJ, de Vos WM,Siezen RJ,Kleerebezem M,Appl.Environ.Microbiol.2005 年3月;71(3):1223-1230)。例如,实施例21中描述了植物乳杆菌中 异丁醇生物合成途径的表达。 屎肠球菌,鹑鸡肠球菌和粪肠球菌中异丁醇生物合成途径的表达 肠球菌属属于乳杆菌目家族并且在乳杆菌,枯草芽孢杆菌和链球 菌的转化中所用的许多质粒和载体可以用于肠球菌。合适载体的非限 制性实例包括pAMβ1及其衍生物(Renault等,Gene 183:175-182 (1996);和O’Sullivan等,Gene 137:227-231(1993));pMBB1和 pHW800,pMBB1的衍生物(Wyckoff等,Appl.Environ.Microbiol.62: 1481-1486(1996));pMG1,一种可缀合质粒(Tanimoto等,J.Bacteriol. 184:5800-5804(2002));pNZ9520(Kleerebezem等,Appl.Environ. Microbiol.63:4581-4584(1997));pAM401(Fujimoto等,Appl.Environ. Microbiol.67:1262-1267(2001));和pAT392(Arthur等,Antimicrob. Agents Chemother.38:1899-1903(1994))。使用来自乳球菌的nisA基 因的用于粪肠球菌的表达载体也可使用(Eichenbaum等,Appl.Environ. Microbiol.64:2763-2769(1998)。此外,可以使用用于屎肠球菌染色 体中基因替代的载体(Nallaapareddy等,Appl.Environ.Microbiol.72: 334-345(2006))。例如,实施例22中描述了粪肠球菌中的异丁醇生物 合成途径的表达。 发酵培养基 本发明中的发酵培养基必须含有合适的碳底物。合适的物质包括, 但不限于,单糖,如葡萄糖和果糖,寡糖,如乳糖或蔗糖,多糖,如 淀粉或纤维素或其混合物,以及来自可再生原料的未纯化混合物,如 干酪乳清渗透物,玉米浆,甜菜糖蜜和麦芽。此外,碳底物还可以是 单碳底物,如二氧化碳,或甲醇,对此已经证明了代谢转化成关键的 生化中间产物。除了单碳和二碳底物,还知道甲醇(methylotrophic) 生物体利用各种其他含碳化合物,如甲胺,葡糖胺和各种氨基酸,用 于代谢活性。例如,已知甲醇酵母利用来自甲胺的碳来形成海藻糖或 甘油(Bellion等,Microb.Growth C1 Compd.,[Int.Symp],7th(1993), 415-32,编辑:Murrell,J.Collin;Kelly,Don P.,出版商:Intercept, Andover,UK)。相似地,假丝酵母的各种物种将代谢丙氨酸或油酸 (Sulter等,Arch.Microbiol.153:485-489(1990))。因此,考虑了本 发明中所用的碳源可以包括各种含碳底物并且只受生物体选择的限 制。 尽管考虑了所有上述的碳底物及其混合物在本发明中是合适的, 但优选的碳底物是葡萄糖,果糖和蔗糖。 除了合适的碳源,发酵培养基必须含有合适的矿物质,盐,辅因 子,缓冲剂和其他成分,这些是本领域技术人员已知的,适于培养物 的生长和促进异丁醇生产需要的酶途径。 培养条件 通常,细胞生长于合适培养基中约25℃至约40℃范围的温度。本 发明中的合适生长培养基是通常的商业制备培养基,如Luria Bertani (LB)培养液,Sabouraud Dextrose(SD)培养液或酵母培养基(YM) 培养液。还可以使用其他限定的或合成的生长培养基,并且用于特定 微生物生长的合适培养基将是微生物学或发酵科学领域中技术人员已 知的。还可以将已知直接或间接调节代谢产物抑制的试剂,例如,环 腺苷2’:3’-单磷酸,掺入发酵培养基中。 用于发酵的合适pH范围为pH5.0至pH9.0,其中优选pH6.0至 pH8.0作为起始条件。 可以在好氧或厌氧条件下进行发酵,其中优选厌氧或微好氧条件。 可以使用本领域已知的各种方法来测定发酵培养基中产生的异丁 醇含量,例如,高性能液相色谱(HPLC)或气相色谱(GC)。 工业分批发酵和连续发酵 本发明使用分批发酵方法。传统的分批发酵是封闭的系统,其中 在发酵开始时设定培养基的组成并且在发酵过程中没有接受人工改 变。因此,在发酵开始时,用所需的微生物接种培养基,并且允许发 酵发生而没有给系统添加任何东西。通常,然而,“分批”发酵对于碳 源的添加是分批的,并且通常努力控制一些因素如pH和氧浓度。在分 批系统中,系统的代谢产物和生物量组成恒定地改变直至停止发酵的 时间。在分批培养内,细胞从静态的停滞阶段调节至高生长对数期, 最后到达静止期,其中生长速率减小或停止。如果未处理,静止期中 的细胞最终将死亡。对数期的细胞通常负责终产物或中间产物的大量 产生。 对标准分批系统的改进是给料-分批系统。给料-分批发酵方法也适 用于本发明并包括典型的分批系统,除了随着发酵进展增量添加底物。 当代谢产物抑制倾向于抑制细胞代谢并且希望在培养基中含有有限量 的底物时,给料-分批系统是有用的。给料-分批系统中实际底物浓度的 测量是困难的,并因此基于测量因素的改变来估算,这些测量因素如 pH,溶解氧和废气如CO2的分压。分批和给料-分批发酵是本领域常见 的和公知的,并且可以在以下找到实例:Thomas D.Brock,在 Biotechnology:A Textbook of Industrial Microbiology(生物技术:工业 微生物学的教科书),第二版(1989)Sinauer Associates,Inc.,Sunderland, MA.,或Deshpande,Mukund V.,Appl.Biochem.Biotechnol.,36:227, (1992),在此引入作为参考。 尽管以分批模式进行本发明,但考虑了该方法也将适于连续发酵 方法。连续发酵是开放的系统,其中将限定的发酵培养基连续加入生 物反应器中,并且同时取出等量的调节过的培养基用于处理。连续发 酵通常将培养物维持于恒定的高密度,其中细胞主要处于对数期生长。 连续发酵允许调节影响细胞生长或终产物浓度的一种因素或任何 数量的因素。例如,一种方法以固定的速率维持限制的营养素如碳源 或氮水平,并允许所有其他参数来调节。在其他系统中,可以连续改 变影响生长的多种因素,而通过培养基浊度测量的细胞浓度保持恒定。 连续系统为维持稳态生长条件而努力,因此由于抽取培养基引起的细 胞损耗必须相对于发酵中的细胞生长率而平衡。调节连续发酵过程的 营养素和生长因子的方法以及用于最大化产品发酵速率的技术是工业 微生物领域中公知的,并且Brock,上文中详细描述了各种方法。 考虑了可以使用分批,给料-分批或连续过程来实践本发明,并且 任何已知的发酵模式将是合适的。此外,考虑了可以将细胞固定于作 为完整细胞催化剂的物质上并接受用于异丁醇生产的发酵条件。 从发酵培养基分离异丁醇的方法 可以使用本领域已知的方法从发酵培养基分离生物产生的异丁 醇。例如,可以通过离心,过滤,滗析等从发酵培养基去除固体。然 后,可以从如上所述已经处理过除去固体的发酵培养基分离异丁醇, 使用如蒸馏,液-液提取或基于膜的分离的方法。因为异丁醇与水形成 低沸点的共沸混合物,蒸馏只可以将混合物分离成共沸组合物。可以 结合另一种分离方法使用蒸馏来获得绕过共沸组成分离。可以结合蒸 馏使用来分离和纯化异丁醇的方法包括,但不限于,滗析,液-液提取, 吸附和基于膜的技术。此外,可以使用共沸蒸馏来分离异丁醇,使用 夹带剂(参见,例如,Doherty和Malone,Conceptual Design of Distillation System,McGraw Hill,New York,2001)。 异丁醇-水混合物形成异质的共沸混合物,使得可以结合滗析使用 蒸馏来分离和纯化异丁醇。在该方法中,将含有异丁醇的发酵培养液 蒸馏至接近共沸组成。然后,冷凝共沸混合物,并通过滗析从发酵培 养基分离异丁醇。可以将滗析过的水相随着回流返回至第一个蒸馏柱。 可以通过在第二个蒸馏柱中蒸馏来进一步纯化富含异丁醇的滗析过的 有机相。 还可以使用液-液提取结合蒸馏从发酵培养基中分离异丁醇。在该 方法中,使用合适的溶剂使用液-液提取从发酵培养液中提取异丁醇。 然后蒸馏含有异丁醇的有机相以从溶剂分离异丁醇。 还可以使用蒸馏结合吸附以从发酵培养液中分离异丁醇。在该方 法中,将含有异丁醇的发酵培养液蒸馏至接近非沸组成,然后使用吸 附剂如分子筛除去剩余的水(Aden等,Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stoveer,Report NREL/TP-510-32438,National Renwable Energy Laboratory,2002年6 月)。 此外,可以使用蒸馏结合全蒸发过程从发酵培养基分离和纯化异 丁醇。在该方法中,将含有异丁醇的发酵培养液蒸馏至接近共沸组成, 然后借由通过亲水性膜的全蒸发过程来除去剩余的水(Guo等,J. Member.Sci.245,199-210(2004))。 实施例 在以下的实施例中进一步限定了本发明。应当理解这些实施例只 是通过说明的方式给出,尽管表示了本发明的优选实施方案。从以上 的讨论和这些实施例,本领域技术人员可以确定本发明的必要特征, 而没有脱离其精神和范围,可以进行本发明的各种改变和改进以使其 适于各种用途和条件。 总的方法 实施例中所用的标准重组DNA和分子克隆技术上本领域公知的并 描述于Sambrook,J.,Fritsch,E.F.和Maniatis,T.Molecular Cloning: A Laboratory Manual(分子克隆:实验室手册);Cold Spring Harbor Laboratory Press:Cold Spring Harbor,NY(1989)(Maniatis)和T.J. Silhavy,M.L.Bennan,和L.W.Enquist,Experiments with Gene Fusions (使用基因融合的实验),Cold Spring Harbor Laboratory Press:Cold Spring Harbor,N.Y.(1984)和Ausubel,F.M.等,Current Protocols in Molecular Biology(分子生物学中的通用实验方案),由Greene Publishing Assoc.和Wiley-Interscience(1987)出版。 适用于细菌培养物的维持和生长的材料和方法是本领域公知的。 适用于以下实施例的技术可以在以下所列的找到,Manual of Methods for General Bacteriology(一般细菌学的方法手册)(Phillipp Gerhardt, R.G.E.Murray,Ralph N.Costilow,Eugene W.Nester,Willis A.Wood, Noel R.Krieg和G.Briggs Phillips编辑),American Society for Microbiology,Washington,DC.(1994))或Thomas D.Brock,在 Biotechnology:A Textbook of Industrial Microbiology(生物技术:工业 微生物学的教科书),第二版,Sinauer Associates,Inc.,Sunderland, MA(1989)。用于细菌细胞生长和维持的所有试剂,限制酶和材料从 Aldrich Chemicals(Milwaukee,WI),BD Diagnostic Systems(Sparks, MD),Life Technologies(Rockville,MD),或Sigma Chemical Company (St.Louis,MO)获得,除非另外指出。 从美国典型培养物中心(ATCC),Manassas,VA获得微生物菌株, 除非另外指出。 表4中给出了以下实施例中使用的寡核苷酸引物。通过 Sigma-Genosys(Woodlands,TX)合成所有寡核苷酸引物。 表4 寡核苷酸克隆,测序和测序引物 名称 序列 描述 SEQ ID NO: N80 CACCATGGACAAACAGTATCCGGTA CGCC budB正向 11 N81 CGAAGGGCGATAGCTTTACCAATCC budB反向 12 N100 CACCATGGCTAACTACTTCAATACA CTGA ilvC正向 13 N101 CCAGGAGAAGGCCTTGAGTGTTTTC TCC ilvC反向 14 N102 CACCATGCCTAAGTACCGTTCCGCC ACCA ilvD正向 15 N103 CGCAGCACTGCTCTTAAATATTCGG C ilvD反向 16 N104 CACCATGAACAACTTTAATCTGCAC ACCC yqhD正向 17 N105 GCTTAGCGGGCGGCTTCGTATATAC GGC yqhD反向 18 N110 GCATGCCTTAAGAAAGGAGGGGGG TCACATGGACAAACAGTATCC budB正向 19 N111 ATGCATTTAATTAATTACAGAATCTG ACTCAGATGCAGC budB反向 20 N112 GTCGACGCTAGCAAAGGAGGGAAT CACCATGGCTAACTACTTCAA ilvC正向 21 N113 TCTAGATTAACCCGCAACAGCAATA CGTTTC ilvC反向 22 N114 TCTAGAAAAGGAGGAATAAAGTATG CCTAAGTACCGTTC ilvD正向 23 N115 GGATCCTTATTAACCCCCCAGTTTCG ATTTA ilvD反向 24 N116 GGATCCAAAGGAGGCTAGACATATG TATACTGTGGGGGA kivD正向 25 N117 GAGCTCTTAGCTTTTATTTTGCTCCG CAAAC kivD反向 26 N118 GAGCTCAAAGGAGGAGCAAGTAAT GAACAACTTTAATCT yqhD正向 27 N119 GAATTCACTAGTCCTAGGTTAGCGG GCGGCTTCGTATATACGG yqhD反向 28 BenNF CAACATTAGCGATTTTCTTTTCTCT Npr正向 29 BenASR CATGAAGCTTACTAGTGGGCTTAAG TTTTGAAAATAATGAAAACT Npr反向 30 N110.2 GAGCTCACTAGTCAATTGTAAGTAA GTAAAAGGAGGTGGGTCACATGGA CAAACAGTATCC budB正向 31 N111.2 GGATCCGATCGACTTAAGCCTCAGC TTACAGAATCTGACTCAGATGCAGC budB反向 32 N112.2 GAGCTCCTTAAGAAGGAGGTAATCA CCATGGCTAACTACTTCAA ilvC正向 33 N113.2 GGATCCGATCGAGCTAGCGCGGCCG CTTAACCCGCAACAGCAATACGTTT C ilvC反向 34 N114.2 GAGCTCGCTAGCAAGGAGGTATAAA GTATGCCTAAGTACCGTTC ilvD正向 35 N115.2 GGATCCGATCGATTAATTAACCTAAG GTTATTAACCCCCCAGTTTCGATTTA ilvD反向 36 N116.2 GAGCTCTTAATTAAAAGGAGGTTAG ACATATGTATACTGTGGGGGA kivD正向 37 N117.2 GGATCCAGATCTCCTAGGACATGTT TAGCTTTTATTTTGCTCCGCAAAC kivD反向 38 N130SeqF1 TGTTCCAACCTGATCACCG 测序引物 40 N130SeqF2 GGAAAACAGCAAGGCGCT 测序引物 41 N130SeqF3 CAGCTGAACCAGTTTGCC 测序引物 42 N130SeqF4 AAAATACCAGCGCCTGTCC 测序引物 43 N130SeqR1 TGAATGGCCACCATGTTG 测序引物 44 N130SeqR2 GAGGATCTCCGCCGCCTG 测序引物 45 N130SeqR3 AGGCCGAGCAGGAAGATC 测序引物 46 N130SeqR4 TGATCAGGTTGGAACAGCC 测序引物 47 N131SeqF1 AAGAACTGATCCCACAGGC 测序引物 48 N131SeqF2 ATCCTGTGCGGTATGTTGC 测序引物 49 N131SeqF3 ATTGCGATGGTGAAAGCG 测序引物 50 N131SeqR1 ATGGTGTTGGCAATCAGCG 测序引物 51 N131SeqR2 GTGCTTCGGTGATGGTTT 测序引物 52 N131SeqR3 TTGAAACCGTGCGAGTAGC 测序引物 53 N132SeqF1 TATTCACTGCCATCTCGCG 测序引物 54 N132SeqF2 CCGTAAGCAGCTGTTCCT 测序引物 55 N132SeqF3 GCTGGAACAATACGACGTTA 测序引物 56 N132SeqF4 TGCTCTACCCAACCAGCTTC 测序引物 57 N132SeqR1 ATGGAAAGACCAGAGGTGCC 测序引物 58 N132SeqR2 TGCCTGTGTGGTACGAAT 测序引物 59 N132SeqR3 TATTACGCGGCAGTGCACT 测序引物 60 N132SeqR4 GGTGATTTTGTCGCAGTTAGAG 测序引物 61 N133SeqF1 TCGAAATTGTTGGGTCGC 测序引物 62 N133SeqF2 GGTCACGCAGTTCATTTCTAAG 测序引物 63 N133SeqF3 TGTGGCAAGCCGTAGAAA 测序引物 64 N133SeqF4 AGGATCGCGTGGTGAGTAA 测序引物 65 N133SeqR1 GTAGCCGTCGTTATTGATGA 测序引物 66 N133SeqR2 GCAGCGAACTAATCAGAGATTC 测序引物 67 N133SeqR3 TGGTCCGATGTATTGGAGG 测序引物 68 N133SeqR4 TCTGCCATATAGCTCGCGT 测序引物 69 Scr1 CCTTTCTTTGTGAATCGG 测序引物 72 Scr2 AGAAACAGGGTGTGATCC 测序引物 73 Scr3 AGTGATCATCACCTGTTGCC 测序引物 74 Scr4 AGCACGGCGAGAGTCGACGG 测序引物 75 T-budB(BamHI) AGATAGATGGATCCGGAGGTGGGTC ACATGGACAAACAGT budB正向 144 B-kivD(BamHI) CTCTAGAGGATCCAGACTCCTAGGA CATG kivD反向 145 T-groE(XhoI) AGATAGATCTCGAGAGCTATTGTAA CATAATCGGTACGGGGGTG PgroE正向 147 B-groEL(SpeI, BamH1) ATTATGTCAGGATCCACTAGTTTCCT CCTTTAATTGGGAATTGTTATCCGC PgroE反向 148 T-groEL AGCTATTGTAACATAATCGGTACGG GGGTG PgroE正向 149 T-ilvCB.s. (BamHI) ACATTGATGGATCCCATAACAAGGG AGAGATTGAAATGGTAAAAG ilvC正向 150 B-ilvCB.s. (SpeIBamHI) TAGACAACGGATCCACTAGTTTAAT TTTGCGCAACGGAGACCACCGC ilvC反向 151 T-BD64(DraIII) TTACCGTGGACTCACCGAGTGGGTA ACTAGCCTCGCCGGAAAGAGCG pBD64正向 152 B-BD64(DraIII) TCACAGTTAAGACACCTGGTGCCGT TAATGCGCCATGACAGCCATGAT pBD64反向 153 T-lacIq(DraIII) ACAGATAGATCACCAGGTGCAAGCT AATTCCGGTGGAAACGAGGTCATC lacIq正向 154 B-lacIq(DraIII) ACAGTACGATACACGGGGTGTCACT GCCCGCTTTCCAGTCGGGAAACC lacIq反向 155 T-groE(DraIII) TCGGATTACGCACCCCGTGAGCTAT TGTAACATAATCGGTACGGGGGTG PgroE正向 156 B-B.silvC (DraIII) CTGCTGATCTCACACCGTGTGTTAA TTTTGCGCAACGGAGACCACCGC ilvC反向 157 T-bdhB(DraIII) TCGATAGCATACACACGGTGGTTAA CAAAGGAGGGGTTAAAATGGTTGAT TTCG bdhB正向 159 B-bdhB (rmBT1DraIII) ATCTACGCACTCGGTGATAAAACGA AAGGCCCAGTCTTTCGACTGAGCCT TTCGTTTTATCTTACACAGATTTTTT GAATATTTGTAGGAC bdhB反向 160 LDH EcoRV F GACGTCATGACCACCCGCCGATCCC TTTT ldhL正向 161 LDH AatIIR GATATCCAACACCAGCGACCGACGT ATTAC ldhL反向 162 Cm F ATTTAAATCTCGAGTAGAGGATCCC AACAAACGAAAATTGGATAAAG Cm正向 163 Cm R ACGCGTTATTATAAAAGCCAGTCATT AGG Cm反向 164 P11 F-StuI CCTAGCGCTATAGTTGTTGACAGAA TGGACATACTATGATATATTGTTGCT ATAGCGA P11启动子 正向 165 P11 R-SpeI CTAGTCGCTATAGCAACAATATATCA TAGTATGTCCATTCTGTCAACAACTA TAGCGCTAGG P11启动子 反向 166 PldhL F-HindIII AAGCTTGTCGACAAACCAACATTAT GACGTGTCTGGGC IdhL正向 167 PldhL R-BamHI GGATCCTCATCCTCTCGTAGTGAAA ATT IdhL反向 168 F-bdhB-AvrII TTCCTAGGAAGGAGGTGGTTAAAAT GGTTGATTTCG bdhB正向 169 R-bdhB-BamHI TTGGATCCTTACACAGATTTTTTGAA TAT bdhB反向 170 F-ilvC(B.s.)-AfIII AACTTAAGAAGGAGGTGATTGAAAT GGTAAAAGTATATT ilvC正向 171 R-ilvC(B.s.)-NotI AAGCGGCCGCTTAATTTTGCGCAAC GGAGACC ivIC反向 172 F-PnisA(HindIII) TTAAGCTTGACATACTTGAATGACCT AGTC nisA启动子 正向 173 R-PnisA(SpeI BamHI) TTGGATCCAAACTAGTATAATTTATT TTGTAGTTCCTTC nisA启动子 反向 174 测定培养基中异丁醇浓度的方法 可以通过本领域已知的各种方法来测定培养基中的异丁醇浓度。 例如,特异性高性能液相色谱(HPLC)方法使用了Shodex SH-1011 柱和Shodex SH-G保护柱,两者都购自Waters Corporation(Milford, MA),具有折射率(RI)检测。使用0.01M H2SO4作为移动相获得色 谱分离,使用0.5mL/分钟的流速和50℃的柱温。异丁醇在所用的条件 下具有46.6分钟的停留时间。或者,可以用气相色谱(GC)方法。例 如,特定的GC方法使用HP-INNOWax柱(30m×0.53mm,同前,1μm 膜厚,Agilent Technologies,Wilmington,DE),使用火焰电离检测仪 (FID)。载气是4.5mL/分钟流速的氦,在150℃测量,使用恒定的排 除压力;注射器分裂是200℃1∶25;烘箱温度是45℃1分钟,以10 ℃/分钟45升至220℃,和220℃5分钟;并在240℃使用FID检测, 使用26mL/分钟氦补充气体。异丁醇的停留时间是4.5分钟。 缩写的意思如下:“s”表示秒,“min”表示分钟,“h”表示小时, “psi”表示每平方英寸的磅数,“nm”表示纳米,“d”表示天,“μL” 表示微升,“mL”表示毫升,“L”表示升,“mm”表示微米,“nm”表 示纳米,“mM”表示毫摩尔,“μM”表示微摩尔,“M”表示摩尔, “mmol”表示毫摩尔,“μmol”表示微摩尔,“g”表示克,“μg”表示 微克和“ng”表示纳克,“PCR”表示聚合酶链式反应,“OD”表示光 密度,“OD600”表示在600nm波长测量的光密度,“kDa”表示千道尔 顿,“g“表示重力常数,“bp”表示碱基对,“kbp”表示千碱基对, “%w/v”表示重量/体积百分比,“%v/v”表示体积/体积百分比, “IPTG”表示异丙基-B-D-硫代半乳糖吡喃糖苷,“RBS”表示核糖体结 合位点,“HPLC”表示高性能液相色谱,和“GC”表示气相色谱。术 语“摩尔选择性”是每摩尔消耗的糖底物产生的产物摩尔数,并记录 为百分比。 实施例1 乙酰乳酸合酶的克隆和表达 该实施例的目的是从肺炎克雷伯氏菌克隆budB基因并在大肠杆菌 BL21-AI中表达。使用PCR从肺炎克雷伯氏菌菌株ATCC 25955基因 组DNA扩增budB基因,获得1.8kbp产物。 使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis, MN;目录编号D-5000A)制备基因组DNA。通过PCR从肺炎克雷伯 氏菌基因组DNA扩增budB基因,使用引物N80和N81(参见表2), 各自如SEQ ID NO:11和12给出。在制造商的试剂盒中提供了其他 PCR扩增试剂,例如,Finnzymes PhusionTM High-Fidelity PCR Master Mix(New England Biolabs Inc.,Beverly,MA;目录no.F-531)并根 据制造商的实验方案来使用。在DNA Thermocycler GeneAmp 9700(PE Applied Biosystems,Foster city,CA)中进行扩增。 为了表达研究,使用Gateway克隆技术(Invitrogen Corp.,Carlsbad, CA)。入门载体pENTRSDD-TOPO允许定向的克隆并提供了目标基因 的Shine-Dalgarno序列。目标载体pDEST14使用了T7启动子,用于 没有标记物的基因的表达。正向引物就在转录起始密码子邻近引入四 个碱基(CACC),以允许定向克隆至pENTRSDD-TOPO(Invitrogen) 中来产生质粒pENTRSDD-TOPObudB。根据制造商的推荐将pENTR 构建体转化至大肠杆菌Top10(Invitrogen)细胞中并涂布。将转化体 生长过夜并根据制造商的推荐使用QIAprep Spin Miniprep试剂盒 (Qiagen,Valencia,CA;目录no.27106)制备质粒DNA。将克隆测 序来证实以正确方向插入了基因并证实序列。SEQ ID NO:1和SEQ ID NO:2各自给出了用于该基因的开放阅读框(ORF)的核苷酸序列和 预测的酶氨基酸序列。 为了形成表达克隆,通过重组将budB基因转移至pDEST14载体 中来产生pDEST14budB。将pDEST14budB载体转化至大肠杆菌 BL21-AI细胞中(Invitrogen)。将转化体接种至补充50μg/mL氨苄青霉 素的Luria Bertani(LB)培养基中并生长过夜。并将等份的过夜培养 物用来接种50mL补充50μg/mL氨苄青霉素的LB。将培养物在37℃振 荡培养直至OD600达到0.6-0.8。将培养物分成两个25-mL培养物,并 将阿拉伯糖加入一个烧瓶中至0.2%w/v的终浓度。阴性对照烧瓶没有 用阿拉伯糖来诱导。将烧瓶在37℃振荡培养4h。通过离心收集细胞并 将细胞沉淀物重悬浮于50mM MOPS中,pH 7.0缓冲液。通过超声波 或通过French Pressure Cell破坏细胞。将全细胞裂解产物离心,产生无 细胞提取物的上清液或不溶性部分。将等份的每个部分(全细胞裂解 产物,无细胞提取物和不溶性部分)重悬浮于SDS(MES)装载缓冲 液中(Invitrogen),加热至85℃10分钟,并接受SDS-PAGE分析 (NuPAGE 4-12%Bis-Tris凝胶,目录no.NP0322Box,Invitrogen)。在 诱导的培养物中存在预期约60kDa分子量的蛋白质,如从核酸序列推 导的,但在未诱导的对照中不存在。 使用Bauerle等(Biochim.Biophys.Acta 92(1):142-149(1964)) 描述的方法来测量无细胞提取物中的乙酰乳酸合酶活性。 实施例2(预测的) 乙酰醇酸还原异构酶的克隆和表达 该预测实施例的目的是描述怎样从大肠杆菌K12克隆ilvC基因并 在大肠杆菌BL21-AI中表达。使用PCR从大肠杆菌基因组DNA扩增 ilvC基因。 以实施例1中所述的budB基因相同的方式克隆和表达ilvC基因。 使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN; 目录编号D-5000A)从大肠杆菌制备基因组DNA。通过PCR扩增ilvC 基因,使用引物N100和N101(参见表2),各自如SEQ ID NO:13 和14给出,产生1.5kbp产物。正向引物就在转录起始密码子邻近引入 四个碱基(CCAC),以允许定向克隆至pENTR/SD/D-TOPO(Invitrogen) 中来产生质粒pENTRSDD-TOPOilvC。将克隆测序来证实以正确方向 插入了基因并证实序列。SEQ ID NO:3和SEQ ID NO:4各自给出了 用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨基酸序 列。 为了形成表达克隆,通过重组将ilvC基因转移至pDEST14 (Invitrogen)载体中来产生pDEST 14ilvC。将pDEST 14ilvC载体转化 至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的表 达。在诱导的培养物中存在预期约54kDa分子量的蛋白质,如从核酸 序列推导的,但在未诱导的对照中不存在。 使用Arfin和Umbarger(J.Biol.Chem.244(5):1118-1127(1969)) 所述的方法来测量无细胞提取物中的乙酰醇酸还原异构酶。 实施例3(预测的) 乙酰醇酸脱水酶的克隆和表达 该预测实施例的目的是描述怎样从大肠杆菌K12克隆ilvD基因并 在大肠杆菌BL21-AI中表达。使用PCR从大肠杆菌基因组DNA扩增 ilvD基因。 以实施例1中所述的budB基因相同的方式克隆和表达ilvD基因。 使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN; 目录编号D-5000A)从大肠杆菌制备基因组DNA。通过PCR扩增ilvD 基因,使用引物N102和N 103(参见表2),各自如SEQ ID NO:15 和16给出,产生1.9kbp产物。正向引物就在转录起始密码子邻近引入 四个碱基(CCAC),以允许定向克隆至pENTR/SD/D-TOPO(Invitrogen) 中来产生质粒pENTRSDD-TOPOilvD。将克隆接受测序来证实以正确 方向插入了基因并证实序列。SEQ ID NO:5和SEQ ID NO:6各自给 出了用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨基 酸序列。 为了形成表达克隆,通过重组将ilvD基因转移至pDEST14 (Invitrogen)载体中来产生pDEST14ilvD。将pDEST14ilvD载体转化 至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的表 达。在诱导的培养物中存在预期约66kDa分子量的蛋白质,如从核酸 序列推导的,但在未诱导的对照中不存在。 使用Flint等(J.Biol.Chem.268(20):14732-14742(1993))描述 的方法来测量无细胞提取物中的乙酰醇酸脱水酶活性。 实施例4(预测的) 支链酮酸脱羧酶的克隆和表达 该预测实施例的目的是描述怎样从乳酸乳球菌克隆kivD基因并在 大肠杆菌BL21-AI中表达。 从GenScript(Piscataway,NJ)获得编码乳酸乳球菌的支链酮酸 脱羧酶(kivD)的DNA序列。获得的序列对于在大肠杆菌和枯草芽孢 杆菌中的表达是密码子最佳化的并克隆至pUC57中,来形成 pUC57-kivD。SEQ ID NO:7和SEQ ID NO:8各自给出了用于该基因 的开放阅读框(ORF)的密码子最佳化核苷酸序列和预测的酶氨基酸 序列。 为了形成表达克隆,使用NdeI和BamHI限制酶位点将来自 pUC57-kivD的1.7kbp kivD片段克隆至载体pET-3a中(Novagen, Madison,WI)。这形成了表达克隆pET-3a-kivD。将pET-3a-kivD载体 转化至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子 的表达。在诱导的培养物中存在预期约61kDa分子量的蛋白质,如从 核酸序列推导的,但在未诱导的对照中不存在。 使用Smit等(Appl.Microbiol.Biotechnol.64:396-402(2003)) 描述的方法来测量无细胞提取物中的支链酮酸脱羧酶活性。 实施例5(预测的) 支链醇脱氢酶的克隆和表达 该预测实施例的目的是描述怎样从大肠杆菌K12克隆yqhD基因并 在大肠杆菌BL21-AI中表达。使用PCR从大肠杆菌基因组DNA扩增 yqhD基因。 以实施例1中所述的budB基因相同的方式克隆和表达yqhD基因。 使用Gentra Puregene试剂盒(Gentra Systems,Inc.,Minneapolis,MN; 目录编号D-5000A)从大肠杆菌制备基因组DNA。通过PCR扩增yqhD 基因,使用引物N104和N105(参见表2),各自如SEQ ID NO:17 和18给出,产生1.2kbp产物。正向引物就在转录起始密码子邻近引入 四个碱基(CCAC),以允许定向克隆至pENTR/SD/D-TOPO(Invitrogen) 中来产生质粒pENTRSDD-TOPOyqhD。将克隆接受测序来证实以正确 方向插入了基因并证实序列。SEQ ID NO:9和SEQ ID NO:10各自 给出了用于该基因的开放阅读框(ORF)的核苷酸序列和预测的酶氨 基酸序列。 为了形成表达克隆,通过重组将yqhD基因转移至pDEST 14 (Invitrogen)载体中来产生pDEST14yqhD。将pDEST14yqhD载体转 化至大肠杆菌BL21-AI细胞中并通过加入阿拉伯糖诱导从T7启动子的 表达。在诱导的培养物中存在预期约42kDa分子量的蛋白质,如从核 酸序列推导的,但在未诱导的对照中不存在。 使用Sulzenbacher等(J.Biol.Chem.342(2):489-502(2004))描 述的方法来测量无细胞提取物中的支链醇脱氢酶活性。 实施例6(预测的) 用于异丁醇生物合成途径中的基因的转化载体的构建 该预测实施例的目的是描述怎样构建转化载体,该载体包括编码 异丁醇生物合成途径中五个步骤的基因。将所有基因置于单个启动子 控制下的单个操纵子中。通过PCR扩增单个基因,使用引入限制位点 的引物,用于之后的克隆,并且正向引物含有最佳化的大肠杆菌核糖 体结合位点(AAAGGAGG)。将PCR产物TOPO克隆至 pCR4Blunt-TOPO载体中并转化至大肠杆菌Top10细胞中(Invitrogen)。 从TOPO克隆制备质粒DNA并证实基因的序列。根据制造商的推荐使 用限制酶和T4DNA连接酶(New England Biolabs,Beverly,MA)。 为了克隆实验,使用QIAquick凝胶提取试剂盒(Qiagen)将限制片段 凝胶纯化。证实序列后,将基因亚克隆至作为克隆平台的修饰pUC19 载体中。通过HindIII/SapI消化来修饰pUC19载体,形成pUC19dHS。 该消化除去了邻接MCS(多克隆位点)的lac启动子,防止操纵子在 载体中的转录。 通过PCR从肺炎克雷伯氏菌ATCC 25955基因组DNA扩增budB 基因,使用引物对N110和N111(参见表2),各自如SEQ ID NO:19 和20给出,形成1.8kbp产物。正向引物引入SphI和AfIII限制位点和 核糖体结合位点(RBS)。反向引物引入PacI和NsiI限制位点。将PCR 产物克隆至pCR4Blunt-TOPO中,形成pCR4Blunt-TOPO-budB。从 TOPO克隆制备质粒DNA并证实基因的序列。 通过PCR从大肠杆菌K12基因组扩增ilvC基因,使用引物对N112 和N113(参见表2),各自如SEQ ID NO:21和22给出,形成1.5kbp 产物。正向引物引入SalI和NheI限制位点和RBS。反向引物引入XbaI 限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成 pCR4Blunt-TOPO-ilvC。从TOPO克隆制备质粒DNA并证实基因的序 列。 通过PCR从大肠杆菌K12基因组扩增ilvD基因,使用引物对N114 和N115(参见表2),各自如SEQ ID NO:23和24给出,形成1.9kbp 产物。正向引物引入XbaI限制位点和RBS。反向引物引入BamHI限 制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成 pCR4Blunt-TOPO-ilvD。从TOPO克隆制备质粒DNA并证实基因的序 列。 通过PCR从pUC57-kivD(实施例4中所述的)扩增kivD基因, 使用引物对N116和N117(参见表2),各自如SEQ ID NO:25和26 给出,形成1.7bp产物。正向引物引入BamHI限制位点和RBS。反向 引物引入SacI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形 成pCR4Blunt-TOPO-kivD。从TOPO克隆制备质粒DNA并证实基因的 序列。 通过PCR从大肠杆菌K12基因组DNA扩增yqhD基因,使用引物 对N118和N119(参见表2),各自如SEQ ID NO:27和28给出,形 成1.2kbp产物。正向引物引入SacI限制位点。反向引物引入SpeI和 EcoRI限制位点。将PCR产物克隆至pCR4Blunt-TOPO中,形成 pCR4Blunt-TOPO-yqhD。从TOPO克隆制备质粒DNA并证实基因的序 列。 为了构建异丁醇途径操纵子,用ScaI和EcoRI从 pCR4Blunt-TOPO-yqhD切除yqhD基因,释放1.2kbp片段。将该片段 连接之前已经用SacI和EcoRI消化过的pUC19dHS。通过限制消化来 证实所得到的克隆,pUC19dHS-yqhD。接着,用SalI和XbaI从 pCR4Blunt-TOPO-ilvC切除ilvC基因,释放1.5kbp片段。将该片段连 接之前已经用SalI和XbaI消化过的pUC19dHS-yqhD。通过限制消化 来证实所得到的克隆,pUC19dHS-ilvC-yqhD。然后用SphI和NsiI从 pCR4Blunt-TOPO-budB切除budB基因,释放1.8kbp片段。用SphI和 PstI消化pUC 19dHS-ilvC-yqhD并连接SphI/NsiI budB片段(NsiI和PstI 产生相容性末端),形成pUC19dHS-budB-ilvC-yqhD。用XbaI和BamHI 从pCR4Blunt-TOPO-ilvD切除1.9kbp含有ilvD基因的片段,并连接用 这些相同的酶消化过的pUC19dHS-budB-ilvC-yqhD,形成 pUC19dHS-budB-ilvC-ilvD-yqhD。最后,用BamHI和SacI从pCR4 Blunt-TOPO-kivD切除kivD,释放1.7kbp片段。将该片段连接之前已 经用BamHI和SacI消化过的pUC 19dHS-budB-ilvC-ilvD-yqhD,形成 pUC19dHS-budB-ilvC-ilvD-kivD-yqhD。 用AfIII和SpeI消化pUC19dHS-budB-ilvC-ilvD-kivD-yqhD来释放 克隆至pBenAS(大肠杆菌-枯草杆菌穿梭载体)中的8.2kbp操纵子片 段。通过修饰pBE93载体来形成质粒,Nagarajan描述了这 (WO93/24631,实施例4)。为了制备pBenAS,用pBE93的NcoI/HindIII 消化除去解淀粉芽孢杆菌(Bacillus amyloliquefaciens)中性蛋白酶启动 子(NPR),信号序列和phoA基因。从pBE93PCR扩增NPR启动子, 使用引物BenNF和BenASR,各自如SEQ ID NO:29和30给出。引 物BenASR在启动子下游引入AfIII,SpeI和HindIII位点。用NcoI和 HindIII消化PCR产物,并将片段克隆至载体中的相应位点中,形成 pBenAS。将操纵子片段亚克隆至pBenAS中的AfIII和SpeI位点中, 形成pBen-budB-ilvC-ilvD-kivD-yqhD。 实施例7(预测的) 大肠杆菌中异丁醇生物合成途径的表达 该预测实施例的目的是描述怎样在大肠杆菌中表达异丁醇生物合 成途径。 将按照实施例6中所述构造的质粒 pBen-budB-ilvC-ilvD-kivD-yqhD转化至大肠杆菌NM522中(ATCC No.47000),来获得大肠杆菌株 NM522/pBen-budB-ilvC-ilvD-kivD-yqhD,并通过SDS-PAGE,酶测试 和蛋白质印迹分析来监控操纵子中的基因表达。对于蛋白质印迹,通 过Sigma-Genosys(The Woodlands,TX)引发对合成肽的抗体。 将大肠杆菌株NM522/pBen-budB-ilvC-ilvD-kivD-yqhD接种至含有 50mL培养基的250mL摇瓶中并在250rpm和35℃振荡。培养基组成如 下:葡萄糖(5g/L),MOPS(0.05M),硫酸铵(0.01M),磷酸钾,单 价碱(0.005M),S10金属混合物(1%(v/v)),酵母提取物(0.1%(w/v)), 酪蛋白氨基酸(0.1%(w/v)),硫胺素(0.1mg/L),脯氨酸(0.05mg/L), 和生物素(0.002mg/L),并用KOH滴定至pH7.0。S10金属混合物含 有:MgCl2(200mM),CaCl2(70mM),MnCl2(5mM),FeCl3(0.1mM), ZnCl2(0.1mM),盐酸硫胺素(0.2mM),CuSO4(172μM),CoCl2(253μM) 和Na2MoCO4(242μM)。18h小时后,通过HPLC或GC分析来检测异 丁醇,使用本领域公知的方法,例如,以上一般方法部分中所述的。 实施例8(预测的) 枯草芽孢杆菌中异丁醇生物合成途径的表达 该预测实施例的目的是描述怎样在枯草芽孢杆菌中表达异丁醇生 物合成途径。使用实施例7中所述的相同方法。 使用按照实施例6中所述构造的质粒 pBen-budB-ilvC-ilvD-kivD-yqhD。将该质粒转化至枯草芽孢杆菌 BE1010中(J.Bacteriol.173:2278-2282(1991))来获得枯草芽孢杆 菌株BE1010/pBen-budB-ilvC-ilvD-kivD-yqhD,并按照实施例7中所述 的来监控每个操纵子中的基因表达。 将枯草芽孢杆菌株BE 1010/pBen-budB-ilvC-ilvD-kivD-yqhD接种 至含有50mL培养基的250mL摇瓶中并在250rpm和35℃振荡18h。培 养基组成如下:葡萄糖(5g/L),MOPS(0.05M),谷氨酸(0.02M), 硫酸铵(0.01M),磷酸钾,单价碱缓冲剂(0.005M),S10金属混合物 (和实施例11中所述的一样,1%(v/v)),酵母提取物(0.1%(w/v)), 酪蛋白氨基酸(0.1%(w/v)),色氨酸(50mg/L),甲硫氨酸(50mg/L) 和赖氨酸(50mg/L),并用KOH滴定至pH7.0。18h小时后,通过HPLC 或GC分析来检测异丁醇,使用本领域公知的方法,例如,以上一般方 法部分中所述的。 实施例9 乙酰乳酸合酶的克隆和表达 为了形成另一个乙酰乳酸合酶表达克隆,将budB基因克隆至载体 pTrc99A中。首先从pENTRSDD-TOPObudB(实施例1中所述的)扩 增budB基因,使用在5’端引入SacI,SpeI和MfeI位点和3’端引入 BbvCI,AflII和BamHI位点的引物(N110.2和N111.2,各自如SEQ ID NO:31和32给出)。将所得到的1.75kbp PCR产物克隆至pCR4-Blunt TOPO(Invitrogen)中,并证实了DNA序列(使用N130Seq测序引物 F1-F4和R1-R4,各自如SEQ ID NO:40-47给出)。然后使用SacI和 BamHI从该载体切除budB基因并克隆至pTrc99A中(Amann等,Gene 69(2):301-315(1988)),产生pTrc99A::budB。将pTrc99A::budB载体 转化至大肠杆菌TOP 10细胞中并将转化体接种至补充50μg/mL氨苄青 霉素的LB培养基中并生长过夜。将等份的过夜培养物用来接种50mL 补充50μg/mL氨苄青霉素的LB。将培养物在37℃振荡培养直至OD600 达到0.6-0.8。然后通过加入0.4mM IPTG来诱导从Trc启动子的budB 表达。还制备了没有用IPTG诱导的阴性对照摇瓶。将摇瓶在37℃振 荡培养4h。按照实施例1中所述的制备无细胞培养物。 按照实施例1中所述的测量无细胞提取物中的乙酰乳酸合酶活性。 用IPTG诱导后三个小时,检测到8单位/mg的乙酰乳酸合酶活性。只 携带pTrc99A质粒的对照菌株呈现出0.03单位/mg的乙酰乳酸合酶活 性。 实施例10 乙酰醇酸还原异构酶的克隆和表达 该实施例的目的是从大肠杆菌K12克隆ilvC基因并在大肠杆菌 TOP10中表达。使用PCR从大肠杆菌菌株FM5(ATCC 53911)扩增 ilvC基因。 以以上实施例2中对ilvC的克隆和表达所述的相同方式克隆和表 达ilvC基因。使用PCR从大肠杆菌FM5基因组扩增ilvC,使用引物 N112.2和N113.2(各自为SEQ ID NO:33和34)。引物在5’端形成SacI 和AfllII位点以及最佳的RBS,在ilvC的3’端形成NotI,NheI和BamHI 位点。根据制造商的实验方案(Invitrogen)将1.5kbp PCR产物克隆至 pCR4Blunt TOPO中,产生pCR4Blunt TOPO::ilvC。使用测序引物 (N131SeqF1-F3,和N131SeqR1-R3,各自如SEQ ID NO:48-53给出) 证实了PCR产物的序列。为了形成表达克隆,使用SacI和BamHI从 pCR4Blunt TOPO::ilvC切除ilvC基因并克隆至pTrc99A中。将 pTrc99A::ilvC载体转化至大肠杆菌TOP10细胞中并通过加入IPTG诱 导从Trc启动子的表达,如实施例9中所述的。按照实施例1中所述的 制备无细胞提取物。 按照实施例2中所述的测量无细胞提取物中的乙酰醇酸还原异构 酶活性。用IPTG诱导三小时后,检测到0.026单位/mg的乙酰醇酸还 原异构酶活性。只携带pTrc99A质粒的对照菌株呈现出低于0.001单位 /mg的乙酰醇酸还原异构酶活性。 实施例11 乙酰醇酸脱水酶的克隆和表达 该实施例的目的是从大肠杆菌K12克隆ilvD基因并在大肠杆菌 TOP10中表达。使用PCR从大肠杆菌菌株FM5(ATCC 53911)扩增 ilvD基因。 以实施例10中所述的ilvC基因相同的方式克隆和表达ilvD基因。 使用PCR从大肠杆菌FM5基因组扩增ilvD,使用引物N114.2和N115.2 (各自为SEQ ID NO:35和36)。引物在5’端形成SacI和NheI位点 以及最佳的RBS,在ilvD的3’端形成Bsu36I,PacI和BamHI位点。 根据制造商的实验方案(Invitrogen)将1.9kbp PCR产物克隆至 pCR4Blunt TOPO中,产生pCR4Blunt TOPO::ilvD。证实了PCR产物 的序列(测序引物N 132SeqF1-F4,和N132SeqR1-R4,各自如SEQ ID NO:54-61给出)。为了形成表达克隆,使用SacI和BamHI从质粒 pCR4Blunt TOPO::ilvD切除ilvD基因并克隆至pTrc99A中。将 pTrc99A::ilvD载体转化至大肠杆菌TOP10细胞中并通过加入IPTG诱 导从Trc启动子的表达,如实施例9中所述的。按照实施例1中所述的 制备无细胞提取物。 按照实施例3中所述的测量无细胞提取物中的乙酰醇酸脱水酶活 性。用IPTG诱导三小时后,测量到46单位/mg的乙酰醇酸脱水酶活 性。只携带pTrc99A质粒的对照菌株没有呈现出乙酰醇酸脱水酶活性。 实施例12 支链酮酸脱羧酶的克隆和表达 该实施例的目的是从乳酸乳球菌克隆kivD基因并在大肠杆菌 TOP10中表达。 以以上实施例10中对ilvC所述的相同方式克隆和表达kivD基因。 使用PCR从质粒pUC57-kivD(参见以上的实施例4)扩增kivD,使用 引物N 116.2和N117.2(各自为SEQ ID NO:37和38)。引物在5’端形 成SacI和PacI位点以及最佳的RBS,在kivD的3’端形成AvrII,BglII 和BamHI位点。根据制造商的实验方案(Invitrogen)将1.7kbp PCR 产物克隆至pCR4Blunt TOPO中,产生pCR4Blunt TOPO::kivD。使用 引物N133SeqF1-F4,和N133SeqR1-R4(各自如SEQ ID NO:62-69给 出)证实了PCR产物的序列。为了形成表达克隆,使用SacI和BamHI 从质粒pCR4Blunt TOPO::kivD切除ilvD基因并克隆至pTrc99A中。将 pTrc99A::kivD载体转化至大肠杆菌TOP10细胞中并通过加入IPTG诱 导从Trc启动子的表达,如实施例9中所述的。按照实施例1中所述的 制备无细胞提取物。 按照实施例4中所述的测量无细胞提取物中的支链酮酸脱羧酶活 性,除了使用Purpald试剂(Al drich,目录No.162892)来检测和定 量醛反应产物。用IPTG诱导三小时后,检测到高于3.7单位/mg的支 链酮酸脱羧酶活性。只携带pTrc99A质粒的对照菌株没有呈现出支链 酮酸脱羧酶活性。 实施例13 支链醇脱氢酶的表达 大肠杆菌含有鉴定为1,3-丙二醇脱氢酶的天然基因(yqhD)(U.S. 专利No.6,514,733)。YqhD蛋白与来自梭菌的AdhB(由adhB编码) 具有40%同一性,AdhB是推定的NADH-依赖性丁醇脱氢酶。使用λRed 技术(Datsenko和Wanner,Proc.Natl.Acad.Sci.U.S.A.97:6640 (2000))将yqhD基因置于大肠杆菌菌株MG1655 1.6yqhD::Cm (WO2004/033646)中的葡萄糖异构酶启动子1.6GI(SEQ ID NO.70) 变体的组成型表达之下。MG1655 1.6yqhD::Cm含有FRT-CmR-FRT盒, 使得可以除去抗生素标记。相似地,由1.5GI启动子(W2003/089621) (SEQ ID NO.71)替代天然启动子,形成菌株MG1655 1.5GI-yqhD::Cm,因此,用1.5GI启动子替代了MG1655 1.6yqhD::Cm 的1.6GI启动子。 将菌株MG 1655 1.5GI-yqhD::Cm在LB培养基中生长至中-对数期 并按照实施例1中所述的制备无细胞提取物。测定细胞提取物时,按 照在pH7.5和35℃在340nm的吸收值降低,发现该菌株具有NAPDH- 依赖性异丁醛还原酶活性。 为了产生第二个含有1.5GI yqhD::Cm的表达菌株,从MG1655 1.5GI yqhD::Cm制备P1裂解物,并通过转导将盒转移至BL21(DE3) (Invitrogen),形成BL21(DE3)1.5GI yqhD::Cm。 实施例14 用于异丁醇生物合成途径中头四个基因的转化载体的构建 该实施例的目的是构建包括异丁醇生物合成途径中头四个基因 (即,budB,ilvC,ilvD和kivD)的转化载体。 为了构建转化载体,首先,通过用AflII和BamHI消化从 pTrc99A::ilvC(实施例10中所述的)获得ilvC基因并克隆至 pTrc99A::budB中(实施例9中所述的),将其用AflII和BamHI消化来 产生质粒pTrc99A::budB-ilvC。接着,各自通过用NheI和PacI(ilvD) 以及PacI和BamHI(kivD)从pTrc99A::ilvD(实施例11中所述的) 和pTrc99A::kivD(实施例12中所述的)获得ilvD和kivD基因。通过 三路(three-way)连接将这些基因引入首先用NheI和BamHI消化的 pTrc99A::budB-ilvC中。通过PCR筛选和限制消化证实了最后的质粒 pTrc99A::budB-ilvC-ilvD-kivD中的所有四个基因。 实施例15 生长于葡萄糖之上的大肠杆菌中异丁醇生物合成途径的表达 为了形成大肠杆菌异丁醇生产菌株,将 pTrc99A::budB-ilvC-ilvD-kivD(实施例14中所述的)转化至大肠杆菌 MG16551.5GI yqhD::Cm和大肠杆菌BL21(DE3)1.5GI yqhD::Cm(实 施例13中所述的)中。最初将转化体生长在含有50μg/mL卡那霉素和 100μg/mL羧苄青霉素的LB培养基中。将来自这些培养物的细胞用来 接种摇瓶(大约175mL总体积),摇瓶含有50或170mL TM3a/葡萄糖 培养基,各自表示高和低氧条件。TM3a/葡萄糖培养基含有(每升): 葡萄糖(10g),KH2PO4(13.6g),柠檬酸一水合物(2.0g),(NH4)2SO4 (3.0g),MgSO4·7H2O(2.0g),CaCl2·2H2O(0.2g),柠檬酸铁铵 (0.33g),·HCl(1.0mg),酵母提取物(0.50g),和10mL微量元素溶 液。用NH4OH将pH调节至6.8。微量元素溶液含有:柠檬酸·H2O (4.0g/L),MnSO4·H2O(3.0g/L),NaCl(1.0g),FeSO4·7H2O(0.10g/L), CoCl2.6H2O(0.10g/L),ZnSO4·7H2O(0.10g/L),CuSO4·5H2O(0.010g/L), H3BO3(0.010g/L),和Na2MoSO4·2H2O(0.010g/L)。 在≤0.01单位的起始OD600接种摇瓶并在34℃300rpm振荡培养。 用0.2μm过滤器帽子密封含有50mL培养基的摇瓶;用密封的帽子封 闭含有150mL培养基的摇瓶。当细胞达到OD600≥0.4单位时,加入 IPTG至0.04mM的终浓度。大约培养18h后,通过HPLC(Shodex Sugar SH1011柱(Showa Denko America,Inc.NY),使用折射率(RI)检测) 和GC(Varian CP-WAX 58(FFAP)CB,0.25mm×0.2μm×25m(Varian, Inc.,Palo Alto,CA,使用火焰电离检测(FID))分析等份培养基的异 丁醇含量,如一般方法部分中所述的。在只携带pTrc99A载体的对照 菌株中没有检测到异丁醇(结果未显示)。表5中显示了通过携带 pTrc99A::budB-ilvC-ilvD-kivD的菌株产生的异丁醇摩尔选择性和滴定 度。在低氧条件下生长的培养物中获得显著较高的异丁醇滴定度。 表5 生长于葡萄糖之上的大肠杆菌菌株产生的异丁醇 菌株 O2条件 异丁醇 mM* 摩尔选择性 (%) MG1655 1.5GI yqhD/ pTrc99A::budB-ilvC-ilvD-kivD 高 0.4 4.2 MG1655 1.5GI yqhD/ pTrc99A::budB-ilvC-ilvD-kivD 低 9.9 39 BL21(DE3)1.5GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD 高 0.3 3.9 BL21(DE3)1.5GI yqhD/pTrc99A::budB-ilvC-ilvD-kivD 低 1.2 12 *通过HPLC测定的 实施例16 生长于蔗糖之上的大肠杆菌中异丁醇生物合成途径的表达 因为实施例15中所述的菌株不能够生长于蔗糖之上,构建另外的 质粒以允许利用蔗糖用于异丁醇生产。从源自ATCC菌株13281的利 用蔗糖的大肠杆菌菌株的基因组DNA分离蔗糖利用基因簇cscBKA, 如SED ID NO:39所给出的。蔗糖利用基因(cscA,cscK和cscB)编 码蔗糖水解酶(CscA),如SEQ ID NO:139所给出的,D-果糖激酶 (CscK),如SEQ ID NO:140所给出的,和蔗糖透性酶(CscB),如 SEQ ID NO:141所给出的。没有包括蔗糖特异性阻遏基因cscR,使得 三个基因cscBKA在大肠杆菌中从它们的天然启动子组成型表达。 用BamHI和EcoRI消化来自蔗糖利用大肠杆菌菌株的基因组 DNA。从琼脂糖凝胶分离具有约4kbp平均大小的片段并连接用BamHI 和EcoRI消化的质粒pLitmus28(New England Biolabs),并转化至超感 受态大肠杆菌TOP10F’细胞中(Invitrogen)。将转化体在含有1%蔗糖 和氨苄青霉素(100μg/mL)的MacConkey琼脂平板上划线并筛选紫色 菌落的外观。从紫色转化体分离质粒DNA,并用M13正向和反向引物 (Invitrogen)以及Scr1-4(各自如SEQ ID NO:72-75中所给出的)测 序。将含有cscB,cscK和cscA(cscBKA)基因的质粒命名为pScr1。 为了形成与异丁醇途径质粒(实施例14)相容的蔗糖利用质粒, 将来自pScr1的操纵子亚克隆至pBHR1中(MoBiTec,Goettingen, Germany)。通过用XhoI消化pScr1(接着用Klenow酶培养来产生钝末 端),然后用AgeI消化来分离cscBKA基因。将所得到的4.2kbp片段连 接已经用NaeI和AgeI消化的pBHR1中,形成9.3kbp质粒 pBHR1::cscBKA。 通过电穿孔将蔗糖质粒pBHR1::cscBKA转化至大肠杆菌BL21 (DE3)1.5yqhD/pTrc99A::budB-ilvC-ilvD-kivD和大肠杆菌MG1655 1.5yqhD/pTrc99A::budB-ilvC-ilvD-kivD中(实施例15中所述的)。首 先在含有100μg/mL氨苄青霉素和50μg/mL卡那霉素的LB培养基上选 择转化体,然后在MacConkey蔗糖(1%)平板上筛选来证实蔗糖操纵 子的功能表达。为了异丁醇的生产,将菌株生长于含有替代葡萄糖的 1%蔗糖的TM3a最小限定培养基中(实施例15中所述的),分析培养 基中所产生的异丁醇含量,如实施例15中所述的,除了在培养14h后 取样。再者,在只携带pTrc99A载体的对照菌株中没有检测到异丁醇 (结果未显示)。表6中显示了通过携带pTrc99A::budB-ilvC-ilvD-kivD 的MG1655 1.5yqhD产生的异丁醇的摩尔选择性和滴定度。 表6 通过生长于蔗糖之上的大肠杆菌菌株MG1655 1.5yqhD/ pTrc99A::budB-ilvC-ilvD-kivD/pBHR1::cScBKA的异丁醇产生 O2条件 IPTG,mM 异丁醇,mM* 摩尔选择性,% 高 0.04 0.17 2 高 0.4 1.59 21 低 0.04 4.03 26 低 0.4 3.95 29 *通过HPLC测定的 实施例17 酿酒酵母中异丁醇途径基因的表达 为了在酿酒酵母中表达异丁醇途径基因,构建多个大肠杆菌-酵母 穿梭载体。使用PCR方法(Yu等,Fungal Genet.Biol.41:973-981 (2004))来融合具有酵母启动子和终止子的基因。特别地,将GPD 启动子(SEQ ID NO:76)和CYC 1终止子(SEQ ID NO:77)融合来 自枯草芽孢杆菌的alsS基因(SEQ ID NO:78),将FBA启动子(SEQ ID NO:79)和CYC1终止子融合来自酿酒酵母的ILV5基因(SEQ ID NO:80),将ADH1启动子(SEQ ID NO:81)和ADH1终止子(SEQ ID NO:82)融合来自酿酒酵母的ILV3基因(SEQ ID NO:83),将 GPM启动子(SEQ ID NO:84)和ADH1终止子融合来自乳酸乳球菌 的kivD基因(SEQ ID NO:7)。将表7中给出的引物设计来包括限制 位点,用于将启动子/基因/终止子产物克隆至来自pRS400系列的大肠 杆菌-酵母穿梭载体中(Christianson等,Gene 110:119-122(1992)), 并用于交换构建体之间的启动子。用于ILV5和ILV3的5’端的引物(各 自为N138和N155,各自如SEQ ID NO:95和107给出的)产生新的 起始密码子来消除这些酶的线粒体靶向。 首先通过TOPO克隆反应(Invitrogen)将所有融合的PCR产物克 隆至pCR4-Blunt中,并证实了序列(使用M13正向和反向引物 (Invitrogen)以及表7中提供的测序引物)。通过TOPO反应将另外两 个启动子(CUP1和GAL1)克隆至pCR4-Blunt中,并通过测序来证实; 表7中显示了引物序列。表8中描述了构建的质粒。将质粒转化至酿 酒酵母BY4743(ATCC 201390)或YJR148w(ATCC 4036939)中, 以使用实施例1-4和实施例9-12中所述的酶测试来测定酶特异性活性。 为了测定酶活性,将培养物在缺少表达质粒选择需要的任何代谢物的 合成完全培养基(Methods in Yeast Genetics,2005,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,pp.201-202)中生长至1.0 的OD600,通过离心收集(在4℃2600×g 8分钟),用缓冲液洗涤,再 次离心,并冷冻于-80℃。将细胞融化,重悬浮于20mM Tris-HCl pH8.0 中至2mL的终体积,然后使用1.2g玻璃珠(0.5mm大小)使用珠子搅 拌器来破坏。将每个样品在高速处理总的3分钟时间(每分钟搅打后 在冰上孵育)。通过离心将提取物清除细胞碎片(在4℃,20,000×g 10 分钟)。 表7 用于酿酒酵母表达载体的克隆和测序的引物序列 名称 序列 描述 SEQ ID NO: N98SeqF1 CGTGTTAGTCACATCAGGAC B.subtilis alsS测序引物 85 N98SeqF2 GGCCATAGCAAAAATCCAAACAGC B.subtilis alsS测序引物 86 N98SeqF3 CCACGATCAATCATATCGAACACG B.subtilis alsS测序引物 87 N98SeqF4 GGTTTCTGTCTCTGGTGACG B.subtilis alsS测序引物 88 N99SeqR1 GTCTGGTGATTCTACGCGCAAG B.subtilis alsS测序引物 89 N99SeqR2 CATCGACTGCATTACGCAACTC B.subtilis alsS测序引物 90 N99SeqR3 CGATCGTCAGAACAACATCTGC B.subtilis alsS测序引物 91 N99SeqR4 CCTTCAGTGTTCGCTGTCAG B.subtilis alsS测序引物 92 N136 CCGCGGATAGATCTGAAATGAATA ACAATACTGACA 具有SacII/Bg1II位点的FBA启动 子正向引物 93 N137 TACCACCGAAGTTGATTTGCTTCA ACATCCTCAGCTCTAGATTTGAATA TGTATTACTTGGTTAT 具有BbvCI位点和ILV5-退火区 的FBA启动子反向引物 94 N138 ATGTTGAAGCAAATCAACTTCGGT GGTA ILV5正向引物(产生另一起始密 码子) 95 N139 TTATTGGTTTTCTGGTCTCAAC ILV5反向引物 96 N140 AAGTTGAGACCAGAAAACCAATA ATTATTAATCATGTAATAGTTATG TCACGCTT 具有PacI位点和ILV5-退火区的 CYC终止子正向引物 97 N141 GCGGCCGCCCGCAAATTAAAGCCT TCGAGC 具有NotI位点的CYC终止子反 向引物 98 N142 GGATCCGCATGCTTGCATTTAGTCG TGC 具有BamHI位点的GPM启动子 正向引物 99 N143 CAGGTAATCCCCCACAGTATACAT CCTCAGCTATTGTAATATGTGTGTT TGTTTGG 具有BbvCI位点和kivD-退火区 的GPM启动子反向引物 100 N144 ATGTATACTGTGGGGGATTACC kivD正向引物 101 N145 TTAGCTTTTATTTTGCTCCGCA kivD反向引物 102 N146 TTTGCGGAGCAAAATAAAAGCTAA TTAATTAAGAGTAAGCGAATTTCTT ATGATTTA ADH终止子正向引物具有PacI 位点和kivD-退火区 103 N147 ACTAGTACCACAGGTGTTGTCCTC TGAG 具有SpeI位点的ADH终止子反 向引物 104 N151 CTAGAGAGCTTTCGTTTTCATG alsS反向引物 105 N152 CTCATGAAAACGAAAGCTCTCTAG TTAATTAATCATGTAATTAGTTATGT CACGCTT 具有PacI位点和alsS-退火区的 CYC终止子正向引物 106 N155 ATGGCAAAGAAGCTCAACAAGTA CT ILV3正向引物(另一起始密码子) 107 N156 TCAAGCATCTAAAACACAACCG ILV3反向引物 108 N157 AACGGTTGTGTTTTAGATGCTTGAT TAATTAAGAGTAAGCGAATTTCTTA TGATTTA 具有PacI位点和ILV3-退火区的 ADH终止子正向引物 109 N158 GGATCCTTTTCTGGCAACCAAACC CATA 具有BamHI位点的ADH启动 子正向引物 110 N159 CGAGTACTTGTTGAGCTTCTTTGC CATCCTCAGCGAGATAGTTGATTGT ATGCTTG 具有BbvCI位点和ILV3-退火区 的ADH启动子反向引物 111 N160SeqF1 GAAAACGTGGCATCCTCTC FBA::ILV5::CYC测序引物 112 N160SeqF2 GCTGACTGGCCAAGAGAAA FBA::ILV5::CYC测序引物 113 N160SeqF3 TGTACTTCTCCCACGGTTTC FBA::ILV5::CYC测序引物 114 N160SeqF4 AGCTACCCAATCTCTATACCCA FBA::ILV5::CYC测序引物 115 N160SeqF5 CCTGAAGTCTAGGTCCCTATTT FBA::ILV5::CYC测序引物 116 N 160SeqR1 GCGTGAATGTAAGCGTGAC FBA::ILV5::CYC测序引物 117 N160SeqR2 CGTCGTATTGAGCCAAGAAC FBA::ILV5::CYC测序引物 118 N160SeqR3 GCATCGGACAACAAGTTCAT FBA::ILV5::CYC测序引物 119 N160SeqR4 TCGTTCTTGAAGTAGTCCAACA FBA::ILV5::CYC测序引物 120 N160SeqR5 TGAGCCCGAAAGAGAGGAT FBA::ILV5::CYC测序引物 121 N161SeqF1 ACGGTATACGGCCTTCCTT ADH::ILV3::ADH测序引物 122 N161SeqF2 GGGTTTGAAAGCTATGCAGT ADH::ILV3::ADH测序引物 123 N161SeqF3 GGTGGTATGTATACTGCCAACA ADH::ILV3::ADH测序引物 124 N161SeqF4 GGTGGTACCCAATCTGTGATTA ADH::ILV3::ADH测序引物 125 N161SeqF5 CGGTTTGGGTAAAGATGTTG ADH::ILV3::ADH测序引物 126 N161SeqF6 AAACGAAAATTCTTATTCTTGA ADH::ILV3::ADH测序引物 127 N161SeqR1 TCGTTTTAAAACCTAAGAGTCA ADH::ILV3::ADH测序引物 128 N161SeqR2 CCAAACCGTAACCCATCAG ADH::ILV3::ADH测序引物 129 N161SeqR3 CACAGATTGGGTACCACCA ADH::ILV3::ADH测序引物 130 N161SeqR4 ACCACAAGAACCAGGACCTG ADH::ILV3::ADH测序引物 131 N161SeqR5 CATAGCTTTCAAACCCGCT ADH::ILV3::ADH测序引物 132 N161SeqR6 CGTATACCGTTGCTCATTAGAG ADH::ILV3::ADH测序引物 133 N162 ATGTTGACAAAAGCAACAAAAGA alsS正向引物 134 N189 ATCCGCGGATAGATCTAGTTCGAG TTTATCATTATCAA 具有SacII/BglII位点的GPD正向 引物 135 N190.1 TTCTTTTGTTGCTTTTGTCAACATC CTCAGCGTTTATGTGTGTTTATTCG AAA 具有BbvCI位点和alsS-退火的区 GPD启动子反向引物 136 N176 ATCCGCGGATAGATCTATTAGAAG CCGCCGAGCGGGCG 具有SacII/BglII位点的GAL1启 动子正向引物 137 N177 ATCCTCAGCTTTTCTCCTTGACGTT AAAGTA 具有BbvCI位点的GAL1启动子 反向 138 N191 ATCCGCGGATAGATCTCCCATTACC GACATTTGGGCGC 具有SacII/BgIII位点的CUP1启 动子正向引物 175 N192 ATCCTCAGCGATGATTGATTGATTG ATTGTA 具有BbvCI位点的CUP1启动子 反向 176 表8 携带异丁醇途径基因的大肠杆菌-酵母穿梭载体 质粒名称 构建 pRS426[ATCC No.77107],URA3选择 - pRS426::GPD::alsS::CYC 用SacII/NotI消化的GPD::alsS::CYC PCR产物 克隆至用相同酶消化的pRS426中 pRS426::FBA::ILV5::CYC 用SacII/NotI消化的FBA::ILV5::CYC PCR产 物克隆至用相同酶消化的pRS426中 pRS425[ATCC No.77106],LEU2选择 - pRS425::ADH::ILV3::ADH 用BamHI/SpeI消化的ADH::ILV3::ADH PCR 产物克隆至用相同酶消化的pRS425中 pRS425::GPM::kivD::ADH 用BamHI/SpeI消化的GPM::kivD::ADH PCR 产物克隆至用相同酶消化的pRS425中 pRS426::CUPI::alsS 来自pRS426::GPD::alsS::CYC的7.7kbp SacII/BbvCI片段连接SacII/BbvCI CUPI片段 pRS426::GAL1::ILV5 来自pRS426::FBA::ILV5::CYC的7kbp SacII/BbvCI片段连接SacII/BbvCI GAL1片段 pRS425::FBA::ILV3 来自pRS425::ADH::ILV3::ADH的8.9kbp BamHI/BbvCI 片段连接来自 pRS426::FBA::ILV5::CYC的0.65kbp BglII/BbvCI FBA片段 pRS425::CPU1-alsS+FBA-ILV3 来自pRS426::CPU1::alsS的2.4kbp SacII/NotI 片段克隆至用SacII/NotI 切割的 pRS425::FBA::ILV3中 pRS426::FBA-ILV5+GPM-kivD 来自pRS425::GMP::kivD::ADH的2.7kbp BamHI/SpeI片段克隆至用BamHI/SpeI切割的 pRS426::FBA::ILV5::CYC中 pRS426::GAL1-FBA+GPM-kivD 来自pRS426::FBA-ILV5+GPM-kivD的8.5kbp SacII/NotI片段连接来自pRS426::GAL1::ILV5 的1.8kbp SacII/NotI片段 pSR423[ATCC No.77104],HIS3选择 - pSR423::CUP1-alsS+FBA-ILV3 来自pRS425::CUP1-alsS+FBA-ILV3的5.2kbp SacI/SalI片段连接用SacI/SalI切割的pSR423 pHR81[ATCC No.87541],URA3和 leu2-d选择 - pHR81::FBA-ILV5+GPM-kivD 来自pRS426::FBA-ILV5+GPM-kivD的4.7kbp SacI/BamH片段连接用SacI/BamHI切割的 pHR81 实施例18 通过重组酿酒酵母生产异丁醇 将质粒pSR423::CUP1-alsS+FBA-ILV3和 pHR81::FBA-ILV5+GPM-kivD(实施例17中所述的)转化至酿酒酵母 YJR148w中来产生菌株YJR148w/ pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kivD。通过将 载体pSR423和pHR81(实施例17中所述的)转化至酿酒酵母YJR148w 中制备对照菌株(YJR148w/pSR423/pHR81)。将菌株维持于含有2% 葡萄糖或蔗糖但缺少尿嘧啶和组氨酸的标准酿酒酵母合成完全培养基 上(Methods in Yeast Genetics,2005,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,pp.201-202),以确保质粒的维持。 为了异丁醇生产,将细胞转移至缺少尿嘧啶,组氨酸和亮氨酸的 合成完全培养基中。由于leu2-d等位基因的转录差,从培养基除去亮 氨酸用来引发pHR81基质粒拷贝数的增加(Erhart和Hollenberg, J.Bacteriol.156:625-635(1983))。将好氧培养物生长于含有50mL培 养基的175mL容量瓶中,在30℃和200rpm的Innova400培养箱中(New Brunswick Scientific,Edison,NJ)。在接种和在30℃保持后,通过将 45mL培养基加入用有波纹的帽子密封的60mL血清瓶中来制备低氧培 养物。按照需要,无菌注射器用来取样和加入诱导剂。接种后大约24h, 将诱导剂CuSO4加入至0.03M终浓度。还制备了每个菌株没有添加 CuSO4的对照培养物。通过GC和HPLC分析CuSO4加入后18或19h 和35h的培养物上清液的异丁醇含量,如以上实施例15中所述的。表 9中呈现了生长于葡萄糖之上的酿酒酵母 YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM -kivD的结果。对于表9中给出的结果,在35h从好氧培养物取样,在 19h从低氧培养物取样,并通过HPLC测量。 表10中呈现了生长于蔗糖之上的酿酒酵母 YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiv D的结果。用18h取的样并通过HPLC测量来获得该表中的结果。 表9 通过生长于葡萄糖之上的酿酒酵母 YJR148w/pSR423::CUJP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiv D的异丁醇生产 菌株 O2水平 异丁醇,mM 摩尔选择 性% YJR148w/pRS423/pHR81(对照) 好氧 0.12 0.04 YJR148w/pRS423/pHR81(对照) 好氧 0.11 0.04 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kiVD a 好氧 0.97 0.34 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kiVD b 好氧 0.93 0.33 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kiVD c 好氧 0.85 0.30 YJR148w/pRS423/pHR81(对照) 低 0.11 0.1 YJR148w/pRS423/pHR81(对照) 低 0.08 0.1 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kiVD a 低 0.28 0.5 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kiVD b 低 0.20 0.3 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kiVD c 低 0.33 0.6 表10 通过生长于蔗糖之上的酿酒酵母 YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiv D的异丁醇生产 菌株 O2水平 异丁醇, mM 摩尔选择性, % YJR148w/pRS423/pHR81(对照) 好氧 0.32 0.6 YJR148w/pRS423/pHR81(对照) 好氧 0.17 0.3 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kivD a 好氧 0.68 1.7 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kivD b 好氧 0.54 1.2 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kivD c 好氧 0.92 2.0 YJR148w/pRS423/pHR81(对照) 低 0.18 0.3 YJR148w/pRS423/pHR81(对照) 低 0.15 0.3 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kivD a 低 0.27 1.2 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kivD b 低 0.30 1.1 YJR148w/pRS423::CUP1-alsS+FBA-ILV3/ pHR81::FBA-ILV5+GPM-kivD c 低 0.21 0.8 菌株后缀“a”,“b”和“c”表示分开的分离物。 结果显示了,在好氧和低氧条件下在葡萄糖或蔗糖上生长,菌株 YJR148w/pSR423::CUP1-alsS+FBA-ILV3/pHR81::FBA-ILV5+GPM-kiv D一致地生产出比对照菌株更高水平的异丁醇。 实施例19 通过重组酿酒酵母的异丁醇生产 将质粒pRS425::CPU1-alsS+FBA-ILV3和 pRS426::GAL1-ILV5+GPM-kivD(实施例17中所述的)转化至酿酒酵 母YJR148w中来产生菌株YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD。通过 将载体pSR425和pRS426(实施例17中所述的)转化至酿酒酵母 YJR148w中制备对照菌株(YJR148w/pSR425/pRS426)。将菌株维持 于合成完全培养基上,如实施例18中所述的。 为了异丁醇生产,将细胞转移至含有2%半乳糖和1%棉子糖而缺 少尿嘧啶和亮氨酸的合成完全培养基中。按照实施例18中所述的制备 好氧和低氧培养物。接种后大约12h,将诱导剂CuSO4加入高达至 0.5mM的终浓度。还制备了每个菌株没有添加CuSO4的对照培养物。 在CuSO4加入后23h将培养物上清液取样,如实施例18中所述的。表 11中呈现了结果。由于观察到的最终光密度非常不同并与定量残留的 碳源相关,表中提供了每OD600单位的异丁醇浓度(替代摩尔选择性), 以允许将含有异丁醇生物合成途径基因的菌株与对照相比较。 表11 通过生长于半乳糖和棉子糖之上的酿酒酵母YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD的异丁 醇生产 菌株 O2水平 CuSO4, mM 异丁醇 mM 每OD单位的 mM异丁醇 YJR148w/pSR425/pRS426(对照) 好氧 0.1 0.12 0.01 YJR148w/pSR425/pRS426(对照) 好氧 0.5 0.13 0.01 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD a 好氧 0 0.20 0.03 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-1LV5+GPM-kivD b 好氧 0.03 0.82 0.09 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD c 好氧 0.1 0.81 0.09 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD d 好氧 0.5 0.16 0.04 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD e 好氧 0.5 0.18 0.01 YJR148w/pSR425/pRS426(对照) 低 0.1 0.042 0.007 YJR148w/pSR425/pRS426(对照) 低 0.5 0.023 0.006 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD a 低 0 0.1 0.04 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD b 低 0.03 0.024 0.02 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD c 低 0.1 0.030 0.04 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD d 低 0.5 0.008 0.02 YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426:: GAL1-ILV5+GPM-kivD e 低 0.5 0.008 0.004 菌株后缀“a”,“b”,“c”,“d”和“e”表示分开的分离物。 结果表示总的说来,在好氧和低氧条件下,与对照菌株相比较, YJR148w/ pRS425::CPU1-alsS+FBA-ILV3/pRS426::GAL1-ILV5+GPM-kivD菌株产 生的每光密度单位的异丁醇水平更高。 实施例20 枯草芽孢杆菌中异丁醇生物合成途径的表达 该实施例的目的是在枯草芽孢杆菌中表达异丁醇生物合成途径。 将异丁醇途径的五个基因(图1中的途径步骤(a)至(e))分成两个 用于表达的操纵子。将各自编码乙酰乳酸合酶,乙酰醇酸脱水酶和支 链酮酸脱羧酶的三个基因budB,ilvD和kivD整合至枯草芽孢杆菌 BE1010的染色体中(Payne和Jackson,J.Bacteriol.173:2278-2282 (1991))。将各自编码乙酰醇酸异构还原酶和丁醇脱氢酶的两个基因 ilvC和bdhB克隆至表达载体中并转化至携带整合的异丁醇基因的芽孢 杆菌菌株中。 三个基因budB,ilvD和kivD整合至枯草芽孢杆菌BE1010的染色 体中。芽孢杆菌整合载体pFP988DssPspac和pFP988DssPgroE用于三 个基因budB(SEQ ID NO:1),ilvD(SEQ ID NO:5)和kivD(SEQ ID NO:7)的染色体整合。两个质粒都含有来自pBR322的大肠杆菌复制 子,用于在大肠杆菌中选择的氨苄青霉素抗生素标记和两个与枯草芽 孢杆菌染色体中的sacB基因同源的指导载体整合的片段,以及通过同 源重组的插入序列。在sacB同源片段之间是pFP988DssPspac上的spac 启动子(PgroE)或pFP988DssPgroE上的groEL启动子(PgroE),和 用于芽孢杆菌的选择标记,红霉素。启动子片段还含有lacO序列,用 于调节lacI阻遏蛋白的表达。pFP988DssPspac(6,341bp)和 pFP988DssPgroE(6,221bp)的序列各自如SEQ ID NO:142和SEQ ID NO:143给出的。 通过从质粒pTrc99a budB-ilvC-ilvD-kivD删除ilvC基因来构建具 有三个基因bubB-ilvD-kivD的盒。实施例14中描述了质粒 pTrc99A::budB-ilvC-ilvD-kivD的构建。用AflII和NheI消化质粒 pTrc99A::budB-ilvC-ilvD-kivD,用DNA聚合酶的Klenow片段处理来 制备钝末端,并凝胶纯化含有pTrc99a载体,budB,ilvD和kivD的所 得到的9.4kbp片段。将9.4kbp载体片段自我连接来形成pTrc99A:: budB-ilvD-kivD,并转化至DH5α感受态细胞(Invitrogen)中。通过限 制性作图证实pTrc99a::budB-ilvD-kivD克隆的ilvC基因删除。用SacI 消化所得到的质粒pTrc99A::budB-ilvD-kivD并用DNA聚合酶的 Klenow片段处理来制备钝末端。然后用BamHI消化质粒并将所得到的 5,297bp budB-ilvD-kivD片段凝胶纯化。将5,297bp budB-ilvD-kivD片段 连接整合载体pFP988DssPspac的SmaI和BamHI位点。将连接混合物 转化至DH5α感受态细胞中。通过5.3kbp budB-ilvD-kivD片段的PCR 扩增来筛选转化体,使用引物T-budB(BamHI)(SEQ ID NO:144) 和B-kivD(BamHI)(SEQ ID NO:145)。将正确的克隆称为 pFP988DssPspac-budB-ilvD-kivD。 从大肠杆菌转化体制备质粒pFP988DssPspac-budB-ilvD-kivD,并 转化至按照Doyle等所述制备的枯草芽孢杆菌BE1010感受态细胞中(J. Bacteriol.144:957(1980))。通过离心收集感受态细胞并将细胞沉淀 物重悬浮于小体积的上清液中。将两体积的SPII-EGTA培养基加入一 体积的感受态细胞中(Methods for General and Molecular Bacteriology, P.Gerhardt等,编辑,American Society for Microbiology,Washington, DC(1994))。将等份(0.3mL)细胞分散于试管中,然后将2至3μg 质粒pFP988DssPspac-budB-ilvD-kivD加入试管中。将试管在37℃振荡 培养30分钟,此后将0.1mL 10%酵母提取物加入每个试管中,然后进 一步培养60分钟。将转化体生长于用于选择的含有红霉素(1.0μg/mL) 的LB平板上,使用双重琼脂覆盖方法(Methods for General and Molecular Bacteriology,上文)。通过PCR扩增筛选转化体,对于budB 使用引物N 130SeqF1(SEQ ID NO:40)和N 130SeqR1(SEQ ID NO: 44),对于kivD使用引物N 133SeqF1(SEQ ID NO:62)和N133SeqR1 (SEQ ID NO:66)。阳性整合体显示出预期的1.7kbp budB和1.7kbp kivD PCR产物。鉴定了两个阳性整合体并命名为枯草芽孢杆菌BE1010 ΔsacB::Pspac-budB-ilvD-kivD #2-3-2和枯草芽孢杆菌BE1010 ΔsacB::Pspac-budB-ilvD-kivD#6-12-7。 整合体枯草芽孢杆菌BE 1010ΔsacB::Pspac-budB-ilvD-kivD#2-3-2 和枯草芽孢杆菌BE1010ΔsacB::Pspac-budB-ilvD-kivD#6-12-7中的酶 活性测定表明BudB,IlvD和KivD在spac启动子(Pspac)的控制下 是低的。为了提高功能性酶的表达,由来自质粒pHT01(MoBitec, Goettingen,Germany)的PgroE启动子替代Pspac启动子。 通过XhoI和BamHI限制性消化从不相关的质粒切除6,039bp pFP988Dss载体片段,如SEQ ID NO:146给出的,并凝胶纯化。从质 粒pHT01PCR扩增PgroE启动子,使用引物T-groE(XhoI)(SEQ ID NO:147)和B-groEL(SpeI,BamH1)(SEQ ID NO:148)。用XhoI 和BamHI消化PCR产物,连接6,039bp pFP988Dss载体片段,并转化 至DH5α感受态细胞中。通过PCR扩增筛选转化体,使用引物T-groE (XhoI)和B-groEL(SpeI,BamH1)。阳性克隆显示出预期的174bp PgroE PCR产物并称为pFP988DssPgroE。还通过DNA测序证实了质粒 pFP988DssPgroE。 用SpeI和PmeI消化质粒pFP988DssPspac-budB-ilvD-kivD并将所 得到的5,313bp budB-ilvD-kivD片段凝胶纯化。将budB-ilvD-kivD片段 连接pFP988DssPgroE的SpeI和PmeI位点并转化至DH5α感受态细胞 中。通过PCR扩增筛选阳性克隆的1,690bp PCR产物,使用引物T-groEL (SEQ ID NO:149)和N 111(SEQ ID NO:20)。将阳性克隆称为 pFP988DssPgroE-budB-ilvD-kivD。 从大肠杆菌转化体制备质粒pFP988DssPgroE-budB-ilvD-kivD,并 转化至枯草芽孢杆菌BE1010感受态细胞中,如上所述。通过PCR扩 增筛选转化体,对于budB使用引物N130SeqF1(SEQ ID NO:40)和 N130SeqR1(SEQ ID NO:44),对于kivD使用引物N133SeqF1(SEQ ID NO:62)和N 133SeqR1(SEQ ID NO:66)。阳性整合体显示出预 期的1.7kbpbudB和1.7kbpkivD PCR产物。分离了两个阳性整合体并 命名为枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7和枯 草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD#8-16。 ilvC和bdhB基因的质粒表达。从质粒表达两个剩余的异丁醇基因, ilvC和bdhB。将质粒pHT01(MoBitec),芽孢杆菌-大肠杆菌穿梭载体, 用于将来自枯草芽孢杆菌的ilvC基因与PgroE启动子融合,使得ilvC 基因从含有lacO序列的PgroE启动子表达。从枯草芽孢杆菌BR151 (ATCC 33677)基因组DNA PCR扩增ilvC基因,如SEQ ID NO:186 给出的,使用引物T-ilvCB.s.(BamHI)(SEQ ID NO:150)和B-ilvCB.s. (SpeI BamHI)(SEQ ID NO:151)。用BamHI消化1,067bp ilvC PCR 产物并连接pHT01的BamHI位点。将连接混合物转化至DH5α感受态 细胞中。通过PCR扩增筛选阳性克隆的1,188bp PCR产物,使用引物 T-groEL和B-ilvB.s(SpeI BamHI)。将阳性克隆称为pHT01-ilvC(B.s)。 将质粒pHT01-ilvC(B.s)用作PgroE-ilvC融合片段的PCR扩增的模板。 质粒pBD64(Minton等,Nucleic Acids Res.18:1651(1990))是 相当稳定的载体,用于在枯草芽孢杆菌中表达外源基因,并含有repB 基因以及氯霉素和卡那霉素抗性基因,用于在枯草芽孢杆菌中的选择。 该质粒用于ilvC和bdhB在PgroE启动子控制下的表达。为了将 PgroE-ilvC,bdhB和lacI阻遏基因克隆至质粒pBD64中,使用一步装 配方法(Tsuge等,Nucleic Acids Res.31:e133(2003))。从pBD64 PCR 扩增3,588bp pBD64片段,该片段含有repB基因,其包括复制功能, 和卡那霉素抗生素标记,使用引物T-BD64(DraIII)(SEQ ID NO:152), 其引入DraIII序列(CACCGAGTG),和B-BD64(DraIII)(SEQ ID NO: 153),其引入DraIII序列(CACCTGGTG)。从pMUTIN4PCR扩增 1,327bp lacI阻遏基因(Vagner等,Microbiol.144:3097-3104(1988)), 使用T-lacIq(DraIII)(SEQ ID NO:154),其引入了DraIII序列 (CACCAGGTG),和B-lacIq(DraIII)(SEQ ID NO:155),其引入了 DraIII序列(CACGGGGTG)。从pHT01-ilvC(B.s)PCR扩增1,224bp PgroE-ilvC融合的盒,使用T-groE(DraIII)(SEQ ID NO:156),其引 入了DraIII序列(CACCCCGTG),和B-B.s.ilvC(DraIII)(SEQ ID NO: 157),其引入了DraIII序列(CACCGTGTG)。从丙醇丁酮梭菌(ATCC 824)基因组DNA PCR扩增1.2kbp bdhB基因(SEQ ID NO:158),使 用引物T-bdhB(DraIII)(SEQ ID NO:159),其引入了DraIII序列 (CACACGGTG),和B-bdhB(rrnBT1DraIII)(SEQ ID NO:160),其 引入了DraIII序列(CACTCGGTG)。DraIII识别序列的可变区中的三 个下划线字母是为了特异性碱基配对而设计的,以 pBD64-lacI-PgroEilvC-bdhB的次序装配四个片段。用DraIII分开消化 两个末端的每个具有DraIII的PCR产物,将所得到的DraIII片段, 3,588bp pBD64,lacI,PgroEilvC和bdhB凝胶纯化,使用QIAGEN凝 胶提取试剂盒(QIAGEN)。制备含有等摩尔浓度的每个片段的混合物, 用于连接,总DNA浓度为30至50μg/100μL。然后将连接溶液在16℃ 孵育过夜。连接产生高分子量串联重复DNA。将连接的长线性DNA 混合物直接转化至感受态枯草芽孢杆菌BE1010中,按照以上所述的来 制备。枯草芽孢杆菌优选采用长的重复的线性DNA形式,而不是环状 DNA来建立质粒。转化后,将培养物涂布在含有10μg/mL卡那霉素的 LB平板上,用于选择。通过DraIII消化筛选阳性重组质粒,获得四个 具有预期大小3,588bp(pBD64),1,327bp(lacI),1,244bp(PgoE-ilvC) 和1,194bp(bdhB)的片段。将阳性质粒称为pBDPgroE-ilvC(B.s.)-bdhB。 通过枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD/pBDPgroE-ilvC (B.s.)-bdhB从葡萄糖或 蔗糖生产异丁醇的证明。为了构建表达异丁醇生物合成途径的五个基 因的重组枯草芽孢杆菌,如上所述制备两个整合体枯草芽孢杆菌 BE1010ΔsacB::PgroE-budB-ilvD-kivD#1-7和枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD#8-16的感受态细胞,并用质粒 pBDPgroE-ilvC(B.s.)-bdhB 转化,产生枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB和枯草 芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD #8-16/pBDPgroE-ilvC(B.s.)-bdhB。 将两个重组菌株在125mL烧瓶中的25mL或100mL含有卡那霉素 (10μg/mL)的葡萄糖培养基中培养,来各自模拟高氧和低氧条件,并 在37℃200rpm振荡好氧生长。培养基由10mM(NH4)2SO4,5mM磷 酸钾缓冲液(pH7.0),100mM MOPS/KOH缓冲液(pH7.0),20mM谷 氨酸/KOH(pH7.0),2%S10金属混合物,1%葡萄糖,0.01%酵母提取 物,0.01%酪蛋白氨基酸以及各自50μg/mL的L-色氨酸,L-甲硫氨酸 和L-赖氨酸组成。S10金属混合物由200mM MgCl2,70mM CaCl2,5mM MnCl2,0.1mM FeCl3,0.1mM ZnCl2,0.2mM盐酸硫胺素,0.172mM CuSO4,0.253mM CoCl2和0.242mM Na2MoCO4组成。在早对数期(大 约0.2的OD600)用1.0mM异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG) 诱导细胞。接种后24h,通过HPLC(Shodex Sugar SH1011柱)使用折 射率(RI)检测分析等份培养液的异丁醇含量,如一般方法部分中所 述。表12中显示了HPLC结果。 表12 通过枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD /pBDPgroE-ilvC(B.s.)-bdhB菌株从葡萄糖生产异丁醇 菌株 O2水平 异丁醇,mM 摩尔选择性,% 枯草芽孢杆菌a(诱导的) 高 1.00 1.8 枯草芽孢杆菌b(诱导的) 高 0.87 1.6 枯草芽孢杆菌a(诱导的) 低 0.06 0.1 枯草芽孢杆菌b(诱导的) 低 0.14 0.3 枯草芽孢杆菌a是枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB 枯草芽孢杆菌b是枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD#8-16/pBDPgroE-ilvC(B.s.)-bdhB 还检测了枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD #1-7/pBDPgroE-ilvC(B.s)-bdhB分离物从蔗糖的异丁醇生产,基本上如 上所述。将重组菌株在125mL烧瓶中的25mL或75mL含有卡那霉素 (10μg/mL)的蔗糖培养基中培养,来模拟高氧和中等氧水平,并在 37℃200rpm振荡生长。除了用10g/L蔗糖替代葡萄糖(10g/L),蔗糖 培养基与葡萄糖培养基相同。细胞没有诱导,或在早对数期(大约0.2 的OD600)用1.0mM异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)诱导。 接种后24h,通过HPLC(Shodex Sugar SH1011柱)使用折射率(RI) 检测分析等份培养液的异丁醇含量,如一般方法部分中所述。表13中 显示了HPLC结果。 表13 通过枯草芽孢杆菌BE1010ΔsacB::PgroE-budB-ilvD-kivD /pBDPgroE-ilvC(B.s.)-bdhB菌株从蔗糖生产异丁醇 菌株 O2水平 异丁醇,mM 摩尔选择性,% 枯草芽孢杆菌a(未诱导) 高 未测出 未测出 枯草芽孢杆菌a(诱导的) 高 0.44 4.9 枯草芽孢杆菌a(诱导的) 中等 0.83 8.6 枯草芽孢杆菌a是枯草芽孢杆菌BE1010 ΔsacB::PgroE-budB-ilvD-kivD#1-7/pBDPgroE-ilvC(B.s.)-bdhB 实施例21(预测的) 植物乳杆菌中异丁醇生物合成途径的表达 该预测实施例的目的是描述怎样在植物乳杆菌中表达异丁醇生物 合成途径。将异丁醇途径的编码五个酶活性的五个基因分至用于表达 的两个操纵子中。通过同源重组将各自编码乙酰乳酸合酶,乙酰醇酸 脱水酶和支链α-酮酸脱羧酶的基因budB,ilvD和kivD整合至植物乳杆 菌的染色体中,使用Hols等所述的方法(Appl.Environ.Microbiol.60: 1401-1413(1994))。将剩余的两个基因(ilvC和bdhB,各自编码乙酰 醇酸还原异构酶和丁醇脱氢酶)克隆至表达质粒中并转化至携带整合 异丁醇基因的乳杆菌菌株中。将植物乳杆菌生长于37℃MRS培养基 (Difco Laboratories,Detroit,MI)中,并分离染色体DNA,如Moreira 等所述的(BMC Microbiol.5:15(2005))。 整合。通过同源重组将合成P11启动子(Rud等,Microbiology 152: 1011-1019(2006))控制下的budB-ilvD-kivD盒在IdhL1基因座整合至 植物乳杆菌ATCC BAA-793(NCIMB 8826)的染色体中。为了构建IdhL 整合靶向载体,将来自植物乳杆菌的具有IdhL同源性的DNA片段 (Genbank NC_004567)PCR扩增,使用引物IDH EcoRV F(SEQ ID NO: 161)和IDH AatIIR(SEQ ID NO:162)。将1986bp PCR片段克隆至 pCR4Blunt-TOPO 中并测序。用EcoRV和AatII消化 pCR4Blunt-TOPO-IdhL1克隆,释放1982bp IdhL1片段,将其凝胶纯化。 用HindIII消化整合载体pFR988,如SEQ ID NO:177给出的,用Klenow DNA聚合酶处理来钝化末端。然后用AatII消化线性化的质粒,并将 2931bp载体片段凝胶纯化。将EcoRV/AatII IdhL1片段连接pFP988载 体片段并转化至大肠杆菌Top10细胞中。在含有氨苄青霉素 (100μg/mL)的LB琼脂平板上选择转化体并通过菌落PCR筛选来证 实pFP988-IdhL的构建。 为了将选择标记添加至整合DNA,从pC194(GenBank NC_002013,SEQ ID NO:267)PCR扩增具有启动子的Cm基因,使 用引物Cm F(SEQ ID NO:163)和Cm R(SEQ ID NO:164),扩增 了836bpPCR产物。将该PCR产物克隆至pCR4Blunt-TOPO中并转化 至大肠杆菌Top10细胞中,形成pCR4Blunt-TOPO-Cm。测序后证实了 通过PCR没有引入错误,从pCR4Blunt-TOPO-Cm将Cm盒消化为 828bpMluI/SwaI片段并凝胶纯化。用MluI和SwaI消化含有IdhL同源 性的整合载体pFP988-IdhL,并将4740bp载体片段凝胶纯化。将Cm 盒片段连接pFP988-IdhL载体,形成pFP988-DIdhL::Cm。 最后,用合成的P11启动子替代淀粉酶启动子来修饰实施例20中 所述的来自pFP988DssPspac-budB-ilvD-kivD的budB-ilvD-kivD盒。然 后,将整个操纵子转移至pFP988-DIdhL::Cm中。通过用引物P11F-StuI (SEQ ID NO:165)和P11R-SpeI(SEQ ID NO:166)退火寡核苷酸 来构建P11启动子。将退火的寡核苷酸在6%Ultra PAGE凝胶上凝胶 纯化(Embi Tec,San Diego,CA)。用StuI和SpeI消化含有淀粉酶启 动子的质粒pFP988DssPspac-budB-ilvD-kivD,并将所得到的10.9kbp 载体片段凝胶纯化。将分离的P11片段连接消化的pFP988DssPspac -budB-ilvD-kivD形成pFP988-P11-budB-ilvD-kivD。然后用StuI和 BamHI消化质粒pFP988DssPspac-budB-ilvD-kivD,并将所得到的 5.4kbp P11-budB-ilvD-kivD片段凝胶纯化。用HpaI和BamHI消化 pFP988-DIdhL::Cm并分离5.5kbp载体片段。将budB-ilvD-kivD操纵子 连接整合载体pFP988-DIdhL::Cm形成pFP988- DIdhL-P11-budB-ilvD-kivD::Cm。 将pFP988-DIdhL-P11-budB-ilvD-kivD::Cm整合至植物乳杆菌 BAA-793中形成包括外源budB,ilvD,kivD基因的植物乳杆菌 ΔIdhL1::budB-ilvD-kivD::Cm。按照Aukrust,T.W.等所述的制备植物乳 杆菌的电感受态细胞(在:Electroporation Protocols for Microorganisms; Nickoloff,J.A编辑;Methods in Molecular Biology,Vol.47;Humana Press,Inc.,Totowa,NJ,1995,pp201-208)。电穿孔后,按照上文中 Aukrust等所述的将细胞在MRSSM培养基中(补充0.5M蔗糖和0.1M MgCl2的MRS培养基)37℃生长2h,没有振荡)。将电穿孔的细胞涂 布于用于选择的含有氯霉素(10μg/mL)的MRS平板上并在37℃培养。 最初通过菌落PCR扩增来筛选转化体以证实整合,然后通过PCR使用 一组引物来更严格地筛选最初的阳性克隆。 ilvC和bdhB基因的质粒表达。在植物乳杆菌IdhL启动子的控制 下(Ferain等,J.Bacteriol.176:596-601(1994))从质粒pTRKH3 (O’Sullivan DJ和Klaenhammer TR,Gene 137:227-231(1993))表 达剩余的两个异丁醇基因。使用引物PIdhL F-HindIII(SEQ ID NO:167) 和PIdhL R-BamHI(SEQ ID NO:168)从植物乳杆菌ATCC BAA-793 的基因组PCR扩增IdhL启动子。将411bp PCR产物克隆至 pCR4Blunt-TOPO中并测序。用HindIII和BamHI消化所得到的质粒 pCR4Blunt-TOPO-PIdhL,释放PIdhL片段。 用HindIII和SphI消化质粒pTRKH3,并将凝胶纯化的载体片段以 三通方式连接PIdhL片段和来自芽孢杆菌表达质粒 pBDPgroE-ilvC(B.s.)-bdhB的凝胶纯化的含有ilvC(B.s.)-bdhB的 2.4kbpBamHI/SphI片段(实施例20)。将连接混合物转化至大肠杆菌 Top 10细胞中并将转化体生长于含有红霉素(150mg/L)的脑心浸液 (BHI,Difco Laboratories,Detroit,MI)平板上。通过PCR筛选转化 体来证实构建。通过电穿孔将所得到的表达质粒 pTRKH3-ilvC(B.s.)-bdhB转化至植物乳杆菌 ΔIdhL1::budB-ilvD-kivD::Cm中,如上所述。 将含有pTRKH3-ilvC(B.s.)-bdhB的植物乳杆菌 ΔIdhL1::budB-ilvD-kivD::Cm接种至含有50mL添加红霉素(10μg/mL) 的MRS培养基的250mL烧瓶中并在37℃生长18至24h,没有振荡, 此后,通过HPLC或GC分析检测异丁醇,如一般方法部分所述的。 实施例22(预测的) 粪肠球菌中异丁醇生物合成途径的表达 该预测实施例的目的是描述怎样在粪肠球菌中表达异丁醇生物合 成途径。已经公开了粪肠球菌菌株V583的完整基因组序列(Paulsen 等,Science 299:2071-2074(2003)),将其用作该实施例中表达异丁 醇生物合成途径的宿主菌株。将大肠杆菌革兰氏阳性穿梭载体,质粒 pTRKH3(O’Sullivan DJ和Klaenhammer TR,Gene 137:227-231 (1993)),用于异丁醇途径的五个基因(bubB,ilvC,ilvD,kivD,bdhB) 在一个操纵子中的表达。pTRKH3含有大肠杆菌质粒p15A复制起点, pAMβ1复制子,以及用于四环素和红霉素的两个抗生素抗性选择标记。 四环素抗性只在大肠杆菌中得到表达,而红霉素抗性在大肠杆菌和革 兰氏阳性细菌中都得到表达。质粒pAMβ1衍生物可以在粪肠球菌中复 制(Poyart等,FEMS Microbiol.Lett.156:193-198(1997))。将诱导 型nisA启动子(PnisA)用于控制编码异丁醇生物合成途径的酶的五个 所需基因的表达,该启动子已经用于基因表达的有效控制,通过各种 革兰氏阳性细菌包括粪肠球菌中的乳酸链球菌肽(Eichenbaum等,Appl. Environ.Microbiol.64:2763-2769(1998))。 将含有异丁醇途径操纵子的质粒pTrc99A::budB-ilvC-ilvD-kivD (实施例14中所述的)进行修饰,用枯草芽孢杆菌ilvC基因(SEQ ID NO:??)替代大肠杆菌ilvC基因(SEQ ID NO:3)。此外,将来自丙 醇丁酮梭菌的bdhB基因(SEQ ID NO:158)添加至操纵子的末端。 首先,使用引物F-bdhB-AvrII(SEQ ID NO:169)和R-bdhB-BamHI (SEQ ID NO:170)扩增来自pBDPgroE-ilvC(B.s.)-bdhB(实施例20 中所述的)的bdh基因,然后TOPO克隆并测序。通过用AvrII和BamHI 消化接着凝胶纯化分离了1194bp bdhB片段。将bdhB片段连接之前已 经用AvrII和BamHI消化的pTrc99A::budB-ilvC-ilvD-kivD,并将所得 到的片段凝胶纯化。通过电穿孔将连接混合物转化至大肠杆菌Top10 细胞中并在含有氨苄青霉素(100μg/mL)的LB琼脂平板上37℃生长 过夜后选择转化体。然后通过菌落PCR筛选转化体以证实含有 pTrc99A::budB-ilvC-ilvD-kivD-bdhB的正确克隆。 接着,使用引物F-ilvC(B.s.)-AflII(SEQ ID NO:171)和 R-ilvC(B.s.)-NotI(SEQ ID NO:172)扩增来自pBDPgroE-ilvC(B.s.)-bdhB (实施例20中所述的)的ilvC(B.s)。将PCR产物克隆并测序。通过用 AflII和NotI消化接着凝胶纯化分离了1051bp ilvC(B.s.)片段。将该片 段连接已经用AflII和NotI切割释放了大肠杆菌ilvC(在连接ilvC(B.s.) 之前凝胶纯化了10.7kbp载体条带)的 pTrc99A::budB-ilvC-ilvD-kivD-bdhB。通过电穿孔将连接混合物转化至 大肠杆菌Top10细胞中并在含有氨苄青霉素(100μg/mL)的LB琼脂 平板上37℃生长过夜后选择转化体。然后通过菌落PCR筛选转化体以 证实含有pTrc99A::budB-ilvC(B.s.)-ilvD-kivD-bdhB的正确克隆。 为了提供用于大肠杆菌革兰氏阳性穿梭载体pTRKH3的启动子, 使用引物F-PnisA(HindIII)(SEQ ID NO:173)和R-PnisA(SpeI BamHI)(SEQ ID NO:174)从乳酸乳球菌基因组DNA PCR扩增nisA 启动子(Chandrapati等,Mol.Microbiol.46(2):467-477(2002)),然 后TOPO克隆。测序后,通过用HindIII和BamHI消化接着凝胶纯化 分离了213bp nisA启动子片段。用HindIII和BamHI消化质粒pTRKH3 并将载体片段凝胶纯化。将线性化pTRKH3连接PnisA片段并通过电 穿孔转化至大肠杆菌Top10细胞中。在含有红霉素(25μg/mL)的LB 琼脂平板上37℃生长过夜后选择转化体。然后通过菌落PCR筛选转化 体以证实pTRKH3-PnisA的正确克隆。 用SpeI和BamHI消化质粒pTRKH3-PnisA,并将载体凝胶纯化。 用SpeI和BamHI消化上述的质粒 pTrc99A::budB-ilvC(B.s.)-ilvD-kivD-bdhB,并将7.5kbp片段凝胶纯化。 将7.5kbp budB-ilvC(B.s.)-ilvD-kivD-bdhB片段在SpeI和BamHI位点连 接pTRKH3-PnisA。通过电穿孔将连接混合物转化至大肠杆菌Top10细 胞中并在含有红霉素(25μg/mL)的LB琼脂平板上37℃生长过夜后选 择转化体。然后通过菌落PCR筛选转化体。将所得到的质粒称为 pTRKH3-PnisA-budB-ilvC(B.s.)-ilvD-kivD-bdhB。从大肠杆菌转化体制 备该质粒并使用本领域已知的方法转化至电感受态粪肠球菌V583细 胞中(Aukrust,T.W.等,在:Electroporation Protocols for Microorganisms (微生物的电穿孔方案);Nickoloff,J.A.编辑;Methods in Molecular Biology(分子生物学方法),Vol.47;Humana Press,Inc.,Totowa, NJ.,1995,pp.217-226),得到粪肠球菌V583/pTRKH3-PnisA- budB-ilvC(B.s.)-ilvD-kivD-bdhB。 通过电穿孔将含有nisA调控基因,nisR和nisK,add9壮观霉素抗 性基因和pSH71复制起点的第二个质粒转化至粪肠球菌 V583/pTRKH3-PnisA-budB-ilvC(B.s.)-ilvD-kivD-bdhB中。含有pSH71 复制起点的质粒与粪肠球菌中的pAMβ1衍生物相容(Eichenbaum等, 上文)。在37℃生长的含有红霉素(25μg/mL)和壮观霉素(100μg/mL) 的LB琼脂平板上选择双药物抗性转化体。 将所得到的带有两个质粒(即表达质粒(pTRKH3-PnisA- budB-ilvC(B.s.)-ilvD-kivD-bdhB)和调控质粒(pSH71-nisRK))的粪肠 球菌株V583B接种于含有50mL补充酵母提取物(0.2%)(Fischetti等, J.Exp.Med.161:1384-1401(1985)),乳酸链球菌肽(20μg/mL) (Eichenbaum等,上文),红霉素(25μg/mL)和壮观霉素(100μg/mL) 的Todd-Hewitt培养液的250mL烧瓶中。将烧瓶在37℃培养18-24h, 没有振荡,该时间后,通过HPLC或GC分析测量异丁醇产生,如一般 方法部分中所述的。 相关申请的交叉参考 依据U.S.C.§119,本申请要求2005年10月26日申请的U.S.临时 申请系列No.60/730290的优先权。 序列表 <110>E.I.du Pont de Nemours and Co. <120>四碳醇的发酵生产 <130>CL3243 PCT <160>267 <170>PatentIn version 3.3 <210>1 <211>1680 <212>DNA <213>肺炎克雷伯氏菌 <400>1 atggacaaac agtatccggt acgccagtgg gcgcacggcg ccgatctcgt cgtcagtcag 60 ctggaagctc agggagtacg ccaggtgttc ggcatccccg gcgccaaaat cgacaaggtc 120 tttgattcac tgctggattc ctccattcgc attattccgg tacgccacga agccaacgcc 180 gcatttatgg ccgccgccgt cggacgcatt accggcaaag cgggcgtggc gctggtcacc 240 tccggtccgg gctgttccaa cctgatcacc ggcatggcca ccgcgaacag cgaaggcgac 300 ccggtggtgg ccctgggcgg cgcggtaaaa cgcgccgata aagcgaagca ggtccaccag 360 agtatggata cggtggcgat gttcagcccg gtcaccaaat acgccatcga ggtgacggcg 420 ccggatgcgc tggcggaagt ggtctccaac gccttccgcg ccgccgagca gggccggccg 480 ggcagcgcgt tcgttagcct gccgcaggat gtggtcgatg gcccggtcag cggcaaagtg 540 ctgccggcca gcggggcccc gcagatgggc gccgcgccgg atgatgccat cgaccaggtg 600 gcgaagctta tcgcccaggc gaagaacccg atcttcctgc tcggcctgat ggccagccag 660 ccggaaaaca gcaaggcgct gcgccgtttg ctggagacca gccatattcc agtcaccagc 720 acctatcagg ccgccggagc ggtgaatcag gataacttct ctcgcttcgc cggccgggtt 780 gggctgttta acaaccaggc cggggaccgt ctgctgcagc tcgccgacct ggtgatctgc 840 atcggctaca gcccggtgga atacgaaccg gcgatgtgga acagcggcaa cgcgacgctg 900 gtgcacatcg acgtgctgcc cgcctatgaa gagcgcaact acaccccgga tgtcgagctg 960 gtgggcgata tcgccggcac tctcaacaag ctggcgcaaa atatcgatca tcggctggtg 1020 ctctccccgc aggcggcgga gatcctccgc gaccgccagc accagcgcga gctgctggac 1080 cgccgcggcg cgcagctcaa ccagtttgcc ctgcatcccc tgcgcatcgt tcgcgccatg 1140 caggatatcg tcaacagcga cgtcacgttg accgtggaca tgggcagctt ccatatctgg 1200 attgcccgct acctgtacac gttccgcgcc cgtcaggtga tgatctccaa cggccagcag 1260 accatgggcg tcgccctgcc ctgggctatc ggcgcctggc tggtcaatcc tgagcgcaaa 1320 gtggtctccg tctccggcga cggcggcttc ctgcagtcga gcatggagct ggagaccgcc 1380 gtccgcctga aagccaacgt gctgcatctt atctgggtcg ataacggcta caacatggtc 1440 gctatccagg aagagaaaaa atatcagcgc ctgtccggcg tcgagtttgg gccgatggat 1500 tttaaagcct atgccgaatc cttcggcgcg aaagggtttg ccgtggaaag cgccgaggcg 1560 ctggagccga ccctgcgcgc ggcgatggac gtcgacggcc cggcggtagt ggccatcccg 1620 gtggattatc gcgataaccc gctgctgatg ggccagctgc atctgagtca gattctgtaa 1680 <210>2 <211>559 <212>PRT <213>肺炎克雷伯氏菌 <400>2 Met Asp Lys Gln Tyr Pro Val Arg Gln Trp Ala His Gly Ala Asp Leu 1 5 10 15 Val Val Ser Gln Leu Glu Ala Gln Gly Val Arg Gln Val Phe Gly Ile 20 25 30 Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Ser Leu Leu Asp Ser Ser 35 40 45 Ile Arg Ile Ile Pro Val Arg His Glu Ala Asn Ala Ala Phe Met Ala 50 55 60 Ala Ala Val Gly Arg Ile Thr Gly Lys Ala Gly Val Ala Leu Val Thr 65 70 75 80 Ser Gly Pro Gly Cys Ser Asn Leu Ile Thr Gly Met Ala Thr Ala Asn 85 90 95 Ser Glu Gly Asp Pro Val Val Ala Leu Gly Gly Ala Val Lys Arg Ala 100 105 110 Asp Lys Ala Lys Gln Val His Gln Ser Met Asp Thr Val Ala Met Phe 115 120 125 Ser Pro Val Thr Lys Tyr Ala Ile Glu Val Thr Ala Pro Asp Ala Leu 130 135 140 Ala Glu Val Val Ser Asn Ala Phe Arg Ala Ala Glu Gln Gly Arg Pro 145 150 155 160 Gly Ser Ala Phe Val Ser Leu Pro Gln Asp Val Val Asp Gly Pro Val 165 170 175 Ser Gly Lys Val Leu Pro Ala Ser Gly Ala Pro Gln Met Gly Ala Ala 180 185 190 Pro Asp Asp Ala Ile Asp Gln Val Ala Lys Leu Ile Ala Gln Ala Lys 195 200 205 Asn Pro Ile Phe Leu Leu Gly Leu Met Ala Ser Gln Pro Glu Asn Ser 210 215 220 Lys Ala Leu Arg Arg Leu Leu Glu Thr Ser His Ile Pro Val Thr Ser 225 230 235 240 Thr Tyr Gln Ala Ala Gly Ala Val Asn Gln Asp Asn Phe Ser Arg Phe 245 250 255 Ala Gly Arg Val Gly Leu Phe Asn Asn Gln Ala Gly Asp Arg Leu Leu 260 265 270 Gln Leu Ala Asp Leu Val Ile Cys Ile Gly Tyr Ser Pro Val Glu Tyr 275 280 285 Glu Pro Ala Met Trp Asn Ser Gly Asn Ala Thr Leu Val His Ile Asp 290 295 300 Val Leu Pro Ala Tyr Glu Glu Arg Asn Tyr Thr Pro Asp Val Glu Leu 305 310 315 320 Val Gly Asp Ile Ala Gly Thr Leu Asn Lys Leu Ala Gln Asn Ile Asp 325 330 335 His Arg Leu Val Leu Ser Pro Gln Ala Ala Glu Ile Leu Arg Asp Arg 340 345 350 Gln His Gln Arg Glu Leu Leu Asp Arg Arg Gly Ala Gln Leu Asn Gln 355 360 365 Phe Ala Leu His Pro Leu Arg Ile Val Arg Ala Met Gln Asp Ile Val 370 375 380 Asn Ser Asp Val Thr Leu Thr Val Asp Met Gly Ser Phe His Ile Trp 385 390 395 400 Ile Ala Arg Tyr Leu Tyr Thr Phe Arg Ala Arg Gln Val Met Ile Ser 405 410 415 Asn Gly Gln Gln Thr Met Gly Val Ala Leu Pro Trp Ala Ile Gly Ala 420 425 430 Trp Leu Val Asn Pro Glu Arg Lys Val Val Ser Val Ser Gly Asp Gly 435 440 445 Gly Phe Leu Gln Ser Ser Met Glu Leu Glu Thr Ala Val Arg Leu Lys 450 455 460 Ala Asn Val Leu His Leu Ile Trp Val Asp Asn Gly Tyr Asn Met Val 465 470 475 480 Ala Ile Gln Glu Glu Lys Lys Tyr Gln Arg Leu Ser Gly Val Glu Phe 485 490 495 Gly Pro Met Asp Phe Lys Ala Tyr Ala Glu Ser Phe Gly Ala Lys Gly 500 505 510 Phe Ala Val Glu Ser Ala Glu Ala Leu Glu Pro Thr Leu Arg Ala Ala 515 520 525 Met Asp Val Asp Gly Pro Ala Val Val Ala Ile Pro Val Asp Tyr Arg 530 535 540 Asp Asn Pro Leu Leu Met Gly Gln Leu His Leu Ser Gln Ile Leu 545 550 555 <210>3 <211>1476 <212>DNA <213>大肠杆菌 <400>3 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggttaa 1476 <210>4 <211>491 <212>PRT <213>大肠杆菌 <400>4 Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln 1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala 20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser 50 55 60 Tyr Ala Leu Arg Lys Glu Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg 65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile 85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser 100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu 115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg 130 135 140 Lys Asp Ile Thr Val Val Met Val Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys 180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser 195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile 210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ala Leu Lys Gln Gly Gly Ile Thr Leu 260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu 275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met 290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln 340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly 355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu 370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu 420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile 435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala 450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly 485 490 <210>5 <211>1851 <212>DNA <213>大肠杆菌 <400>5 atgcctaagt accgttccgc caccaccact catggtcgta atatggcggg tgctcgtgcg 60 ctgtggcgcg ccaccggaat gaccgacgcc gatttcggta agccgattat cgcggttgtg 120 aactcgttca cccaatttgt accgggtcac gtccatctgc gcgatctcgg taaactggtc 180 gccgaacaaa ttgaagcggc tggcggcgtt gccaaagagt tcaacaccat tgcggtggat 240 gatgggattg ccatgggcca cggggggatg ctttattcac tgccatctcg cgaactgatc 300 gctgattccg ttgagtatat ggtcaacgcc cactgcgccg acgccatggt ctgcatctct 360 aactgcgaca aaatcacccc ggggatgctg atggcttccc tgcgcctgaa tattccggtg 420 atctttgttt ccggcggccc gatggaggcc gggaaaacca aactttccga tcagatcatc 480 aagctcgatc tggttgatgc gatgatccag ggcgcagacc cgaaagtatc tgactcccag 540 agcgatcagg ttgaacgttc cgcgtgtccg acctgcggtt cctgctccgg gatgtttacc 600 gctaactcaa tgaactgcct gaccgaagcg ctgggcctgt cgcagccggg caacggctcg 660 ctgctggcaa cccacgccga ccgtaagcag ctgttcctta atgctggtaa acgcattgtt 720 gaattgacca aacgttatta cgagcaaaac gacgaaagtg cactgccgcg taatatcgcc 780 agtaaggcgg cgtttgaaaa cgccatgacg ctggatatcg cgatgggtgg atcgactaac 840 accgtacttc acctgctggc ggcggcgcag gaagcggaaa tcgacttcac catgagtgat 900 atcgataagc tttcccgcaa ggttccacag ctgtgtaaag ttgcgccgag cacccagaaa 960 taccatatgg aagatgttca ccgtgctggt ggtgttatcg gtattctcgg cgaactggat 1020 cgcgcggggt tactgaaccg tgatgtgaaa aacgtacttg gcctgacgtt gccgcaaacg 1080 ctggaacaat acgacgttat gctgacccag gatgacgcgg taaaaaatat gttccgcgca 1140 ggtcctgcag gcattcgtac cacacaggca ttctcgcaag attgccgttg ggatacgctg 1200 gacgacgatc gcgccaatgg ctgtatccgc tcgctggaac acgcctacag caaagacggc 1260 ggcctggcgg tgctctacgg taactttgcg gaaaacggct gcatcgtgaa aacggcaggc 1320 gtcgatgaca gcatcctcaa attcaccggc ccggcgaaag tgtacgaaag ccaggacgat 1380 gcggtagaag cgattctcgg cggtaaagtt gtcgccggag atgtggtagt aattcgctat 1440 gaaggcccga aaggcggtcc ggggatgcag gaaatgctct acccaaccag cttcctgaaa 1500 tcaatgggtc tcggcaaagc ctgtgcgctg atcaccgacg gtcgtttctc tggtggcacc 1560 tctggtcttt ccatcggcca cgtctcaccg gaagcggcaa gcggcggcag cattggcctg 1620 attgaagatg gtgacctgat cgctatcgac atcccgaacc gtggcattca gttacaggta 1680 agcgatgccg aactggcggc gcgtcgtgaa gcgcaggacg ctcgaggtga caaagcctgg 1740 acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc gtgcttatgc cagcctggca 1800 accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac tggggggtta a 1851 <210>6 <211>616 <212>PRT <213>大肠杆菌 <400>6 Met Pro Lys Tyr Arg Ser Ala Thr Thr Thr His Gly Arg Asn Met Ala 1 5 10 15 Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Met Thr Asp Ala Asp Phe 20 25 30 Gly Lys Pro Ile Ile Ala Val Val Asn Ser Phe Thr Gln Phe Val Pro 35 40 45 Gly His Val His Leu Arg Asp Leu Gly Lys Leu Val Ala Glu Gln Ile 50 55 60 Glu Ala Ala Gly Gly Val Ala Lys Glu Phe Asn Thr Ile Ala Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser 85 90 95 Arg Glu Leu Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Cys 100 105 110 Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly 115 120 125 Met Leu Met Ala Ser Leu Arg Leu Asn Ile Pro Val Ile Phe Val Ser 130 135 140 Gly Gly Pro Met Glu Ala Gly Lys Thr Lys Leu Ser Asp Gln Ile Ile 145 150 155 160 Lys Leu Asp Leu Val Asp Ala Met Ile Gln Gly Ala Asp Pro Lys Val 165 170 175 Ser Asp Ser Gln Ser Asp Gln Val Glu Arg Ser Ala Cys Pro Thr Cys 180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Thr 195 200 205 Glu Ala Leu Gly Leu Ser Gln Pro Gly Asn Gly Ser Leu Leu Ala Thr 210 215 220 His Ala Asp Arg Lys Gln Leu Phe Leu Asn Ala Gly Lys Arg Ile Val 225 230 235 240 Glu Leu Thr Lys Arg Tyr Tyr Glu Gln Asn Asp Glu Ser Ala Leu Pro 245 250 255 Arg Asn Ile Ala Ser Lys Ala Ala Phe Glu Asn Ala Met Thr Leu Asp 260 265 270 Ile Ala Met Gly Gly Ser Thr Asn Thr Val Leu His Leu Leu Ala Ala 275 280 285 Ala Gln Glu Ala Glu Ile Asp Phe Thr Met Ser Asp Ile Asp Lys Leu 290 295 300 Ser Arg Lys Val Pro Gln Leu Cys Lys Val Ala Pro Ser Thr Gln Lys 305 310 315 320 Tyr His Met Glu Asp Val His Arg Ala Gly Gly Val Ile Gly Ile Leu 325 330 335 Gly Glu Leu Asp Arg Ala Gly Leu Leu Asn Arg Asp Val Lys Asn Val 340 345 350 Leu Gly Leu Thr Leu Pro Gln Thr Leu Glu Gln Tyr Asp Val Met Leu 355 360 365 Thr Gln Asp Asp Ala Val Lys Asn Met Phe Arg Ala Gly Pro Ala Gly 370 375 380 Ile Arg Thr Thr Gln Ala Phe Ser Gln Asp Cys Arg Trp Asp Thr Leu 385 390 395 400 Asp Asp Asp Arg Ala Asn Gly Cys Ile Arg Ser Leu Glu His Ala Tyr 405 410 415 Ser Lys Asp Gly Gly Leu Ala Val Leu Tyr Gly Asn Phe Ala Glu Asn 420 425 430 Gly Cys Ile Val Lys Thr Ala Gly Val Asp Asp Ser Ile Leu Lys Phe 435 440 445 Thr Gly Pro Ala Lys Val Tyr Glu Ser Gln Asp Asp Ala Val Glu Ala 450 455 460 Ile Leu Gly Gly Lys Val Val Ala Gly Asp Val Val Val Ile Arg Tyr 465 470 475 480 Glu Gly Pro Lys Gly Gly Pro Gly Met Gln Glu Met Leu Tyr Pro Thr 485 490 495 Ser Phe Leu Lys Ser Met Gly Leu Gly Lys Ala Cys Ala Leu Ile Thr 500 505 510 Asp Gly Arg Phe Ser Gly Gly Thr Ser Gly Leu Ser Ile Gly His Val 515 520 525 Ser Pro Glu Ala Ala Ser Gly Gly Ser Ile Gly Leu Ile Glu Asp Gly 530 535 540 Asp Leu Ile Ala Ile Asp Ile Pro Asn Arg Gly Ile Gln Leu Gln Val 545 550 555 560 Ser Asp Ala Glu Leu Ala Ala Arg Arg Glu Ala Gln Asp Ala Arg Gly 565 570 575 Asp Lys Ala Trp Thr Pro Lys Asn Arg Glu Arg Gln Val Ser Phe Ala 580 585 590 Leu Arg Ala Tyr Ala Ser Leu Ala Thr Ser Ala Asp Lys Gly Ala Val 595 600 605 Arg Asp Lys Ser Lys Leu Gly Gly 610 615 <210>7 <211>1662 <212>DNA <213>乳酸乳球菌 <400>7 tctagacata tgtatactgt gggggattac ctgctggatc gcctgcacga actggggatt 60 gaagaaattt tcggtgtgcc aggcgattat aacctgcagt tcctggacca gattatctcg 120 cacaaagata tgaagtgggt cggtaacgcc aacgaactga acgcgagcta tatggcagat 180 ggttatgccc gtaccaaaaa agctgctgcg tttctgacga cctttggcgt tggcgaactg 240 agcgccgtca acggactggc aggaagctac gccgagaacc tgccagttgt cgaaattgtt 300 gggtcgccta cttctaaggt tcagaatgaa ggcaaatttg tgcaccatac tctggctgat 360 ggggatttta aacattttat gaaaatgcat gaaccggtta ctgcggcccg cacgctgctg 420 acagcagaga atgctacggt tgagatcgac cgcgtcctgt ctgcgctgct gaaagagcgc 480 aagccggtat atatcaatct gcctgtcgat gttgccgcag cgaaagccga aaagccgtcg 540 ctgccactga aaaaagaaaa cagcacctcc aatacatcgg accaggaaat tctgaataaa 600 atccaggaat cactgaagaa tgcgaagaaa ccgatcgtca tcaccggaca tgagatcatc 660 tcttttggcc tggaaaaaac ggtcacgcag ttcatttcta agaccaaact gcctatcacc 720 accctgaact tcggcaaatc tagcgtcgat gaagcgctgc cgagttttct gggtatctat 780 aatggtaccc tgtccgaacc gaacctgaaa gaattcgtcg aaagcgcgga ctttatcctg 840 atgctgggcg tgaaactgac ggatagctcc acaggcgcat ttacccacca tctgaacgag 900 aataaaatga tttccctgaa tatcgacgaa ggcaaaatct ttaacgagcg catccagaac 960 ttcgattttg aatctctgat tagttcgctg ctggatctgt ccgaaattga gtataaaggt 1020 aaatatattg ataaaaaaca ggaggatttt gtgccgtcta atgcgctgct gagtcaggat 1080 cgtctgtggc aagccgtaga aaacctgaca cagtctaatg aaacgattgt tgcggaacag 1140 ggaacttcat ttttcggcgc ctcatccatt tttctgaaat ccaaaagcca tttcattggc 1200 caaccgctgt gggggagtat tggttatacc tttccggcgg cgctgggttc acagattgca 1260 gataaggaat cacgccatct gctgtttatt ggtgacggca gcctgcagct gactgtccag 1320 gaactggggc tggcgatccg tgaaaaaatc aatccgattt gctttatcat caataacgac 1380 ggctacaccg tcgaacgcga aattcatgga ccgaatcaaa gttacaatga catcccgatg 1440 tggaactata gcaaactgcc ggaatccttt ggcgcgacag aggatcgcgt ggtgagtaaa 1500 attgtgcgta cggaaaacga atttgtgtcg gttatgaaag aagcgcaggc tgacccgaat 1560 cgcatgtatt ggattgaact gatcctggca aaagaaggcg caccgaaagt tctgaaaaag 1620 atggggaaac tgtttgcgga gcaaaataaa agctaaggat cc 1662 <210>8 <211>548 <212>PRT <213>乳酸乳球菌 <400>8 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys Ser 545 <210>9 <211>1164 <212>DNA <213>大肠杆菌 <400>9 atgaacaactt taatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60 ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120 gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180 gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240 gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300 accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360 caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420 gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480 caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540 tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600 gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660 ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720 cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780 ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840 cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900 cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960 gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020 acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080 gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140 cgtatatacg aagccgcccg ctaa 1164 <210>10 <211>387 <212>PRT <213>大肠杆菌 <400>10 Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys 1 5 10 15 Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val 20 25 30 Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp 35 40 45 Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly 50 55 60 Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu 65 70 75 80 Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser 85 90 95 Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu 100 105 110 Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys 115 120 125 Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser 130 135 140 Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys 145 150 155 160 Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp 165 170 175 Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val 180 185 190 Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val 195 200 205 Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu 210 215 220 Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val 225 230 235 240 Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile 245 250 255 Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu 260 265 270 Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val 275 280 285 Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu 290 295 300 Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp 305 310 315 320 Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln 325 330 335 Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser 340 345 350 Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu 355 360 365 Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu 370 375 380 Ala Ala Arg 385 <210>11 <211>29 <212>DNA <213>人造序列 <220> <223>引物 <400>11 caccatggac aaacagtatc cggtacgcc 29 <210>12 <211>25 <212>DNA <213>人造序列 <220> <223>引物 <400>12 cgaagggcga tagctttacc aatcc 25 <210>13 <211>29 <212>DNA <213>人造序列 <220> <223>引物 <400>13 caccatggct aactacttca atacactga 29 <210>14 <211>28 <212>DNA <213>人造序列 <220> <223>引物 <400>14 ccaggagaag gccttgagtg ttttctcc 28 <210>15 <211>29 <212>DNA <213>人造序列 <220> <223>引物 <400>15 caccatgcct aagtaccgtt ccgccacca 29 <210>16 <211>26 <212>DNA <213>人造序列 <220> <223>引物 <400>16 cgcagcactg ctcttaaata ttcggc 26 <210>17 <211>29 <212>DNA <213>人造序列 <220> <223>引物 <400>17 caccatgaac aactttaatc tgcacaccc 29 <210>18 <211>29 <212>DNA <213>人造序列 <220> <223>引物 <400>18 caccatgaac aactttaatc tgcacaccc 29 <210>19 <211>45 <212>DNA <213>人造序列 <220> <223>引物 <400>19 gcatgcctta agaaaggagg ggggtcacat ggacaaacag tatcc 45 <210>20 <211>39 <212>DNA <213>人造序列 <220> <223>引物 <400>20 atgcatttaa ttaattacag aatctgactc agatgcagc 39 <210>21 <211>45 <212>DNA <213>人造序列 <220> <223>引物 <400>21 gtcgacgcta gcaaaggagg gaatcaccat ggctaactac ttcaa 45 <210>22 <211>31 <212>DNA <213>人造序列 <220> <223>引物 <400>22 tctagattaa cccgcaacag caatacgttt c 31 <210>23 <211>39 <212>DNA <213>人造序列 <220> <223>引物 <400>23 tctagaaaag gaggaataaa gtatgcctaa gtaccgttc 39 <210>24 <211>31 <212>DNA <213>人造序列 <220> <223>引物 <400>24 ggatccttat taacccccca gtttcgattt a 31 <210>25 <211>39 <212>DNA <213>人造序列 <220> <223>引物 <400>25 ggatccaaag gaggctagac atatgtatac tgtggggga 39 <210>26 <211>31 <212>DNA <213>人造序列 <220> <223>引物 <400>26 gagctcttag cttttatttt gctccgcaaa c 31 <210>27 <211>39 <212>DNA <213>人造序列 <220> <223>引物 <400>27 gagctcaaag gaggagcaag taatgaacaa ctttaatct 39 <210>28 <211>43 <212>DNA <213>人造序列 <220> <223>引物 <400>28 gaattcacta gtcctaggtt agcgggcggc ttcgtatata cgg 43 <210>29 <211>25 <212>DNA <213>人造序列 <220> <223>引物 <400>29 caacattagc gattttcttt tctct 25 <210>30 <211>45 <212>DNA <213>人造序列 <220> <223>引物 <400>30 catgaagctt actagtgggc ttaagttttg aaaataatga aaact 45 <210>31 <211>61 <212>DNA <213>人造序列 <220> <223>引物N110.2 <400>31 gagctcacta gtcaattgta agtaagtaaa aggaggtggg tcacatggac aaacagtatc 60 c 61 <210>32 <211>50 <212>DNA <213>人造序列 <220> <223>引物N111.2 <400>32 ggatccgatc gacttaagcc tcagcttaca gaatctgact cagatgcagc 50 <210>33 <211>44 <212>DNA <213>人造序列 <220> <223>引物N112.2 <400>33 gagctcctta agaaggaggt aatcaccatg gctaactact tcaa 44 <210>34 <211>51 <212>DNA <213>人造序列 <220> <223>引物N113.2 <400>34 ggatccgatc gagctagcgc ggccgcttaa cccgcaacag caatacgttt c 51 <210>35 <211>44 <212>DNA <213>人造序列 <220> <223>引物N114.2 <400>35 gagctcgcta gcaaggaggt ataaagtatg cctaagtacc gttc 44 <210>36 <211>52 <212>DNA <213>人造序列 <220> <223>引物N115.2 <400>36 ggatccgatc gattaattaa cctaaggtta ttaacccccc agtttcgatt ta 52 <210>37 <211>46 <212>DNA <213>人造序列 <220> <223>引物N116.2 <400>37 gagctcttaa ttaaaaggag gttagacata tgtatactgt ggggga 46 <210>38 <211>49 <212>DNA <213>人造序列 <220> <223>引物117.2 <400>38 ggatccagat ctcctaggac atgtttagct tttattttgc tccgcaaac 49 <210>39 <211>3883 <212>DNA <213>大肠杆菌 <400>39 ctatattgct gaaggtacag gcgtttccat aactatttgc tcgcgttttt tactcaagaa 60 gaaaatgcca aatagcaaca tcaggcagac aatacccgaa attgcgaaga aaactgtctg 120 gtagcctgcg tggtcaaaga gtatcccagt cggcgttgaa agcagcacaa tcccaagcga 180 actggcaatt tgaaaaccaa tcagaaagat cgtcgacgac aggcgcttat caaagtttgc 240 cacgctgtat ttgaagacgg atatgacaca aagtggaacc tcaatggcat gtaacaactt 300 cactaatgaa ataatccagg ggttaacgaa cagcgcgcag gaaaggatac gcaacgccat 360 aatcacaact ccgataagta atgcattttt tggccctacc cgattcacaa agaaaggaat 420 aatcgccatg cacagcgctt cgagtaccac ctggaatgag ttgagataac catacaggcg 480 cgttcctaca tcgtgtgatt cgaataaacc tgaataaaag acaggaaaaa gttgttgatc 540 aaaaatgtta tagaaagacc acgtccccac aataaatatg acgaaaaccc agaagtttcg 600 atccttgaaa actgcgataa aatcctcttt ttttacccct cccgcatctg ccgctacgca 660 ctggtgatcc ttatctttaa aacgcatgtt gatcatcata aatacagcgc caaatagcga 720 gaccaaccag aagttgatat ggggactgat actaaaaaat atgccggcaa agaacgcgcc 780 aatagcatag ccaaaagatc cccaggcgcg cgctgttcca tattcgaaat gaaaatttcg 840 cgccattttt tcggtgaagc tatcaagcaa accgcatccc gccagatacc ccaagccaaa 900 aaatagcgcc cccagaatta gacctacaga aaaattgctt tgcagtaacg gttcataaac 960 gtaaatcata aacggtccgg tcaagaccag gatgaaactc atacaccaga tgagcggttt 1020 cttcagaccg agtttatcct gaacgatgcc gtagaacatc ataaatagaa tgctggtaaa 1080 ctggttgacc gaataaagtg tacctaattc cgtccctgtc aaccctagat gtcctttcag 1140 ccaaatagcg tataacgacc accacagcga ccaggaaata aaaaagagaa atgagtaact 1200 ggatgcaaaa cgatagtacg catttctgaa tggaatattc agtgccataa ttacctgcct 1260 gtcgttaaaa aattcacgtc ctatttagag ataagagcga cttcgccgtt tacttctcac 1320 tattccagtt cttgtcgaca tggcagcgct gtcattgccc ctttcgccgt tactgcaagc 1380 gctccgcaac gttgagcgag atcgataatt cgtcgcattt ctctctcatc tgtagataat 1440 cccgtagagg acagacctgt gagtaacccg gcaacgaacg catctcccgc ccccgtgcta 1500 tcgacacaat tcacagacat tccagcaaaa tggtgaactt gtcctcgata acagaccacc 1560 accccttctg cacctttagt caccaacagc atggcgatct catactcttt tgccagggcg 1620 catatatcct gatcgttctg tgtttttcca ctgataagtc gccattcttc ttccgagagc 1680 ttgacgacat ccgccagttg tagcgcctgc cgcaaacaca agcggagcaa atgctcgtct 1740 tgccatagat cttcacgaat attaggatcg aagctgacaa aacctccggc atgccggatc 1800 gccgtcatcg cagtaaatgc gctggtacgc gaaggctcgg cagacaacgc aattgaacag 1860 agatgtaacc attcgccatg tcgccagcag ggcaagtctg tcgtctctaa aaaaagatcg 1920 gcactggggc ggaccataaa cgtaaatgaa cgttcccctt gatcgttcag atcgacaagc 1980 accgtggatg tccggtgcca ttcatcttgc ttcagatacg tgatatcgac tccctcagtt 2040 agcagcgttc tttgcattaa cgcaccaaaa ggatcatccc ccacccgacc tataaaccca 2100 cttgttccgc ctaatctggc gattcccacc gcaacgttag ctggcgcgcc gccaggacaa 2160 ggcagtaggc gcccgtctga ttctggcaag agatctacga ccgcatcccc taaaacccat 2220 actttggctg acattttttt cccttaaatt catctgagtt acgcatagtg ataaacctct 2280 ttttcgcaaa atcgtcatgg atttactaaa acatgcatat tcgatcacaa aacgtcatag 2340 ttaacgttaa catttgtgat attcatcgca tttatgaaag taagggactt tatttttata 2400 aaagttaacg ttaacaattc accaaatttg cttaaccagg atgattaaaa tgacgcaatc 2460 tcgattgcat gcggcgcaaa acgccctagc aaaacttcat gagcaccggg gtaacacttt 2520 ctatccccat tttcacctcg cgcctcctgc cgggtggatg aacgatccaa acggcctgat 2580 ctggtttaac gatcgttatc acgcgtttta tcaacatcat ccgatgagcg aacactgggg 2640 gccaatgcac tggggacatg ccaccagcga cgatatgatc cactggcagc atgagcctat 2700 tgcgctagcg ccaggagacg ataatgacaa agacgggtgt ttttcaggta gtgctgtcga 2760 tgacaatggt gtcctctcac ttatctacac cggacacgtc tggctcgatg gtgcaggtaa 2820 tgacgatgca attcgcgaag tacaatgtct ggctaccagt cgggatggta ttcatttcga 2880 gaaacagggt gtgatcctca ctccaccaga aggaatcatg cacttccgcg atcctaaagt 2940 gtggcgtgaa gccgacacat ggtggatggt agtcggggcg aaagatccag gcaacacggg 3000 gcagatcctg ctttatcgcg gcagttcgtt gcgtgaatgg accttcgatc gcgtactggc 3060 ccacgctgat gcgggtgaaa gctatatgtg ggaatgtccg gactttttca gccttggcga 3120 tcagcattat ctgatgtttt ccccgcaggg aatgaatgcc gagggataca gttaccgaaa 3180 tcgctttcaa agtggcgtaa tacccggaat gtggtcgcca ggacgacttt ttgcacaatc 3240 cgggcatttt actgaacttg ataacgggca tgacttttat gcaccacaaa gctttttagc 3300 gaaggatggt cggcgtattg ttatcggctg gatggatatg tgggaatcgc caatgccctc 3360 aaaacgtgaa ggatgggcag gctgcatgac gctggcgcgc gagctatcag agagcaatgg 3420 caaacttcta caacgcccgg tacacgaagc tgagtcgtta cgccagcagc atcaatctgt 3480 ctctccccgc acaatcagca ataaatatgt tttgcaggaa aacgcgcaag cagttgagat 3540 tcagttgcag tgggcgctga agaacagtga tgccgaacat tacggattac agctcggcac 3600 tggaatgcgg ctgtatattg ataaccaatc tgagcgactt gttttgtggc ggtattaccc 3660 acacgagaat ttagacggct accgtagtat tcccctcccg cagcgtgaca cgctcgccct 3720 aaggatattt atcgatacat catccgtgga agtatttatt aacgacgggg aagcggtgat 3780 gagtagtcga atctatccgc agccagaaga acgggaactg tcgctttatg cctcccacgg 3840 agtggctgtg ctgcaacatg gagcactctg gctactgggt taa 3883 <210>40 <211>19 <212>DNA <213>人造序列 <220> <223>引物N130SeqF1 <400>40 tgttccaacc tgatcaccg 19 <210>41 <211>18 <212>DNA <213>人造序列 <220> <223>引物N130SeqF2 <400>41 ggaaaacagc aaggcgct 18 <210>42 <211>18 <212>DNA <213>人造序列 <220> <223>引物N130SeqF3 <400>42 cagctgaacc agtttgcc 18 <210>43 <211>19 <212>DNA <213>人造序列 <220> <223>引物N130SeqF4 <400>43 aaaataccag cgcctgtcc 19 <210>44 <211>18 <212>DNA <213>人造序列 <220> <223>引物N130SeqR1 <400>44 tgaatggcca ccatgttg 18 <210>45 <211>18 <212>DNA <213>人造序列 <220> <223>引物N130SeqR2 <400>45 gaggatctcc gccgcctg 18 <210>46 <211>18 <212>DNA <213>人造序列 <220> <223>引物N130SeqR3 <400>46 aggccgagca ggaagatc 18 <210>47 <211>19 <212>DNA <213>人造序列 <220> <223>引物N130SeqR4 <400>47 tgatcaggtt ggaacagcc 19 <210>48 <211>19 <212>DNA <213>人造序列 <220> <223>引物N131SeqF1 <400>48 aagaactgat cccacaggc 19 <210>49 <211>19 <212>DNA <213>人造序列 <220> <223>引物N131SeqF2 <400>49 atcctgtgcg gtatgttgc 19 <210>50 <211>18 <212>DNA <213>人造序列 <220> <223>引物N131Seqf3 <400>50 attgcgatgg tgaaagcg 18 <210>51 <211>19 <212>DNA <213>人造序列 <220> <223>引物N131SeqR1 <400>51 atggtgttgg caatcagcg 19 <210>52 <211>18 <212>DNA <213>人造序列 <220> <223>引物N131SeqR2 <400>52 gtgcttcggt gatggttt 18 <210>53 <211>19 <212>DNA <213>人造序列 <220> <223>引物N131SeqR3 <400>53 ttgaaaccgt gcgagtagc 19 <210>54 <211>19 <212>DNA <213>人造序列 <220> <223>引物N132SeqF1 <400>54 tattcactgc catctcgcg 19 <210>55 <211>18 <212>DNA <213>人造序列 <220> <223>引物N132SeqF2 <400>55 ccgtaagcag ctgttcct 18 <210>56 <211>20 <212>DNA <213>人造序列 <220> <223>引物N132SeqF3 <400>56 gctggaacaa tacgacgtta 20 <210>57 <211>20 <212>DNA <213>人造序列 <220> <223>引物N132SeqF4 <400>57 tgctctaccc aaccagcttc 20 <210>58 <211>20 <212>DNA <213>人造序列 <220> <223>引物N132SeqR1 <400>58 atggaaagac cagaggtgcc 20 <210>59 <211>18 <212>DNA <213>人造序列 <220> <223>引物N132SeqR2 <400>59 tgcctgtgtg gtacgaat 18 <210>60 <211>19 <212>DNA <213>人造序列 <220> <223>引物N132SeqR3 <400>60 tattacgcgg cagtgcact 19 <210>61 <211>22 <212>DNA <213>人造序列 <220> <223>引物N132SeqR4 <400>61 ggtgattttg tcgcagttag ag 22 <210>62 <211>18 <212>DNA <213>人造序列 <220> <223>引物N133SeqF1 <400>62 tcgaaattgt tgggtcgc 18 <210>63 <211>22 <212>DNA <213>人造序列 <220> <223>引物N133SeqF2 <400>63 ggtcacgcag ttcatttcta ag 22 <210>64 <211>18 <212>DNA <213>人造序列 <220> <223>引物N133SeqF3 <400>64 tgtggcaagc cgtagaaa 18 <210>65 <211>19 <212>DNA <213>人造序列 <220> <223>引物N133SeqF4 <400>65 aggatcgcgt ggtgagtaa 19 <210>66 <211>20 <212>DNA <213>人造序列 <220> <223>引物N133SeqR1 <400>66 gtagccgtcg ttattgatga 20 <210>67 <211>22 <212>DNA <213>人造序列 <220> <223>引物N133SeqR2 <400>67 gcagcgaact aatcagagat tc 22 <210>68 <211>19 <212>DNA <213>人造序列 <220> <223>引物N133SeqR3 <400>68 tggtccgatg tattggagg 19 <210>69 <211>19 <212>DNA <213>人造序列 <220> <223>引物N133SeqR4 <400>69 tctgccatat agctcgcgt 19 <210>70 <211>42 <212>DNA <213>人造序列 <220> <223>启动子1.6GI变体 <400>70 gcccttgaca atgccacatc ctgagcaaat aattcaacca ct 42 <210>71 <211>42 <212>DNA <213>人造序列 <220> <223>启动子1.5GI <400>71 gcccttgact atgccacatc ctgagcaaat aattcaacca ct 42 <210>72 <211>18 <212>DNA <213>人造序列 <220> <223>引物Scr1 <400>72 cctttctttg tgaatcgg 18 <210>73 <211>18 <212>DNA <213>人造序列 <220> <223>引物Scr2 <400>73 agaaacaggg tgtgatcc 18 <210>74 <211>20 <212>DNA <213>人造序列 <220> <223>引物Scr3 <400>74 agtgatcatc acctgttgcc 20 <210>75 <211>20 <212>DNA <213>人造序列 <220> <223>引物Scr4 <400>75 agcacggcga gagtcgacgg 20 <210>76 <211>672 <212>DNA <213>酿酒酵母 <400>76 agttcgagtt tatcattatc aatactgcca tttcaaagaa tacgtaaata attaatagta 60 gtgattttcc taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta 120 catgcccaaa atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa 180 cagtttattc ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga 240 aaaaaaaaga atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc 300 cattctctta gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc 360 tcaatggagt gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca 420 tgtatctatc tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa 480 gctgaaaaaa aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta 540 tataaagacg gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt 600 ctacttttat agttagtctt ttttttagtt ttaaaacacc aagaacttag tttcgaataa 660 acacacataa ac 672 <210>77 <211>270 <212>DNA <213>酿酒酵母 <400>77 gacctcgagt catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 60 ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 120 tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 180 gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 240 cgaaggcttt aatttgcggc cggtacccaa 270 <210>78 <211>1716 <212>DNA <213>枯草芽孢杆菌 <400>78 atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60 gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120 attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180 gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240 gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300 actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360 cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420 gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480 gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540 aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600 atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660 aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720 ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780 tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840 gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900 ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960 cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020 gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080 catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140 gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200 cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260 aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320 ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380 ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440 tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500 ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560 tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620 atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680 gaattcgggg aactcatgaa aacgaaagct ctctag 1716 <210>79 <211>643 <212>DNA <213>酿酒酵母 <400>79 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210>80 <211>1188 <212>DNA <213>酿酒酵母 <400>80 atgttgagaa ctcaagccgc cagattgatc tgcaactccc gtgtcatcac tgctaagaga 60 acctttgctt tggccacccg tgctgctgct tacagcagac cagctgcccg tttcgttaag 120 ccaatgatca ctacccgtgg tttgaagcaa atcaacttcg gtggtactgt tgaaaccgtc 180 tacgaaagag ctgactggcc aagagaaaag ttgttggact acttcaagaa cgacactttt 240 gctttgatcg gttacggttc ccaaggttac ggtcaaggtt tgaacttgag agacaacggt 300 ttgaacgtta tcattggtgt ccgtaaagat ggtgcttctt ggaaggctgc catcgaagac 360 ggttgggttc caggcaagaa cttgttcact gttgaagatg ctatcaagag aggtagttac 420 gttatgaact tgttgtccga tgccgctcaa tcagaaacct ggcctgctat caagccattg 480 ttgaccaagg gtaagacttt gtacttctcc cacggtttct ccccagtctt caaggacttg 540 actcacgttg aaccaccaaa ggacttagat gttatcttgg ttgctccaaa gggttccggt 600 agaactgtca gatctttgtt caaggaaggt cgtggtatta actcttctta cgccgtctgg 660 aacgatgtca ccggtaaggc tcacgaaaag gcccaagctt tggccgttgc cattggttcc 720 ggttacgttt accaaaccac tttcgaaaga gaagtcaact ctgacttgta cggtgaaaga 780 ggttgtttaa tgggtggtat ccacggtatg ttcttggctc aatacgacgt cttgagagaa 840 aacggtcact ccccatctga agctttcaac gaaaccgtcg aagaagctac ccaatctcta 900 tacccattga tcggtaagta cggtatggat tacatgtacg atgcttgttc caccaccgcc 960 agaagaggtg ctttggactg gtacccaatc ttcaagaatg ctttgaagcc tgttttccaa 1020 gacttgtacg aatctaccaa gaacggtacc gaaaccaaga gatctttgga attcaactct 1080 caacctgact acagagaaaa gctagaaaag gaattagaca ccatcagaaa catggaaatc 1140 tggaaggttg gtaaggaagt cagaaagttg agaccagaaa accaataa 1188 <210>81 <211>760 <212>DNA <213>酿酒酵母 <400>81 tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt tgtttccggg 60 tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat 120 accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata 180 ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg 240 gtacataacg aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc 300 actacccttt ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc 360 tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa 420 tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt 480 tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc 540 acactactct ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat 600 aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg 660 tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca 720 agctatacca agcatacaat caactatctc atatacaatg 760 <210>82 <211>316 <212>DNA <213>酿酒酵母 <400>82 gagtaagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata 60 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 120 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 180 cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cacccaattg 240 tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta tgtcctcaga 300 ggacaacacc tgtggt 316 <210>83 <211>1758 <212>DNA <213>酿酒酵母 <400>83 atgggcttgt taacgaaagt tgctacatct agacaattct ctacaacgag atgcgttgca 60 aagaagctca acaagtactc gtatatcatc actgaaccta agggccaagg tgcgtcccag 120 gccatgcttt atgccaccgg tttcaagaag gaagatttca agaagcctca agtcggggtt 180 ggttcctgtt ggtggtccgg taacccatgt aacatgcatc tattggactt gaataacaga 240 tgttctcaat ccattgaaaa agcgggtttg aaagctatgc agttcaacac catcggtgtt 300 tcagacggta tctctatggg tactaaaggt atgagatact cgttacaaag tagagaaatc 360 attgcagact cctttgaaac catcatgatg gcacaacact acgatgctaa catcgccatc 420 ccatcatgtg acaaaaacat gcccggtgtc atgatggcca tgggtagaca taacagacct 480 tccatcatgg tatatggtgg tactatcttg cccggtcatc caacatgtgg ttcttcgaag 540 atctctaaaa acatcgatat cgtctctgcg ttccaatcct acggtgaata tatttccaag 600 caattcactg aagaagaaag agaagatgtt gtggaacatg catgcccagg tcctggttct 660 tgtggtggta tgtatactgc caacacaatg gcttctgccg ctgaagtgct aggtttgacc 720 attccaaact cctcttcctt cccagccgtt tccaaggaga agttagctga gtgtgacaac 780 attggtgaat acatcaagaa gacaatggaa ttgggtattt tacctcgtga tatcctcaca 840 aaagaggctt ttgaaaacgc cattacttat gtcgttgcaa ccggtgggtc cactaatgct 900 gttttgcatt tggtggctgt tgctcactct gcgggtgtca agttgtcacc agatgatttc 960 caaagaatca gtgatactac accattgatc ggtgacttca aaccttctgg taaatacgtc 1020 atggccgatt tgattaacgt tggtggtacc caatctgtga ttaagtatct atatgaaaac 1080 aacatgttgc acggtaacac aatgactgtt accggtgaca ctttggcaga acgtgcaaag 1140 aaagcaccaa gcctacctga aggacaagag attattaagc cactctccca cccaatcaag 1200 gccaacggtc acttgcaaat tctgtacggt tcattggcac caggtggagc tgtgggtaaa 1260 attaccggta aggaaggtac ttacttcaag ggtagagcac gtgtgttcga agaggaaggt 1320 gcctttattg aagccttgga aagaggtgaa atcaagaagg gtgaaaaaac cgttgttgtt 1380 atcagatatg aaggtccaag aggtgcacca ggtatgcctg aaatgctaaa gccttcctct 1440 gctctgatgg gttacggttt gggtaaagat gttgcattgt tgactgatgg tagattctct 1500 ggtggttctc acgggttctt aatcggccac attgttcccg aagccgctga aggtggtcct 1560 atcgggttgg tcagagacgg cgatgagatt atcattgatg ctgataataa caagattgac 1620 ctattagtct ctgataagga aatggctcaa cgtaaacaaa gttgggttgc acctccacct 1680 cgttacacaa gaggtactct atccaagtat gctaagttgg tttccaacgc ttccaacggt 1740 tgtgttttag atgcttga 1758 <210>84 <211>753 <212>DNA <213>酿酒酵母 <400>84 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300 gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360 cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420 ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480 agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540 aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600 ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660 gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720 ttaataatcc aaacaaacac acatattaca ata 753 <210>85 <211>20 <212>DNA <213>人造序列 <220> <223>引物N98SeqF1 <400>85 cgtgttagtc acatcaggac 20 <210>86 <211>24 <212>DNA <213>人造序列 <220> <223>引物N98SeqF2 <400>86 ggccatagca aaaatccaaa cagc 24 <210>87 <211>24 <212>DNA <213>人造序列 <220> <223>引物N98SeqF3 <400>87 ccacgatcaa tcatatcgaa cacg 24 <210>88 <211>20 <212>DNA <213>人造序列 <220> <223>引物N98SeqF4 <400>88 ggtttctgtc tctggtgacg 20 <210>89 <211>22 <212>DNA <213>人造序列 <220> <223>引物N99SeqR1 <400>89 gtctggtgat tctacgcgca ag 22 <210>90 <211>22 <212>DNA <213>人造序列 <220> <223>引物N99SeqR2 <400>90 catcgactgc attacgcaac tc 22 <210>91 <211>22 <212>DNA <213>人造序列 <220> <223>引物N99SeqR3 <400>91 cgatcgtcag aacaacatct gc 22 <210>92 <211>20 <212>DNA <213>人造序列 <220> <223>引物N99SeqR4 <400>92 ccttcagtgt tcgctgtcag 20 <210>93 <211>36 <212>DNA <213>人造序列 <220> <223>引物N136 <400>93 ccgcggatag atctgaaatg aataacaata ctgaca 36 <210>94 <211>65 <212>DNA <213>人造序列 <220> <223>引物N137 <400>94 taccaccgaa gttgatttgc ttcaacatcc tcagctctag atttgaatat gtattacttg 60 gttat 65 <210>95 <211>28 <212>DNA <213>人造序列 <220> <223>引物N138 <400>95 atgttgaagc aaatcaactt cggtggta 28 <210>96 <211>22 <212>DNA <213>人造序列 <220> <223>引物N139 <400>96 ttattggttt tctggtctca ac 22 <210>97 <211>57 <212>DNA <213>人造序列 <220> <223>引物N140 <400>97 aagttgagac cagaaaacca ataattaatt aatcatgtaa ttagttatgt cacgctt 57 <210>98 <211>30 <212>DNA <213>人造序列 <220> <223>引物N141 <400>98 gcggccgccc gcaaattaaa gccttcgagc 30 <210>99 <211>28 <212>DNA <313>人造序列 <220> <223>引物N142 <400>99 ggatccgcat gcttgcattt agtcgtgc 28 <210>100 <211>56 <212>DNA <213>人造序列 <220> <223>N143 <400>100 caggtaatcc cccacagtat acatcctcag ctattgtaat atgtgtgttt gtttgg 56 <210>101 <211>22 <212>DNA <213>人造序列 <220> <223>引物N144 <400>101 atgtatactg tgggggatta cc 22 <210>102 <211>22 <212>DNA <213>人造序列 <220> <223>引物N145 <400>102 ttagctttta ttttgctccg ca 22 <210>103 <211>57 <212>DNA <213>人造序列 <220> <223>引物N146 <400>103 tttgcggagc aaaataaaag ctaattaatt aagagtaagc gaatttctta tgattta 57 <210>104 <211>28 <212>DNA <213>人造序列 <220> <223>引物N147 <400>104 actagtacca caggtgttgt cctctgag 28 <210>105 <211>22 <212>DNA <213>人造序列 <220> <223>引物N151 <400>105 ctagagagct ttcgttttca tg 22 <210>106 <211>57 <212>DNA <213>人造序列 <220> <223>引物N152 <400>106 ctcatgaaaa cgaaagctct ctagttaatt aatcatgtaa ttagttatgt cacgctt 57 <210>107 <211>25 <212>DNA <213>人造序列 <220> <223>引物N155 <400>107 atggcaaaga agctcaacaa gtact 25 <210>108 <211>22 <212>DNA <213>人造序列 <220> <223>引物N156 <400>108 tcaagcatct aaaacacaac cg 22 <210>109 <211>57 <212>DNA <213>人造序列 <220> <223>引物N157 <400>109 aacggttgtg ttttagatgc ttgattaatt aagagtaagc gaatttctta tgattta 57 <210>110 <211>28 <212>DNA <213>人造序列 <220> <223>引物N158 <400>110 ggatcctttt ctggcaacca aacccata 28 <210>111 <211>56 <212>DNA <213>人造序列 <220> <223>引物N159 <400>111 cgagtacttg ttgagcttct ttgccatcct cagcgagata gttgattgta tgcttg 56 <210>112 <211>19 <212>DNA <213>人造序列 <220> <223>引物N160SeqF1 <400>112 gaaaacgtgg catcctctc 19 <210>113 <211>19 <212>DNA <213>人造序列 <220> <223>引物N160SeqF2 <400>113 gctgactggc caagagaaa 19 <210>114 <211>20 <212>DNA <213>人造序列 <220> <223>引物N160SeqF3 <400>114 tgtacttctc ccacggtttc 20 <210>115 <211>22 <212>DNA <213>人造序列 <220> <223>引物N160SeqF4 <400>115 agctacccaa tctctatacc ca 22 <210>116 <211>22 <212>DNA <213>人造序列 <220> <223>引物N160SeqF5 <400>116 cctgaagtct aggtccctat tt 22 <210>117 <211>19 <212>DNA <213>人造序列 <220> <223>N160SeqR1 <400>117 gcgtgaatgt aagcgtgac 19 <210>118 <211>20 <212>DNA <213>人造序列 <220> <223>引物N160SeqR2 <400>118 cgtcgtattg agccaagaac 20 <210>119 <211>20 <212>DNA <213>人造序列 <220> <223>引物N160SeqR3 <400>119 gcatcggaca acaagttcat 20 <210>120 <211>22 <212>DNA <213>人造序列 <220> <223>引物N160SeqR4 <400>120 tcgttcttga agtagtccaa ca 22 <210>121 <211>19 <212>DNA <213>人造序列 <220> <223>引物N160SeqR5 <400>121 tgagcccgaa agagaggat 19 <210>122 <211>19 <212>DNA <213>人造序列 <220> <223>引物N161SeqF1 <400>122 acggtatacg gccttcctt 19 <210>123 <211>20 <212>DNA <213>人造序列 <220> <223>引物N161SeqF2 <400>123 gggtttgaaa gctatgcagt 20 <210>124 <211>22 <212>DNA <213>人造序列 <220> <223>引物N161SeqF3 <400>124 ggtggtatgt atactgccaa ca 22 <210>125 <211>22 <212>DNA <213>人造序列 <220> <223>引物N161SeqF4 <400>125 ggtggtaccc aatctgtgat ta 22 <210>126 <211>20 <212>DNA <213>人造序列 <220> <223>引物N161SeqF5 <400>126 cggtttgggt aaagatgttg 20 <210>127 <211>22 <212>DNA <213>人造序列 <220> <223>引物N161SeqF6 <400>127 aaacgaaaat tcttattctt ga 22 <210>128 <211>22 <212>DNA <213>人造序列 <220> <223>引物N161SeqR1 <400>128 tcgttttaaa acctaagagt ca 22 <210>129 <211>19 <212>DNA <213>人造序列 <220> <223>引物N161SeqR2 <400>129 ccaaaccgta acccatcag 19 <210>130 <211>19 <212>DNA <213>人造序列 <220> <223>引物N161SeqR3 <400>130 cacagattgg gtaccacca 19 <210>131 <211>20 <212>DNA <213>人造序列 <220> <223>引物N161Seqr4 <400>131 accacaagaa ccaggacctg 20 <210>132 <211>19 <212>DNA <213>人造序列 <220> <223>引物N161SeqR5 <400>132 catagctttc aaacccgct 19 <210>133 <211>22 <212>DNA <213>人造序列 <220> <223>引物N161SeqR6 <400>133 cgtataccgt tgctcattagag 22 <210>134 <211>23 <212>DNA <213>人造序列 <220> <223>引物N162 <400>134 atgttgacaa aagcaacaaa aga 23 <210>135 <211>38 <212>DNA <213>人造序列 <220> <223>引物N189 <400>135 atccgcggat agatctagtt cgagtttatc attatcaa 38 <210>136 <211>53 <212>DNA <213>人造序列 <220> <223>引物N190.1 <400>136 ttcttttgtt gcttttgtca acatcctcag cgtttatgtg tgtttattcg aaa 53 <210>137 <211>38 <212>DNA <213>人造序列 <220> <223>引物N176 <400>137 atccgcggat agatctatta gaagccgccg agcgggcg 38 <210>138 <211>31 <212>DNA <213>人造序列 <220> <223>引物N177 <400>138 atcctcagct tttctccttg acgttaaagt a 31 <210>139 <211>477 <212>PRT <213>大肠杆菌 <400>139 Met Thr Gln Ser Arg Leu His Ala Ala Gln Asn Ala Leu Ala Lys Leu 1 5 10 15 His Glu His Arg Gly Asn Thr Phe Tyr Pro His Phe His Leu Ala Pro 20 25 30 Pro Ala Gly Trp Met Asn Asp Pro Asn Gly Leu Ile Trp Phe Asn Asp 35 40 45 Arg Tyr His Ala Phe Tyr Gln His His Pro Met Ser Glu His Trp Gly 50 55 60 Pro Met His Trp Gly His Ala Thr Ser Asp Asp Met Ile His Trp Gln 65 70 75 80 His Glu Pro Ile Ala Leu Ala Pro Gly Asp Asp Asn Asp Lys Asp Gly 85 90 95 Cys Phe Ser Gly Ser Ala Val Asp Asp Asn Gly Val Leu Ser Leu Ile 100 105 110 Tyr Thr Gly His Val Trp Leu Asp Gly Ala Gly Asn Asp Asp Ala Ile 115 120 125 Arg Glu Val Gln Cys Leu Ala Thr Ser Arg Asp Gly Ile His Phe Glu 130 135 140 Lys Gln Gly Val Ile Leu Thr Pro Pro Glu Gly Ile Met His Phe Arg 145 150 155 160 Asp Pro Lys Val Trp Arg Glu Ala Asp Thr Trp Trp Met Val Val Gly 165 170 175 Ala Lys Asp Pro Gly Asn Thr Gly Gln Ile Leu Leu Tyr Arg Gly Ser 180 185 190 Ser Leu Arg Glu Trp Thr Phe Asp Arg Val Leu Ala His Ala Asp Ala 195 200 205 Gly Glu Ser Tyr Met Trp Glu Cys Pro Asp Phe Phe Ser Leu Gly Asp 210 215 220 Gln His Tyr Leu Met Phe Ser Pro Gln Gly Met Asn Ala Glu Gly Tyr 225 230 235 240 Ser Tyr Arg Asn Arg Phe Gln Ser Gly Val Ile Pro Gly Met Trp Ser 245 250 255 Pro Gly Arg Leu Phe Ala Gln Ser Gly His Phe Thr Glu Leu Asp Asn 260 265 270 Gly His Asp Phe Tyr Ala Pro Gln Ser Phe Leu Ala Lys Asp Gly Arg 275 280 285 Arg Ile Val Ile Gly Trp Met Asp Met Trp Glu Ser Pro Met Pro Ser 290 295 300 Lys Arg Glu Gly Trp Ala Gly Cys Met Thr Leu Ala Arg Glu Leu Ser 305 310 315 320 Glu Ser Asn Gly Lys Leu Leu Gln Arg Pro Val His Glu Ala Glu Ser 325 330 335 Leu Arg Gln Gln His Gln Ser Val Ser Pro Arg Thr Ile Ser Asn Lys 340 345 350 Tyr Val Leu Gln Glu Asn Ala Gln Ala Val Glu Ile Gln Leu Gln Trp 355 360 365 Ala Leu Lys Asn Ser Asp Ala Glu His Tyr Gly Leu Gln Leu Gly Thr 370 375 380 Gly Met Arg Leu Tyr Ile Asp Asn Gln Ser Glu Arg Leu Val Leu Trp 385 390 395 400 Arg Tyr Tyr Pro His Glu Asn Leu Asp Gly Tyr Arg Ser Ile Pro Leu 405 410 415 Pro Gln Arg Asp Thr Leu Ala Leu Arg Ile Phe Ile Asp Thr Ser Ser 420 425 430 Val Glu Val Phe Ile Asn Asp Gly Glu Ala Val Met Ser Ser Arg Ile 435 440 445 Tyr Pro Gln Pro Glu Glu Arg Glu Leu Ser Leu Tyr Ala Ser His Gly 450 455 460 Val Ala Val Leu Gln His Gly Ala Leu Trp Leu Leu Gly 465 470 475 <210>140 <211>304 <212>PRT <213>大肠杆菌 <400>140 Met Ser Ala Lys Val Trp Val Leu Gly Asp Ala Val Val Asp Leu Leu 1 5 10 15 Pro Glu Ser Asp Gly Arg Leu Leu Pro Cys Pro Gly Gly Ala Pro Ala 20 25 30 Asn Val Ala Val Gly Ile Ala Arg Leu Gly Gly Thr Ser Gly Phe Ile 35 40 45 Gly Arg Val Gly Asp Asp Pro Phe Gly Ala Leu Met Gln Arg Thr Leu 50 55 60 Leu Thr Glu Gly Val Asp Ile Thr Tyr Leu Lys Gln Asp Glu Trp His 65 70 75 80 Arg Thr Ser Thr Val Leu Val Asp Leu Asn Asp Gln Gly Glu Arg Ser 85 90 95 Phe Thr Phe Met Val Arg Pro Ser Ala Asp Leu Phe Leu Glu Thr Thr 100 105 110 Asp Leu Pro Cys Trp Arg His Gly Glu Trp Leu His Leu Cys Ser Ile 115 120 125 Ala Leu Ser Ala Glu Pro Ser Arg Thr Ser Ala Phe Thr Ala Met Thr 130 135 140 Ala Ile Arg His Ala Gly Gly Phe Val Ser Phe Asp Pro Asn Ile Arg 145 150 155 160 Glu Asp Leu Trp Gln Asp Glu His Leu Leu Arg Leu Cys Leu Arg Gln 165 170 175 Ala Leu Gln Leu Ala Asp Val Val Lys Leu Ser Glu Glu Glu Trp Arg 180 185 190 Leu Ile Ser Gly Lys Thr Gln Asn Asp Gln Asp Ile Cys Ala Leu Ala 195 200 205 Lys Glu Tyr Glu Ile Ala Met Leu Leu Val Thr Lys Gly Ala Glu Gly 210 215 220 Val Val Val Cys Tyr Arg Gly Gln Val His His Phe Ala Gly Met Ser 225 230 235 240 Val Asn Cys Val Asp Ser Thr Gly Ala Gly Asp Ala Phe Val Ala Gly 245 250 255 Leu Leu Thr Gly Leu Ser Ser Thr Gly Leu Ser Thr Asp Glu Arg Glu 260 265 270 Met Arg Arg Ile Ile Asp Leu Ala Gln Arg Cys Gly Ala Leu Ala Val 275 280 285 Thr Ala Lys Gly Ala Met Thr Ala Leu Pro Cys Arg Gln Glu Leu Glu 290 295 300 <210>141 <211>415 <212>PRT <213>大肠杆菌 <400>141 Met Ala Leu Asn Ile Pro Phe Arg Asn Ala Tyr Tyr Arg Phe Ala Ser 1 5 10 15 Ser Tyr Ser Phe Leu Phe Phe Ile Ser Trp Ser Leu Trp Trp Ser Leu 20 25 30 Tyr Ala Ile Trp Leu Lys Gly His Leu Gly Leu Thr Gly Thr Glu Leu 35 40 45 Gly Thr Leu Tyr Ser Val Asn Gln Phe Thr Ser Ile Leu Phe Met Met 50 55 60 Phe Tyr Gly Ile Val Gln Asp Lys Leu Gly Leu Lys Lys Pro Leu Ile 65 70 75 80 Trp Cys Met Ser Phe Ile Leu Val Leu Thr Gly Pro Phe Met Ile Tyr 85 90 95 Val Tyr Glu Pro Leu Leu Gln Ser Asn Phe Ser Val Gly Leu Ile Leu 100 105 110 Gly Ala Leu Phe Phe Gly Leu Gly Tyr Leu Ala Gly Cys Gly Leu Leu 115 120 125 Asp Ser Phe Thr Glu Lys Met Ala Arg Asn Phe His Phe Glu Tyr Gly 130 135 140 Thr Ala Arg Ala Trp Gly Ser Phe Gly Tyr Ala Ile Gly Ala Phe Phe 145 150 155 160 Ala Gly Ile Phe Phe Ser Ile Ser Pro His Ile Asn Phe Trp Leu Val 165 170 175 Ser Leu Phe Gly Ala Val Phe Met Met Ile Asn Met Arg Phe Lys Asp 180 185 190 Lys Asp His Gln Cys Val Ala Ala Asp Ala Gly Gly Val Lys Lys Glu 195 200 205 Asp Phe Ile Ala Val Phe Lys Asp Arg Asn Phe Trp Val Phe Val Ile 210 215 220 Phe Ile Val Gly Thr Trp Ser Phe Tyr Asn Ile Phe Asp Gln Gln Leu 225 230 235 240 Phe Pro Val Phe Tyr Ser Gly Leu Phe Glu Ser His Asp Val Gly Thr 245 250 255 Arg Leu Tyr Gly Tyr Leu Asn Ser Phe Gln Val Val Leu Glu Ala Leu 260 265 270 Cys Met Ala Ile Ile Pro Phe Phe Val Asn Arg Val Gly Pro Lys Asn 275 280 285 Ala Leu Leu Ile Gly Val Val Ile Met Ala Leu Arg Ile Leu Ser Cys 290 295 300 Ala Leu Phe Val Asn Pro Trp Ile Ile Ser Leu Val Lys Leu Leu His 305 310 315 320 Ala Ile Glu Val Pro Leu Cys Val Ile Ser Val Phe Lys Tyr Ser Val 325 330 335 Ala Asn Phe Asp Lys Arg Leu Ser Ser Thr Ile Phe Leu Ile Gly Phe 340 345 350 Gln Ile Ala Ser Ser Leu Gly Ile Val Leu Leu Ser Thr Pro Thr Gly 355 360 365 Ile Leu Phe Asp His Ala Gly Tyr Gln Thr Val Phe Phe Ala Ile Ser 370 375 380 Gly Ile Val Cys Leu Met Leu Leu Phe Gly Ile Phe Phe Leu Ser Lys 385 390 395 400 Lys Arg Glu Gln Ile Val Met Glu Thr Pro Val Pro Ser Ala Ile 405 410 415 <210>142 <211>6341 <212>DNA <213>人造序列 <220> <223>质粒pFP988DssPspac <400>142 gatccaagtt taaactgtac actagatatt tcttctccgc ttaaatcatc aaagaaatct 60 ttatcacttg taaccagtcc gtccacatgt cgaattgcat ctgaccgaat tttacgtttc 120 cctgaataat tctcatcaat cgtttcatca attttatctt tatactttat attttgtgcg 180 ttaatcaaat cataattttt atatgtttcc tcatgattta tgtctttatt attatagttt 240 ttattctctc tttgattatg tctttgtatc ccgtttgtat tacttgatcc tttaactctg 300 gcaaccctca aaattgaatg agacatgcta cacctccgga taataaatat atataaacgt 360 atatagattt cataaagtct aacacactag acttatttac ttcgtaatta agtcgttaaa 420 ccgtgtgctc tacgaccaaa actataaaac ctttaagaac tttctttttt tacaagaaaa 480 aagaaattag ataaatctct catatctttt attcaataat cgcatccgat tgcagtataa 540 atttaacgat cactcatcat gttcatattt atcagagctc gtgctataat tatactaatt 600 ttataaggag gaaaaaatat gggcattttt agtatttttg taatcagcac agttcattat 660 caaccaaaca aaaaataagt ggttataatg aatcgttaat aagcaaaatt catataacca 720 aattaaagag ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt 780 caaaacataa tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct 840 ttgaaatcgg ctcaggaaaa ggccatttta cccttgaatt agtaaagagg tgtaatttcg 900 taactgccat tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc 960 acgataattt ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat 1020 cctataaaat atatggtaat ataccttata acataagtac ggatataata cgcaaaattg 1080 tttttgatag tatagctaat gagatttatt taatcgtgga atacgggttt gctaaaagat 1140 tattaaatac aaaacgctca ttggcattac ttttaatggc agaagttgat atttctatat 1200 taagtatggt tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca 1260 gattaagtag aaaaaaatca agaatatcac acaaagataa acaaaagtat aattatttcg 1320 ttatgaaatg ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt 1380 ccttaaaaca tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc 1440 ttttcaatag ctataaatta tttaataagt aagttaaggg atgcagttca tcgatgaagg 1500 caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg 1560 ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga 1620 atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca 1680 aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat 1740 gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa 1800 cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct 1860 gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta 1920 catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg 1980 ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc 2040 tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt 2100 ctacgcagac aaacaatcaa cgtttgcgcc aagcttgcat gcgagagtag ggaactgcca 2160 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 2220 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga 2280 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta 2340 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt tttgtttatt 2400 tttctaaata cattcaaata tgtatccgct catgctccgg atctgcatcg caggatgctg 2460 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt 2520 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag 2580 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg 2640 tatcattacc cccatgaaca gaaattcccc cttacacgga ggcatcaagt gaccaaacag 2700 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag 2760 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac 2820 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 2880 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2940 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 3000 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 3060 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 3120 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3180 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3240 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3300 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3360 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 3420 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 3480 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3540 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3600 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 3660 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 3720 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 3780 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 3840 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3900 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3960 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4020 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4080 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 4140 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 4200 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 4260 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 4320 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 4380 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 4440 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 4500 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 4560 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 4620 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 4680 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 4740 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 4800 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 4860 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4920 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 4980 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 5040 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 5100 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5160 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 5220 aagcccgtca gggcgcgtca gcgggtgttc atgtgcgtaa ctaacttgcc atcttcaaac 5280 aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg 5340 aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca 5400 ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac 5460 ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa 5520 aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc 5580 ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc aaactatcac 5640 ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt 5700 tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc 5760 gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa 5820 gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat 5880 ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca 5940 tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac 6000 ggaaaaacgt atcaaaatgt acagaattcg agctctcgag taattctaca cagcccagtc 6060 cagactattc ggcactgaaa ttatgggtga agtggtcaag acctcactag gcaccttaaa 6120 aatagcgcac cctgaagaag atttatttga ggtagccctt gcctacctag cttccaagaa 6180 agatatccta acagcacaag agcggaaaga tgttttgttc tacatccaga acaacctctg 6240 ctaaaattcc tgaaaaattt tgcaaaaagt tgttgacttt atctacaagg tgtggcataa 6300 tgtgtggaat tgtgagcgct cacaattaag cttgaattcc c 6341 <210>143 <211>6221 <212>DNA <213>人造序列 <220> <223>质粒pFP988DssPgroE <400>143 tcgagagcta ttgtaacata atcggtacgg gggtgaaaaa gctaacggaa aagggagcgg 60 aaaagaatga tgtaagcgtg aaaaattttt tatcttatca cttgaaattg gaagggagat 120 tctttattat aagaattgtg gaattgtgag cggataacaa ttcccaatta aaggaggaaa 180 ctagtggatc caagtttaaa ctgtacacta gatatttctt ctccgcttaa atcatcaaag 240 aaatctttat cacttgtaac cagtccgtcc acatgtcgaa ttgcatctga ccgaatttta 300 cgtttccctg aataattctc atcaatcgtt tcatcaattt tatctttata ctttatattt 360 tgtgcgttaa tcaaatcata atttttatat gtttcctcat gatttatgtc tttattatta 420 tagtttttat tctctctttg attatgtctt tgtatcccgt ttgtattact tgatccttta 480 actctggcaa ccctcaaaat tgaatgagac atgctacacc tccggataat aaatatatat 540 aaacgtatat agatttcata aagtctaaca cactagactt atttacttcg taattaagtc 600 gttaaaccgt gtgctctacg accaaaacta taaaaccttt aagaactttc tttttttaca 660 agaaaaaaga aattagataa atctctcata tcttttattc aataatcgca tccgattgca 720 gtataaattt aacgatcact catcatgttc atatttatca gagctcgtgc tataattata 780 ctaattttat aaggaggaaa aaatatgggc atttttagta tttttgtaat cagcacagtt 840 cattatcaac caaacaaaaa ataagtggtt ataatgaatc gttaataagc aaaattcata 900 taaccaaatt aaagagggtt ataatgaacg agaaaaatat aaaacacagt caaaacttta 960 ttacttcaaa acataatata gataaaataa tgacaaatat aagattaaat gaacatgata 1020 atatctttga aatcggctca ggaaaaggcc attttaccct tgaattagta aagaggtgta 1080 atttcgtaac tgccattgaa atagaccata aattatgcaa aactacagaa aataaacttg 1140 ttgatcacga taatttccaa gttttaaaca aggatatatt gcagtttaaa tttcctaaaa 1200 accaatccta taaaatatat ggtaatatac cttataacat aagtacggat ataatacgca 1260 aaattgtttt tgatagtata gctaatgaga tttatttaat cgtggaatac gggtttgcta 1320 aaagattatt aaatacaaaa cgctcattgg cattactttt aatggcagaa gttgatattt 1380 ctatattaag tatggttcca agagaatatt ttcatcctaa acctaaagtg aatagctcac 1440 ttatcagatt aagtagaaaa aaatcaagaa tatcacacaa agataaacaa aagtataatt 1500 atttcgttat gaaatgggtt aacaaagaat acaagaaaat atttacaaaa aatcaattta 1560 acaattcctt aaaacatgca ggaattgacg atttaaacaa tattagcttt gaacaattct 1620 tatctctttt caatagctat aaattattta ataagtaagt taagggatgc agttcatcga 1680 tgaaggcaac tacagctcag gcgacaacca tacgctgaga gatcctcact acgtagaaga 1740 taaaggccac aaatacttag tatttgaagc aaacactgga actgaagatg gctaccaagg 1800 cgaagaatct ttatttaaca aagcatacta tggcaaaagc acatcattct tccgtcaaga 1860 aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct gagttagcaa acggcgctct 1920 cggtatgatt gagctaaacg atgattacac actgaaaaaa gtgatgaaac cgctgattgc 1980 atctaacaca gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg 2040 gtacctgttc actgactccc gcggatcaaa aatgacgatt gacggcatta cgtctaacga 2100 tatttacatg cttggttatg tttctaattc tttaactggc ccatacaagc cgctgaacaa 2160 aactggcctt gtgttaaaaa tggatcttga tcctaacgat gtaaccttta cttactcaca 2220 cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt acaagctata tgacaaacag 2280 aggattctac gcagacaaac aatcaacgtt tgcgccaagc ttgcatgcga gagtagggaa 2340 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct 2400 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg 2460 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc 2520 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actctttttg 2580 tttatttttc taaatacatt caaatatgta tccgctcatg ctccggatct gcatcgcagg 2640 atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc 2700 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg 2760 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt 2820 catcggtatc attaccccca tgaacagaaa ttccccctta cacggaggca tcaagtgacc 2880 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt 2940 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac 3000 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac 3060 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc 3120 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc 3180 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 3240 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 3300 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 3360 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 3420 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 3480 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3540 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 3600 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 3660 tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt 3720 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3780 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3840 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3900 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 3960 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 4020 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 4080 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4140 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 4200 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 4260 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 4320 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 4380 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4440 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 4500 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 4560 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 4620 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 4680 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 4740 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 4800 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 4860 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 4920 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4980 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 5040 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 5100 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 5160 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 5220 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 5280 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa 5340 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 5400 gcagacaagc ccgtcagggc gcgtcagcgg gtgttcatgt gcgtaactaa cttgccatct 5460 tcaaacagga gggctggaag aagcagaccg ctaacacagt acataaaaaa ggagacatga 5520 acgatgaaca tcaaaaagtt tgcaaaacaa gcaacagtat taacctttac taccgcactg 5580 ctggcaggag gcgcaactca agcgtttgcg aaagaaacga accaaaagcc atataaggaa 5640 acatacggca tttcccatat tacacgccat gatatgctgc aaatccctga acagcaaaaa 5700 aatgaaaaat atcaagttcc tgaattcgat tcgtccacaa ttaaaaatat ctcttctgca 5760 aaaggcctgg acgtttggga cagctggcca ttacaaaacg ctgacggcac tgtcgcaaac 5820 tatcacggct accacatcgt ctttgcatta gccggagatc ctaaaaatgc ggatgacaca 5880 tcgatttaca tgttctatca aaaagtcggc gaaacttcta ttgacagctg gaaaaacgct 5940 ggccgcgtct ttaaagacag cgacaaattc gatgcaaatg attctatcct aaaagaccaa 6000 acacaagaat ggtcaggttc agccacattt acatctgacg gaaaaatccg tttattctac 6060 actgatttct ccggtaaaca ttacggcaaa caaacactga caactgcaca agttaacgta 6120 tcagcatcag acagctcttt gaacatcaac ggtgtagagg attataaatc aatctttgac 6180 ggtgacggaa aaacgtatca aaatgtacag aattcgagct c 6221 <210>144 <211>40 <212>DNA <213>人造序列 <220> <223>引物T-budB(BamHI) <400>144 agatagatgg atccggaggt gggtcacatg gacaaacagt 40 <210>145 <211>29 <212>DNA <213>人造序列 <220> <223>引物B-kivD(BamHI) <400>145 ctctagagga tccagactcc taggacatg 29 <210>146 <211>6039 <212>DNA <213>人造序列 <220> <223>载体片段pFP988Dss <400>146 gatccaagtt taaactgtac actagatatt tcttctccgc ttaaatcatc aaagaaatct 60 ttatcacttg taaccagtcc gtccacatgt cgaattgcat ctgaccgaat tttacgtttc 120 cctgaataat tctcatcaat cgtttcatca attttatctt tatactttat attttgtgcg 180 ttaatcaaat cataattttt atatgtttcc tcatgattta tgtctttatt attatagttt 240 ttattctctc tttgattatg tctttgtatc ccgtttgtat tacttgatcc tttaactctg 300 gcaaccctca aaattgaatg agacatgcta cacctccgga taataaatat atataaacgt 360 atatagattt cataaagtct aacacactag acttatttac ttcgtaatta agtcgttaaa 420 ccgtgtgctc tacgaccaaa actataaaac ctttaagaac tttctttttt tacaagaaaa 480 aagaaattag ataaatctct catatctttt attcaataat cgcatccgat tgcagtataa 540 atttaacgat cactcatcat gttcatattt atcagagctc gtgctataat tatactaatt 600 ttataaggag gaaaaaatat gggcattttt agtatttttg taatcagcac agttcattat 660 caaccaaaca aaaaataagt ggttataatg aatcgttaat aagcaaaatt catataacca 720 aattaaagag ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt 780 caaaacataa tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct 840 ttgaaatcgg ctcaggaaaa ggccatttta cccttgaatt agtaaagagg tgtaatttcg 900 taactgccat tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc 960 acgataattt ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat 1020 cctataaaat atatggtaat ataccttata acataagtac ggatataata cgcaaaattg 1080 tttttgatag tatagctaat gagatttatt taatcgtgga atacgggttt gctaaaagat 1140 tattaaatac aaaacgctca ttggcattac ttttaatggc agaagttgat atttctatat 1200 taagtatggt tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca 1260 gattaagtag aaaaaaatca agaatatcac acaaagataa acaaaagtat aattatttcg 1320 ttatgaaatg ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt 1380 ccttaaaaca tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc 1440 ttttcaatag ctataaatta tttaataagt aagttaaggg atgcagttca tcgatgaagg 1500 caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg 1560 ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga 1620 atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca 1680 aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat 1740 gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa 1800 cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct 1860 gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta 1920 catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg 1980 ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc 2040 tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt 2100 ctacgcagac aaacaatcaa cgtttgcgcc aagcttgcat gcgagagtag ggaactgcca 2160 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 2220 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga 2280 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta 2340 agcagaaggc catcctgacg gatggccttt ttgcgtttct acaaactctt tttgtttatt 2400 tttctaaata cattcaaata tgtatccgct catgctccgg atctgcatcg caggatgctg 2460 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt 2520 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag 2580 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg 2640 tatcattacc cccatgaaca gaaattcccc cttacacgga ggcatcaagt gaccaaacag 2700 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag 2760 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac 2820 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 2880 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 2940 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 3000 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 3060 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 3120 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3180 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3240 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3300 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3360 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 3420 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 3480 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3540 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3600 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 3660 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 3720 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 3780 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 3840 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3900 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3960 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4020 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4080 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 4140 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 4200 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 4260 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 4320 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 4380 ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 4440 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 4500 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 4560 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 4620 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 4680 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 4740 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 4800 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 4860 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4920 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 4980 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 5040 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 5100 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 5160 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 5220 aagcccgtca gggcgcgtca gcgggtgttc atgtgcgtaa ctaacttgcc atcttcaaac 5280 aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac atgaacgatg 5340 aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc actgctggca 5400 ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa ggaaacatac 5460 ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca aaaaaatgaa 5520 aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa atatctcttc tgcaaaaggc 5580 ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc aaactatcac 5640 ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga cacatcgatt 5700 tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa cgctggccgc 5760 gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga ccaaacacaa 5820 gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt ctacactgat 5880 ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa cgtatcagca 5940 tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt tgacggtgac 6000 ggaaaaacgt atcaaaatgt acagaattcg agctctcga 6039 <210>147 <211>44 <212>DNA <213>人造序列 <220> <223>引物T-grcE(XhoI) <400>147 agatagatct cgagagctat tgtaacataa tcggtacggg ggtg 44 <210>148 <211>51 <212>DNA <213>人造序列 <220> <223>引物B-groEL(SpeI BamH1) <400>148 attatgtcag gatccactag tttcctcctt taattgggaa ttgttatccg c 51 <210>149 <211>30 <212>DNA <213>人造序列 <220> <223>引物T-groEL <400>149 agctattgta acataatcgg tacgggggtg 30 <210>150 <211>45 <212>DNA <213>人造序列 <220> <223>引物T-ilvCB.s.(BamHI) <400>150 acattgatgg atcccataac aagggagaga ttgaaatggt aaaag 45 <210>151 <211>47 <212>DNA <213>人造序列 <220> <223>引物B-ilvCB.s.(SpeIBamHI) <400>151 tagacaacgg atccactagt ttaattttgc gcaacggaga ccaccgc 47 <210>152 <211>47 <212>DNA <213>人造序列 <220> <223>引物T-BD64(DraIII) <400>152 ttaccgtgga ctcaccgagt gggtaactag cctcgccgga aagagcg 47 <210>153 <211>48 <212>DNA <213>人造序列 <220> <223>引物B-BD64(DraIII) <400>153 tcacagttaa gacacctggt gccgttaatg cgccatgaca gccatgat 48 <210>154 <211>49 <212>DNA <213>人造序列 <220> <223>引物T-lacIq(DraIII) <400>154 acagatagat caccaggtgc aagctaattc cggtggaaac gaggtcatc 49 <210>155 <211>48 <212>DNA <213>人造序列 <220> <223>引物B-lacIq(DraIII) <400>155 acagtacgat acacggggtg tcactgcccg ctttccagtc gggaaacc 48 <210>156 <211>49 <212>DNA <213>人造序列 <220> <223>引物T-groE(DraIII) <400>156 tcggattacg caccccgtga gctattgtaa cataatcggt acgggggtg 49 <210>157 <211>48 <212>DNA <213>人造序列 <220> <223>引物B-B.s.ilvC(DraIII) <400>157 ctgctgatct cacaccgtgt gttaattttg cgcaacggag accaccgc 48 <210>158 <211>1221 <212>DNA <213>丙醇丁酮梭菌 <400>158 cacacggtgt aaataataat ctaaacagga ggggttaaaa tggttgattt cgaatattca 60 ataccaacta gaattttttt cggtaaagat aagataaatg tacttggaag agagcttaaa 120 aaatatggtt ctaaagtgct tatagtttat ggtggaggaa gtataaagag aaatggaata 180 tatgataaag ctgtaagtat acttgaaaaa aacagtatta aattttatga acttgcagga 240 gtagagccaa atccaagagt aactacagtt gaaaaaggag ttaaaatatg tagagaaaat 300 ggagttgaag tagtactagc tataggtgga ggaagtgcaa tagattgcgc aaaggttata 360 gcagcagcat gtgaatatga tggaaatcca tgggatattg tgttagatgg ctcaaaaata 420 aaaagggtgc ttcctatagc tagtatatta accattgctg caacaggatc agaaatggat 480 acgtgggcag taataaataa tatggataca aacgaaaaac taattgcggc acatccagat 540 atggctccta agttttctat attagatcca acgtatacgt ataccgtacc taccaatcaa 600 acagcagcag gaacagctga tattatgagt catatatttg aggtgtattt tagtaataca 660 aaaacagcat atttgcagga tagaatggca gaagcgttat taagaacttg tattaaatat 720 ggaggaatag ctcttgagaa gccggatgat tatgaggcaa gagccaatct aatgtgggct 780 tcaagtcttg cgataaatgg acttttaaca tatggtaaag acactaattg gagtgtacac 840 ttaatggaac atgaattaag tgcttattac gacataacac acggcgtagg gcttgcaatt 900 ttaacaccta attggatgga gtatatttta aataatgata cagtgtacaa gtttgttgaa 960 tatggtgtaa atgtttgggg aatagacaaa gaaaaaaatc actatgacat agcacatcaa 1020 gcaatacaaa aaacaagaga ttactttgta aatgtactag gtttaccatc tagactgaga 1080 gatgttggaa ttgaagaaga aaaattggac ataatggcaa aggaatcagt aaagcttaca 1140 ggaggaacca taggaaacct aagaccagta aacgcctccg aagtcctaca aatattcaaa 1200 aaatctgtgt aacaccgagt g 1221 <210>159 <211>54 <212>DNA <213>人造序列 <220> <223>引物T-bdhB(DraIII) <400>159 tcgatagcat acacacggtg gttaacaaag gaggggttaa aatggttgat ttcg 54 <210>160 <211>91 <212>DNA <213>人造序列 <220> <223>引物B-bdhB(rrnBT1DraIII) <400>160 atctacgcac tcggtgataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat 60 cttacacaga ttttttgaat atttgtagga c 91 <210>161 <211>29 <212>DNA <213>人造序列 <220> <223>引物LDH EcoRV F <400>161 gacgtcatga ccacccgccg atccctttt 29 <210>162 <211>30 <212>DNA <213>人造序列 <220> <223>引物LDH AatIIR <400>162 gatatccaac accagcgacc gacgtattac 30 <210>163 <211>47 <212>DNA <213>人造序列 <220> <223>引物Cm F <400>163 atttaaatct cgagtagagg atcccaacaa acgaaaattg gataaag 47 <210>164 <211>29 <212>DNA <213>人造序列 <220> <223>引物Cm R <400>164 acgcgttatt ataaaagcca gtcattagg 29 <210>165 <211>58 <212>DNA <213>人造序列 <220> <223>引物P11 F-StuI <400>165 cctagcgcta tagttgttga cagaatggac atactatgat atattgttgc tatagcga 58 <210>166 <211>62 <212>DNA <213>人造序列 <220> <223>引物P11 R-SpeI <400>166 ctagtcgcta tagcaacaat atatcatagt atgtccattc tgtcaacaac tatagcgcta 60 gg 62 <210>167 <211>38 <212>DNA <213>人造序列 <220> <223>引物PldhL F-HindIII <400>167 aagcttgtcg acaaaccaac attatgacgt gtctgggc 38 <210>168 <211>28 <212>DNA <213>人造序列 <220> <223>引物PldhL R-BamHI <400>168 ggatcctcat cctctcgtag tgaaaatt 28 <210>169 <211>36 <212>DNA <213>人造序列 <220> <223>引物F-bdhB-AvrII <400>169 ttcctaggaa ggaggtggtt aaaatggttg atttcg 36 <210>170 <211>29 <212>DNA <213>人造序列 <220> <223>引物R-bdhB-BamHI <400>170 ttggatcctt acacagattt tttgaatat 29 <210>171 <211>39 <212>DNA <213>人造序列 <220> <223>引物F-ilvC(B.s.)-AflII <400>171 aacttaagaa ggaggtgatt gaaatggtaa aagtatatt 39 <210>172 <211>32 <212>DNA <213>人造序列 <220> <223>引物R-ilvC(B.s.)-NotI <400>172 aagcggccgc ttaattttgc gcaacggaga cc 32 <210>173 <211>30 <212>DNA <213>人造序列 <220> <223>引物F-PnisA(HindIII) <400>173 ttaagcttga catacttgaa tgacctagtc 30 <210>174 <211>39 <212>DNA <213>人造序列 <220> <223>引物R-PnisA(SpeI BamHI) <400>174 ttggatccaa actagtataa tttattttgt agttccttc 39 <210>175 <211>38 <212>DNA <213>人造序列 <220> <223>引物N191 <400>175 atccgcggat agatctccca ttaccgacat ttgggcgc 38 <210>176 <211>31 <212>DNA <213>人造序列 <220> <223>引物N192 <400>176 atcctcagcg atgattgatt gattgattgt a 31 <210>177 <211>6509 <212>DNA <213>人造序列 <220> <223>载体pFP988 <400>177 tcgaggcccc gcacatacga aaagactggc tgaaaacatt gagcctttga tgactgatga 60 tttggctgaa gaagtggatc gattgtttga gaaaagaaga agaccataaa aataccttgt 120 ctgtcatcag acagggtatt ttttatgctg tccagactgt ccgctgtgta aaaaatagga 180 ataaaggggg gttgttatta ttttactgat atgtaaaata taatttgtat aaggaattgt 240 gagcggataa caattcctac gaaaatgaga gggagaggaa acatgattca aaaacgaaag 300 cggacagttt cgttcagact tgtgcttatg tgcacgctgt tatttgtcag tttgccgatt 360 acaaaaacat cagccggatc ccaccatcac catcaccatt aagaattcct agaaactcca 420 agctatcttt aaaaaatcta gtaaatgcac gagcaacatc ttttgttgct cagtgcattt 480 tttattttgt acactagata tttcttctcc gcttaaatca tcaaagaaat ctttatcact 540 tgtaaccagt ccgtccacat gtcgaattgc atctgaccga attttacgtt tccctgaata 600 attctcatca atcgtttcat caattttatc tttatacttt atattttgtg cgttaatcaa 660 atcataattt ttatatgttt cctcatgatt tatgtcttta ttattatagt ttttattctc 720 tctttgatta tgtctttgta tcccgtttgt attacttgat cctttaactc tggcaaccct 780 caaaattgaa tgagacatgc tacacctccg gataataaat atatataaac gtatatagat 840 ttcataaagt ctaacacact agacttattt acttcgtaat taagtcgtta aaccgtgtgc 900 tctacgacca aaactataaa acctttaaga actttctttt tttacaagaa aaaagaaatt 960 agataaatct ctcatatctt ttattcaata atcgcatccg attgcagtat aaatttaacg 1020 atcactcatc atgttcatat ttatcagagc tcgtgctata attatactaa ttttataagg 1080 aggaaaaaat atgggcattt ttagtatttt tgtaatcagc acagttcatt atcaaccaaa 1140 caaaaaataa gtggttataa tgaatcgtta ataagcaaaa ttcatataac caaattaaag 1200 agggttataa tgaacgagaa aaatataaaa cacagtcaaa actttattac ttcaaaacat 1260 aatatagata aaataatgac aaatataaga ttaaatgaac atgataatat ctttgaaatc 1320 ggctcaggaa aaggccattt tacccttgaa ttagtaaaga ggtgtaattt cgtaactgcc 1380 attgaaatag accataaatt atgcaaaact acagaaaata aacttgttga tcacgataat 1440 ttccaagttt taaacaagga tatattgcag tttaaatttc ctaaaaacca atcctataaa 1500 atatatggta atatacctta taacataagt acggatataa tacgcaaaat tgtttttgat 1560 agtatagcta atgagattta tttaatcgtg gaatacgggt ttgctaaaag attattaaat 1620 acaaaacgct cattggcatt acttttaatg gcagaagttg atatttctat attaagtatg 1680 gttccaagag aatattttca tcctaaacct aaagtgaata gctcacttat cagattaagt 1740 agaaaaaaat caagaatatc acacaaagat aaacaaaagt ataattattt cgttatgaaa 1800 tgggttaaca aagaatacaa gaaaatattt acaaaaaatc aatttaacaa ttccttaaaa 1860 catgcaggaa ttgacgattt aaacaatatt agctttgaac aattcttatc tcttttcaat 1920 agctataaat tatttaataa gtaagttaag ggatgcagtt catcgatgaa ggcaactaca 1980 gctcaggcga caaccatacg ctgagagatc ctcactacgt agaagataaa ggccacaaat 2040 acttagtatt tgaagcaaac actggaactg aagatggcta ccaaggcgaa gaatctttat 2100 ttaacaaagc atactatggc aaaagcacat cattcttccg tcaagaaagt caaaaacttc 2160 tgcaaagcga taaaaaacgc acggctgagt tagcaaacgg cgctctcggt atgattgagc 2220 taaacgatga ttacacactg aaaaaagtga tgaaaccgct gattgcatct aacacagtaa 2280 cagatgaaat tgaacgcgcg aacgtcttta aaatgaacgg caaatggtac ctgttcactg 2340 actcccgcgg atcaaaaatg acgattgacg gcattacgtc taacgatatt tacatgcttg 2400 gttatgtttc taattcttta actggcccat acaagccgct gaacaaaact ggccttgtgt 2460 taaaaatgga tcttgatcct aacgatgtaa cctttactta ctcacacttc gctgtacctc 2520 aagcgaaagg aaacaatgtc gtgattacaa gctatatgac aaacagagga ttctacgcag 2580 acaaacaatc aacgtttgcg ccaagcttgc atgcgagagt agggaactgc caggcatcaa 2640 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg 2700 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg 2760 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag 2820 gccatcctga cggatggcct ttttgcgttt ctacaaactc tttttgttta tttttctaaa 2880 tacattcaaa tatgtatccg ctcatgctcc ggatctgcat cgcaggatgc tgctggctac 2940 cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc 3000 tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg 3060 catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta 3120 cccccatgaa cagaaattcc cccttacacg gaggcatcaa gtgaccaaac aggaaaaaac 3180 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa 3240 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga 3300 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 3360 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 3420 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga 3480 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac 3540 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct 3600 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3660 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3720 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3780 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3840 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3900 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3960 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4020 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4080 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4140 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4200 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 4260 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4320 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4380 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4440 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4500 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4560 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4620 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4680 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4740 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4800 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc 4860 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4920 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4980 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 5040 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 5100 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 5160 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 5220 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 5280 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 5340 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 5400 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 5460 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5520 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5580 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 5640 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 5700 cagggcgcgt cagcgggtgt tcatgtgcgt aactaacttg ccatcttcaa acaggagggc 5760 tggaagaagc agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa 5820 aaagtttgca aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc 5880 aactcaagcg tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc 5940 ccatattaca cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca 6000 agttcctgaa ttcgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt 6060 ttgggacagc tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca 6120 catcgtcttt gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt 6180 ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa 6240 agacagcgac aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc 6300 aggttcagcc acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg 6360 taaacattac ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag 6420 ctctttgaac atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac 6480 gtatcaaaat gtacagcatg ccacgcgtc 6509 <210>178 <211>571 <212>PRT <213>枯草芽孢杆菌 <400>178 Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg 1 5 10 15 Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His 20 25 30 Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu 35 40 45 Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala 50 55 60 Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val 65 70 75 80 Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu 85 90 95 Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn 100 105 110 Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn 115 120 125 Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp 130 135 140 Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser 145 150 155 160 Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val 165 170 175 Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys 180 185 190 Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile 195 200 205 Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg 210 215 220 Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu 225 230 235 240 Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu 245 250 255 Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly 260 265 270 Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp 275 280 285 Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr 290 295 300 Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln 305 310 315 320 Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile 325 330 335 Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile 340 345 350 Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala 355 360 365 Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu 370 375 380 Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser 385 390 395 400 His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr 405 410 415 Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp 420 425 430 Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val 435 440 445 Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala 450 455 460 Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr 465 470 475 480 Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser 485 490 495 Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe 500 505 510 Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val 515 520 525 Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro 530 535 540 Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys 545 550 555 560 Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu 565 570 <210>179 <211>1665 <212>DNA <213>乳酸乳球菌 <400>179 atgtctgaga aacaatttgg ggcgaacttg gttgtcgata gtttgattaa ccataaagtg 60 aagtatgtat ttgggattcc aggagcaaaa attgaccggg tttttgattt attagaaaat 120 gaagaaggcc ctcaaatggt cgtgactcgt catgagcaag gagctgcttt catggctcaa 180 gctgtcggtc gtttaactgg cgaacctggt gtagtagttg ttacgagtgg gcctggtgta 240 tcaaaccttg cgactccgct tttgaccgcg acatcagaag gtgatgctat tttggctatc 300 ggtggacaag ttaaacgaag tgaccgtctt aaacgtgcgc accaatcaat ggataatgct 360 ggaatgatgc aatcagcaac aaaatattca gcagaagttc ttgaccctaa tacactttct 420 gaatcaattg ccaacgctta tcgtattgca aaatcaggac atccaggtgc aactttctta 480 tcaatccccc aagatgtaac ggatgccgaa gtatcaatca aagccattca accactttca 540 gaccctaaaa tggggaatgc ctctattgat gacattaatt atttagcaca agcaattaaa 600 aatgctgtat tgccagtaat tttggttgga gctggtgctt cagatgctaa agtcgcttca 660 tccttgcgta atctattgac tcatgttaat attcctgtcg ttgaaacatt ccaaggtgca 720 ggggttattt cacatgattt agaacatact ttttatggac gtatcggtct tttccgcaat 780 caaccaggcg atatgcttct gaaacgttct gaccttgtta ttgctgttgg ttatgaccca 840 attgaatatg aagctcgtaa ctggaatgca gaaattgata gtcgaattat cgttattgat 900 aatgccattg ctgaaattga tacttactac caaccagagc gtgaattaat tggtgatatc 960 gcagcaacat tggataatct tttaccagct gttcgtggct acaaaattcc aaaaggaaca 1020 aaagattatc tcgatggcct tcatgaagtt gctgagcaac acgaatttga tactgaaaat 1080 actgaagaag gtagaatgca ccctcttgat ttggtcagca ctttccaaga aatcgtcaag 1140 gatgatgaaa cagtaaccgt tgacgtaggt tcactctaca tttggatggc acgtcatttc 1200 aaatcatacg aaccacgtca tctcctcttc tcaaacggaa tgcaaacact cggagttgca 1260 cttccttggg caattacagc cgcattgttg cgcccaggta aaaaagttta ttcacactct 1320 ggtgatggag gcttcctttt cacagggcaa gaattggaaa cagctgtacg tttgaatctt 1380 ccaatcgttc aaattatctg gaatgacggc cattatgata tggttaaatt ccaagaagaa 1440 atgaaatatg gtcgttcagc agccgttgat tttggctatg ttgattacgt aaaatatgct 1500 gaagcaatga gagcaaaagg ttaccgtgca cacagcaaag aagaacttgc tgaaattctc 1560 aaatcaatcc cagatactac tggaccggtg gtaattgacg ttcctttgga ctattctgat 1620 aacattaaat tagcagaaaa attattgcct gaagagtttt attga 1665 <210>180 <211>554 <212>PRT <213>乳酸乳球菌 <400>180 Met Ser Glu Lys Gln Phe Gly Ala Asn Leu Val Val Asp Ser Leu Ile 1 5 10 15 Asn His Lys Val Lys Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile Asp 20 25 30 Arg Val Phe Asp Leu Leu Glu Asn Glu Glu Gly Pro Gln Met Val Val 35 40 45 Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala Gln Ala Val Gly Arg 50 55 60 Leu Thr Gly Glu Pro Gly Val Val Val Val Thr Ser Gly Pro Gly Val 65 70 75 80 Ser Asn Leu Ala Thr Pro Leu Leu Thr Ala Thr Ser Glu Gly Asp Ala 85 90 95 Ile Leu Ala Ile Gly Gly Gln Val Lys Arg Ser Asp Arg Leu Lys Arg 100 105 110 Ala His Gln Ser Met Asp Asn Ala Gly Met Met Gln Ser Ala Thr Lys 115 120 125 Tyr Ser Ala Glu Val Leu Asp Pro Asn Thr Leu Ser Glu Ser Ile Ala 130 135 140 Asn Ala Tyr Arg Ile Ala Lys Ser Gly His Pro Gly Ala Thr Phe Leu 145 150 155 160 Ser Ile Pro Gln Asp Val Thr Asp Ala Glu Val Ser Ile Lys Ala Ile 165 170 175 Gln Pro Leu Ser Asp Pro Lys Met Gly Asn Ala Ser Ile Asp Asp Ile 180 185 190 Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val Ile Leu 195 200 205 Val Gly Ala Gly Ala Ser Asp Ala Lys Val Ala Ser Ser Leu Arg Asn 210 215 220 Leu Leu Thr His Val Asn Ile Pro Val Val Glu Thr Phe Gln Gly Ala 225 230 235 240 Gly Val Ile Ser His Asp Leu Glu His Thr Phe Tyr Gly Arg Ile Gly 245 250 255 Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg Ser Asp Leu 260 265 270 Val Ile Ala Val Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Arg Asn Trp 275 280 285 Asn Ala Glu Ile Asp Ser Arg Ile Ile Val Ile Asp Asn Ala Ile Ala 290 295 300 Glu Ile Asp Thr Tyr Tyr Gln Pro Glu Arg Glu Leu Ile Gly Asp Ile 305 310 315 320 Ala Ala Thr Leu Asp Asn Leu Leu Pro Ala Val Arg Gly Tyr Lys Ile 325 330 335 Pro Lys Gly Thr Lys Asp Tyr Leu Asp Gly Leu His Glu Val Ala Glu 340 345 350 Gln His Glu Phe Asp Thr Glu Asn Thr Glu Glu Gly Arg Met His Pro 355 360 365 Leu Asp Leu Val Ser Thr Phe Gln Glu Ile Val Lys Asp Asp Glu Thr 370 375 380 Val Thr Val Asp Val Gly Ser Leu Tyr Ile Trp Met Ala Arg His Phe 385 390 395 400 Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr 405 410 415 Leu Gly Val Ala Leu Pro Trp Ala Ile Thr Ala Ala Leu Leu Arg Pro 420 425 430 Gly Lys Lys Val Tyr Ser His Ser Gly Asp Gly Gly Phe Leu Phe Thr 435 440 445 Gly Gln Glu Leu Glu Thr Ala Val Arg Leu Asn Leu Pro Ile Val Gln 450 455 460 Ile Ile Trp Asn Asp Gly His Tyr Asp Met Val Lys Phe Gln Glu Glu 465 470 475 480 Met Lys Tyr Gly Arg Ser Ala Ala Val Asp Phe Gly Tyr Val Asp Tyr 485 490 495 Val Lys Tyr Ala Glu Ala Met Arg Ala Lys Gly Tyr Arg Ala His Ser 500 505 510 Lys Glu Glu Leu Ala Glu Ile Leu Lys Ser Ile Pro Asp Thr Thr Gly 515 520 525 Pro Val Val Ile Asp Val Pro Leu Asp Tyr Ser Asp Asn Ile Lys Leu 530 535 540 Ala Glu Lys Leu Leu Pro Glu Glu Phe Tyr 545 550 <210>181 <211>395 <212>PRT <213>酿酒酵母 <400>181 Met Leu Arg Thr Gln Ala Ala Arg Leu Ile Cys Asn Ser Arg Val Ile 1 5 10 15 Thr Ala Lys Arg Thr Phe Ala Leu Ala Thr Arg Ala Ala Ala Tyr Ser 20 25 30 Arg Pro Ala Ala Arg Phe Val Lys Pro Met Ile Thr Thr Arg Gly Leu 35 40 45 Lys Gln Ile Asn Phe Gly Gly Thr Val Glu Thr Val Tyr Glu Arg Ala 50 55 60 Asp Trp Pro Arg Glu Lys Leu Leu Asp Tyr Phe Lys Asn Asp Thr Phe 65 70 75 80 Ala Leu Ile Gly Tyr Gly Ser Gln Gly Tyr Gly Gln Gly Leu Asn Leu 85 90 95 Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val Arg Lys Asp Gly Ala 100 105 110 Ser Trp Lys Ala Ala Ile Glu Asp Gly Trp Val Pro Gly Lys Asn Leu 115 120 125 Phe Thr Val Glu Asp Ala Ile Lys Arg Gly Ser Tyr Val Met Asn Leu 130 135 140 Leu Ser Asp Ala Ala Gln Ser Glu Thr Trp Pro Ala Ile Lys Pro Leu 145 150 155 160 Leu Thr Lys Gly Lys Thr Leu Tyr Phe Ser His Gly Phe Ser Pro Val 165 170 175 Phe Lys Asp Leu Thr His Val Glu Pro Pro Lys Asp Leu Asp Val Ile 180 185 190 Leu Val Ala Pro Lys Gly Ser Gly Arg Thr Val Arg Ser Leu Phe Lys 195 200 205 Glu Gly Arg Gly Ile Asn Ser Ser Tyr Ala Val Trp Asn Asp Val Thr 210 215 220 Gly Lys Ala His Glu Lys Ala Gln Ala Leu Ala Val Ala Ile Gly Ser 225 230 235 240 Gly Tyr Val Tyr Gln Thr Thr Phe Glu Arg Glu Val Asn Ser Asp Leu 245 250 255 Tyr Gly Glu Arg Gly Cys Leu Met Gly Gly Ile His Gly Met Phe Leu 260 265 270 Ala Gln Tyr Asp Val Leu Arg Glu Asn Gly His Ser Pro Ser Glu Ala 275 280 285 Phe Asn Glu Thr Val Glu Glu Ala Thr Gln Ser Leu Tyr Pro Leu Ile 290 295 300 Gly Lys Tyr Gly Met Asp Tyr Met Tyr Asp Ala Cys Ser Thr Thr Ala 305 310 315 320 Arg Arg Gly Ala Leu Asp Trp Tyr Pro Ile Phe Lys Asn Ala Leu Lys 325 330 335 Pro Val Phe Gln Asp Leu Tyr Glu Ser Thr Lys Asn Gly Thr Glu Thr 340 345 350 Lys Arg Ser Leu Glu Phe Asn Ser Gln Pro Asp Tyr Arg Glu Lys Leu 355 360 365 Glu Lys Glu Leu Asp Thr Ile Arg Asn Met Glu Ile Trp Lys Val Gly 370 375 380 Lys Glu Val Arg Lys Leu Arg Pro Glu Asn Gln 385 390 395 <210>182 <211>993 <212>DNA <213>海藻甲烷球菌 <400>182 atgaaggtat tctatgactc agattttaaa ttagatgctt taaaagaaaa aacaattgca 60 gtaatcggtt atggaagtca aggtagggca cagtccttaa acatgaaaga cagcggatta 120 aacgttgttg ttggtttaag aaaaaacggt gcttcatgga acaacgctaa agcagacggt 180 cacaatgtaa tgaccattga agaagctgct gaaaaagcgg acatcatcca catcttaata 240 cctgatgaat tacaggcaga agtttatgaa agccagataa aaccatacct aaaagaagga 300 aaaacactaa gcttttcaca tggttttaac atccactatg gattcattgt tccaccaaaa 360 ggagttaacg tggttttagt tgctccaaaa tcacctggaa aaatggttag aagaacatac 420 gaagaaggtt tcggtgttcc aggtttaatc tgtattgaaa ttgatgcaac aaacaacgca 480 tttgatattg tttcagcaat ggcaaaagga atcggtttat caagagctgg agttatccag 540 acaactttca aagaagaaac agaaactgac cttttcggtg aacaagctgt tttatgcggt 600 ggagttaccg aattaatcaa ggcaggattt gaaacactcg ttgaagcagg atacgcacca 660 gaaatggcat actttgaaac ctgccacgaa ttgaaattaa tcgttgactt aatctaccaa 720 aaaggattca aaaacatgtg gaacgatgta agtaacactg cagaatacgg cggacttaca 780 agaagaagca gaatcgttac agctgattca aaagctgcaa tgaaagaaat cttaagagaa 840 atccaagatg gaagattcac aaaagaattc cttctcgaaa aacaggtaag ctatgctcat 900 ttaaaatcaa tgagaagact cgaaggagac ttacaaatcg aagaagtcgg cgcaaaatta 960 agaaaaatgt gcggtcttga aaaagaagaa taa 993 <210>183 <211>330 <212>PRT <213>海藻甲烷球菌 <400>183 Met Lys Val Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25 30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys 35 40 45 Asn Gly Ala Ser Trp Asn Asn Ala Lys Ala Asp Gly His Asn Val Met 50 55 60 Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile Ile His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Glu Ser Gln Ile Lys Pro Tyr 85 90 95 Leu Lys Glu Gly Lys Thr Leu Ser Phe Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Val Pro Pro Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser Pro Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150 155 160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala 165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu Leu Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270 Ala Met Lys Glu Ile Leu Arg Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280 285 Glu Phe Leu Leu Glu Lys Gln Val Ser Tyr Ala His Leu Lys Ser Met 290 295 300 Arg Arg Leu Glu Gly Asp Leu Gln Ile Glu Glu Val Gly Ala Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu 325 330 <210>184 <211>1476 <212>DNA <213>枯草芽孢杆菌 <400>184 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggttaa 1476 <210>185 <211>342 <212>PRT <213>枯草芽孢杆菌 <400>185 Met Val Lys Val Tyr Tyr Asn Gly Asp Ile Lys Glu Asn Val Leu Ala 1 5 10 15 Gly Lys Thr Val Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala His 20 25 30 Ala Leu Asn Leu Lys Glu Ser Gly Val Asp Val Ile Val Gly Val Arg 35 40 45 Gln Gly Lys Ser Phe Thr Gln Ala Gln Glu Asp Gly His Lys Val Phe 50 55 60 Ser Val Lys Glu Ala Ala Ala Gln Ala Glu Ile Ile Met Val Leu Leu 65 70 75 80 Pro Asp Glu Gln Gln Gln Lys Val Tyr Glu Ala Glu Ile Lys Asp Glu 85 90 95 Leu Thr Ala Gly Lys Ser Leu Val Phe Ala His Gly Phe Asn Val His 100 105 110 Phe His Gln Ile Val Pro Pro Ala Asp Val Asp Val Phe Leu Val Ala 115 120 125 Pro Lys Gly Pro Gly His Leu Val Arg Arg Thr Tyr Glu Gln Gly Ala 130 135 140 Gly Val Pro Ala Leu Phe Ala Ile Tyr Gln Asp Val Thr Gly Glu Ala 145 150 155 160 Arg Asp Lys Ala Leu Ala Tyr Ala Lys Gly Ile Gly Gly Ala Arg Ala 165 170 175 Gly Val Leu Glu Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Ser Ala Leu Val Lys Ala 195 200 205 Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Gln Pro Glu Leu Ala Tyr 210 215 220 Phe Glu Cys Leu His Glu Leu Lys Leu Ile Val Asp Leu Met Tyr Glu 225 230 235 240 Glu Gly Leu Ala Gly Met Arg Tyr Ser Ile Ser Asp Thr Ala Gln Trp 245 250 255 Gly Asp Phe Val Ser Gly Pro Arg Val Val Asp Ala Lys Val Lys Glu 260 265 270 Ser Met Lys Glu Val Leu Lys Asp Ile Gln Asn Gly Thr Phe Ala Lys 275 280 285 Glu Trp Ile Val Glu Asn Gln Val Asn Arg Pro Arg Phe Asn Ala Ile 290 295 300 Asn Ala Ser Glu Asn Glu His Gln Ile Glu Val Val Gly Arg Lys Leu 305 310 315 320 Arg Glu Met Met Pro Phe Val Lys Gln Gly Lys Lys Lys Glu Ala Val 325 330 335 Val Ser Val Ala Gln Asn 340 <210>186 <211>585 <212>PRT <213>酿酒酵母 <400>186 Met Gly Leu Leu Thr Lys Val Ala Thr Ser Arg Gln Phe Ser Thr Thr 1 5 10 15 Arg Cys Val Ala Lys Lys Leu Asn Lys Tyr Ser Tyr Ile Ile Thr Glu 20 25 30 Pro Lys Gly Gln Gly Ala Ser Gln Ala Met Leu Tyr Ala Thr Gly Phe 35 40 45 Lys Lys Glu Asp Phe Lys Lys Pro Gln Val Gly Val Gly Ser Cys Trp 50 55 60 Trp Ser Gly Asn Pro Cys Asn Met His Leu Leu Asp Leu Asn Asn Arg 65 70 75 80 Cys Ser Gln Ser Ile Glu Lys Ala Gly Leu Lys Ala Met Gln Phe Asn 85 90 95 Thr Ile Gly Val Ser Asp Gly Ile Ser Met Gly Thr Lys Gly Met Arg 100 105 110 Tyr Ser Leu Gln Ser Arg Glu Ile Ile Ala Asp Ser Phe Glu Thr Ile 115 120 125 Met Met Ala Gln His Tyr Asp Ala Asn Ile Ala Ile Pro Ser Cys Asp 130 135 140 Lys Asn Met Pro Gly Val Met Met Ala Met Gly Arg His Asn Arg Pro 145 150 155 160 Ser Ile Met Val Tyr Gly Gly Thr Ile Leu Pro Gly His Pro Thr Cys 165 170 175 Gly Ser Ser Lys Ile Ser Lys Asn Ile Asp Ile Val Ser Ala Phe Gln 180 185 190 Ser Tyr Gly Glu Tyr Ile Ser Lys Gln Phe Thr Glu Glu Glu Arg Glu 195 200 205 Asp Val Val Glu His Ala Cys Pro Gly Pro Gly Ser Cys Gly Gly Met 210 215 220 Tyr Thr Ala Asn Thr Met Ala Ser Ala Ala Glu Val Leu Gly Leu Thr 225 230 235 240 Ile Pro Asn Ser Ser Ser Phe Pro Ala Val Ser Lys Glu Lys Leu Ala 245 250 255 Glu Cys Asp Asn Ile Gly Glu Tyr Ile Lys Lys Thr Met Glu Leu Gly 260 265 270 Ile Leu Pro Arg Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn Ala Ile 275 280 285 Thr Tyr Val Val Ala Thr Gly Gly Ser Thr Asn Ala Val Leu His Leu 290 295 300 Val Ala Val Ala His Ser Ala Gly Val Lys Leu Ser Pro Asp Asp Phe 305 310 315 320 Gln Arg Ile Ser Asp Thr Thr Pro Leu Ile Gly Asp Phe Lys Pro Ser 325 330 335 Gly Lys Tyr Val Met Ala Asp Leu Ile Asn Val Gly Gly Thr Gln Ser 340 345 350 Val Ile Lys Tyr Leu Tyr Glu Asn Asn Met Leu His Gly Asn Thr Met 355 360 365 Thr Val Thr Gly Asp Thr Leu Ala Glu Arg Ala Lys Lys Ala Pro Ser 370 375 380 Leu Pro Glu Gly Gln Glu Ile Ile Lys Pro Leu Ser His Pro Ile Lys 385 390 395 400 Ala Asn Gly His Leu Gln Ile Leu Tyr Gly Ser Leu Ala Pro Gly Gly 405 410 415 Ala Val Gly Lys Ile Thr Gly Lys Glu Gly Thr Tyr Phe Lys Gly Arg 420 425 430 Ala Arg Val Phe Glu Glu Glu Gly Ala Phe Ile Glu Ala Leu Glu Arg 435 440 445 Gly Glu Ile Lys Lys Gly Glu Lys Thr Val Val Val Ile Arg Tyr Glu 450 455 460 Gly Pro Arg Gly Ala Pro Gly Met Pro Glu Met Leu Lys Pro Ser Ser 465 470 475 480 Ala Leu Met Gly Tyr Gly Leu Gly Lys Asp Val Ala Leu Leu Thr Asp 485 490 495 Gly Arg Phe Ser Gly Gly Ser His Gly Phe Leu Ile Gly His Ile Val 500 505 510 Pro Glu Ala Ala Glu Gly Gly Pro Ile Gly Leu Val Arg Asp Gly Asp 515 520 525 Glu Ile Ile Ile Asp Ala Asp Asn Asn Lys Ile Asp Leu Leu Val Ser 530 535 540 Asp Lys Glu Met Ala Gln Arg Lys Gln Ser Trp Val Ala Pro Pro Pro 545 550 555 560 Arg Tyr Thr Arg Gly Thr Leu Ser Lys Tyr Ala Lys Leu Val Ser Asn 565 570 575 Ala Ser Asn Gly Cys Val Leu Asp Ala 580 585 <210>187 <211>1653 <212>DNA <213>海藻甲烷球菌 <400>187 atgataagtg ataacgtcaa aaagggagtt ataagaactc caaaccgagc tcttttaaag 60 gcttgcggat atacagacga agacatggaa aaaccattta ttggaattgt aaacagcttt 120 acagaagttg ttcccggcca cattcactta agaacattat cagaagcggc taaacatggt 180 gtttatgcaa acggtggaac accatttgaa tttaatacca ttggaatttg cgacggtatt 240 gcaatgggcc acgaaggtat gaaatactct ttaccttcaa gagaaattat tgcagacgct 300 gttgaatcaa tggcaagagc acatggattt gatggtcttg ttttaattcc tacgtgtgat 360 aaaatcgttc ctggaatgat aatgggtgct ttaagactaa acattccatt tattgtagtt 420 actggaggac caatgcttcc cggagaattc caaggtaaaa aatacgaact tatcagcctt 480 tttgaaggtg tcggagaata ccaagttgga aaaattactg aagaagagtt aaagtgcatt 540 gaagactgtg catgttcagg tgctggaagt tgtgcagggc tttacactgc aaacagtatg 600 gcctgcctta cagaagcttt gggactctct cttccaatgt gtgcaacaac gcatgcagtt 660 gatgcccaaa aagttaggct tgctaaaaaa agtggctcaa aaattgttga tatggtaaaa 720 gaagacctaa aaccaacaga catattaaca aaagaagctt ttgaaaatgc tattttagtt 780 gaccttgcac ttggtggatc aacaaacaca acattacaca ttcctgcaat tgcaaatgaa 840 attgaaaata aattcataac tctcgatgac tttgacaggt taagcgatga agttccacac 900 attgcatcaa tcaaaccagg tggagaacac tacatgattg atttacacaa tgctggaggt 960 attcctgcgg tattgaacgt tttaaaagaa aaaattagag atacaaaaac agttgatgga 1020 agaagcattt tggaaatcgc agaatctgtt aaatacataa attacgacgt tataagaaaa 1080 gtggaagctc cggttcacga aactgctggt ttaagggttt taaagggaaa tcttgctcca 1140 aacggttgcg ttgtaaaaat cggtgcagta catccgaaaa tgtacaaaca cgatggacct 1200 gcaaaagttt acaattccga agatgaagca atttctgcga tacttggcgg aaaaattgta 1260 gaaggggacg ttatagtaat cagatacgaa ggaccatcag gaggccctgg aatgagagaa 1320 atgctctccc caacttcagc aatctgtgga atgggtcttg atgacagcgt tgcattgatt 1380 actgatggaa gattcagtgg tggaagtagg ggcccatgta tcggacacgt ttctccagaa 1440 gctgcagctg gcggagtaat tgctgcaatt gaaaacgggg atatcatcaa aatcgacatg 1500 attgaaaaag aaataaatgt tgatttagat gaatcagtca ttaaagaaag actctcaaaa 1560 ctgggagaat ttgagcctaa aatcaaaaaa ggctatttat caagatactc aaaacttgtc 1620 tcatctgctg acgaaggggc agttttaaaa taa 1653 <210>188 <211>550 <212>PRT <213>海藻甲烷球菌 <400>188 Met Ile Ser Asp Asn Val Lys Lys Gly Val Ile Arg Thr Pro Asn Arg 1 5 10 15 Ala Leu Leu Lys Ala Cys Gly Tyr Thr Asp Glu Asp Met Glu Lys Pro 20 25 30 Phe Ile Gly Ile Val Asn Ser Phe Thr Glu Val Val Pro Gly His Ile 35 40 45 His Leu Arg Thr Leu Ser Glu Ala Ala Lys His Gly Val Tyr Ala Asn 50 55 60 Gly Gly Thr Pro Phe Glu Phe Asn Thr Ile Gly Ile Cys Asp Gly Ile 65 70 75 80 Ala Met Gly His Glu Gly Met Lys Tyr Ser Leu Pro Ser Arg Glu Ile 85 90 95 Ile Ala Asp Ala Val Glu Ser Met Ala Arg Ala His Gly Phe Asp Gly 100 105 110 Leu Val Leu Ile Pro Thr Cys Asp Lys Ile Val Pro Gly Met Ile Met 115 120 125 Gly Ala Leu Arg Leu Asn Ile Pro Phe Ile Val Val Thr Gly Gly Pro 130 135 140 Met Leu Pro Gly Glu Phe Gln Gly Lys Lys Tyr Glu Leu Ile Ser Leu 145 150 155 160 Phe Glu Gly Val Gly Glu Tyr Gln Val Gly Lys Ile Thr Glu Glu Glu 165 170 175 Leu Lys Cys Ile Glu Asp Cys Ala Cys Ser Gly Ala Gly Ser Cys Ala 180 185 190 Gly Leu Tyr Thr Ala Asn Ser Met Ala Cys Leu Thr Glu Ala Leu Gly 195 200 205 Leu Ser Leu Pro Met Cys Ala Thr Thr His Ala Val Asp Ala Gln Lys 210 215 220 Val Arg Leu Ala Lys Lys Ser Gly Ser Lys Ile Val Asp Met Val Lys 225 230 235 240 Glu Asp Leu Lys Pro Thr Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn 245 250 255 Ala Ile Leu Val Asp Leu Ala Leu Gly Gly Ser Thr Asn Thr Thr Leu 260 265 270 His Ile Pro Ala Ile Ala Asn Glu Ile Glu Asn Lys Phe Ile Thr Leu 275 280 285 Asp Asp Phe Asp Arg Leu Ser Asp Glu Val Pro His Ile Ala Ser Ile 290 295 300 Lys Pro Gly Gly Glu His Tyr Met Ile Asp Leu His Asn Ala Gly Gly 305 310 315 320 Ile Pro Ala Val Leu Asn Val Leu Lys Glu Lys Ile Arg Asp Thr Lys 325 330 335 Thr Val Asp Gly Arg Ser Ile Leu Glu Ile Ala Glu Ser Val Lys Tyr 340 345 350 Ile Asn Tyr Asp Val Ile Arg Lys Val Glu Ala Pro Val His Glu Thr 355 360 365 Ala Gly Leu Arg Val Leu Lys Gly Asn Leu Ala Pro Asn Gly Cys Val 370 375 380 Val Lys Ile Gly Ala Val His Pro Lys Met Tyr Lys His Asp Gly Pro 385 390 395 400 Ala Lys Val Tyr Asn Ser Glu Asp Glu Ala Ile Ser Ala Ile Leu Gly 405 410 415 Gly Lys Ile Val Glu Gly Asp Val Ile Val Ile Arg Tyr Glu Gly Pro 420 425 430 Ser Gly Gly Pro Gly Met Arg Glu Met Leu Ser Pro Thr Ser Ala Ile 435 440 445 Cys Gly Met Gly Leu Asp Asp Ser Val Ala Leu Ile Thr Asp Gly Arg 450 455 460 Phe Ser Gly Gly Ser Arg Gly Pro Cys Ile Gly His Val Ser Pro Glu 465 470 475 480 Ala Ala Ala Gly Gly Val Ile Ala Ala Ile Glu Asn Gly Asp Ile Ile 485 490 495 Lys Ile Asp Met Ile Glu Lys Glu Ile Asn Val Asp Leu Asp Glu Ser 500 505 510 Val Ile Lys Glu Arg Leu Ser Lys Leu Gly Glu Phe Glu Pro Lys Ile 515 520 525 Lys Lys Gly Tyr Leu Ser Arg Tyr Ser Lys Leu Val Ser Ser Ala Asp 530 535 540 Glu Gly Ala Val Leu Lys 545 550 <210>189 <211>1677 <212>DNA <213>枯草芽孢杆菌 <400>189 atggcagaat tacgcagtaa tatgatcaca caaggaatcg atagagctcc gcaccgcagt 60 ttgcttcgtg cagcaggggt aaaagaagag gatttcggca agccgtttat tgcggtgtgt 120 aattcataca ttgatatcgt tcccggtcat gttcacttgc aggagtttgg gaaaatcgta 180 aaagaagcaa tcagagaagc agggggcgtt ccgtttgaat ttaataccat tggggtagat 240 gatggcatcg caatggggca tatcggtatg agatattcgc tgccaagccg tgaaattatc 300 gcagactctg tggaaacggt tgtatccgca cactggtttg acggaatggt ctgtattccg 360 aactgcgaca aaatcacacc gggaatgctt atggcggcaa tgcgcatcaa cattccgacg 420 atttttgtca gcggcggacc gatggcggca ggaagaacaa gttacgggcg aaaaatctcc 480 ctttcctcag tattcgaagg ggtaggcgcc taccaagcag ggaaaatcaa cgaaaacgag 540 cttcaagaac tagagcagtt cggatgccca acgtgcgggt cttgctcagg catgtttacg 600 gcgaactcaa tgaactgtct gtcagaagca cttggtcttg ctttgccggg taatggaacc 660 attctggcaa catctccgga acgcaaagag tttgtgagaa aatcggctgc gcaattaatg 720 gaaacgattc gcaaagatat caaaccgcgt gatattgtta cagtaaaagc gattgataac 780 gcgtttgcac tcgatatggc gctcggaggt tctacaaata ccgttcttca tacccttgcc 840 cttgcaaacg aagccggcgt tgaatactct ttagaacgca ttaacgaagt cgctgagcgc 900 gtgccgcact tggctaagct ggcgcctgca tcggatgtgt ttattgaaga tcttcacgaa 960 gcgggcggcg tttcagcggc tctgaatgag ctttcgaaga aagaaggagc gcttcattta 1020 gatgcgctga ctgttacagg aaaaactctt ggagaaacca ttgccggaca tgaagtaaag 1080 gattatgacg tcattcaccc gctggatcaa ccattcactg aaaagggagg ccttgctgtt 1140 ttattcggta atctagctcc ggacggcgct atcattaaaa caggcggcgt acagaatggg 1200 attacaagac acgaagggcc ggctgtcgta ttcgattctc aggacgaggc gcttgacggc 1260 attatcaacc gaaaagtaaa agaaggcgac gttgtcatca tcagatacga agggccaaaa 1320 ggcggacctg gcatgccgga aatgctggcg ccaacatccc aaatcgttgg aatgggactc 1380 gggccaaaag tggcattgat tacggacgga cgtttttccg gagcctcccg tggcctctca 1440 atcggccacg tatcacctga ggccgctgag ggcgggccgc ttgcctttgt tgaaaacgga 1500 gaccatatta tcgttgatat tgaaaaacgc atcttggatg tacaagtgcc agaagaagag 1560 tgggaaaaac gaaaagcgaa ctggaaaggt tttgaaccga aagtgaaaac cggctacctg 1620 gcacgttatt ctaaacttgt gacaagtgcc aacaccggcg gtattatgaa aatctag 1677 <210>190 <211>558 <212>PRT <213>枯草芽孢杆菌 <400>190 Met Ala Glu Leu Arg Ser Asn Met Ile Thr Gln Gly Ile Asp Arg Ala 1 5 10 15 Pro His Arg Ser Leu Leu Arg Ala Ala Gly Val Lys Glu Glu Asp Phe 20 25 30 Gly Lys Pro Phe Ile Ala Val Cys Asn Ser Tyr Ile Asp Ile Val Pro 35 40 45 Gly His Val His Leu Gln Glu Phe Gly Lys Ile Val Lys Glu Ala Ile 50 55 60 Arg Glu Ala Gly Gly Val Pro Phe Glu Phe Asn Thr Ile Gly Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Ile Gly Met Arg Tyr Ser Leu Pro Ser 85 90 95 Arg Glu Ile Ile Ala Asp Ser Val Glu Thr Val Val Ser Ala His Trp 100 105 110 Phe Asp Gly Met Val Cys Ile Pro Asn Cys Asp Lys Ile Thr Pro Gly 115 120 125 Met Leu Met Ala Ala Met Arg Ile Asn Ile Pro Thr Ile Phe Val Ser 130 135 140 Gly Gly Pro Met Ala Ala Gly Arg Thr Ser Tyr Gly Arg Lys Ile Ser 145 150 155 160 Leu Ser Ser Val Phe Glu Gly Val Gly Ala Tyr Gln Ala Gly Lys Ile 165 170 175 Asn Glu Asn Glu Leu Gln Glu Leu Glu Gln Phe Gly Cys Pro Thr Cys 180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Ser 195 200 205 Glu Ala Leu Gly Leu Ala Leu Pro Gly Asn Gly Thr Ile Leu Ala Thr 210 215 220 Ser Pro Glu Arg Lys Glu Phe Val Arg Lys Ser Ala Ala Gln Leu Met 225 230 235 240 Glu Thr Ile Arg Lys Asp Ile Lys Pro Arg Asp Ile Val Thr Val Lys 245 250 255 Ala Ile Asp Asn Ala Phe Ala Leu Asp Met Ala Leu Gly Gly Ser Thr 260 265 270 Asn Thr Val Leu His Thr Leu Ala Leu Ala Asn Glu Ala Gly Val Glu 275 280 285 Tyr Ser Leu Glu Arg Ile Asn Glu Val Ala Glu Arg Val Pro His Leu 290 295 300 Ala Lys Leu Ala Pro Ala Ser Asp ValPhe Ile Glu Asp Leu His Glu 305 310 315 320 Ala Gly Gly Val Ser Ala Ala Leu Asn Glu Leu Ser Lys Lys Glu Gly 325 330 335 Ala Leu His Leu Asp Ala Leu Thr Val Thr Gly Lys Thr Leu Gly Glu 340 345 350 Thr Ile Ala Gly His Glu Val Lys Asp Tyr Asp Val Ile His Pro Leu 355 360 365 Asp Gln Pro Phe Thr Glu Lys Gly Gly Leu Ala Val Leu Phe Gly Asn 370 375 380 Leu Ala Pro Asp Gly Ala Ile Ile Lys Thr Gly Gly Val Gln Asn Gly 385 390 395 400 Ile Thr Arg His Glu Gly Pro Ala Val Val Phe Asp Ser Gln Asp Glu 405 410 415 Ala Leu Asp Gly Ile Ile Asn Arg Lys Val Lys Glu Gly Asp Val Val 420 425 430 Ile Ile Arg Tyr Glu Gly Pro Lys Gly Gly Pro Gly Met Pro Glu Met 435 440 445 Leu Ala Pro Thr Ser Gln Ile Val Gly Met Gly Leu Gly Pro Lys Val 450 455 460 Ala Leu Ile Thr Asp Gly Arg Phe Ser Gly Ala Ser Arg Gly Leu Ser 465 470 475 480 Ile Gly His Val Ser Pro Glu Ala Ala Glu Gly Gly Pro Leu Ala Phe 485 490 495 Val Glu Asn Gly Asp His Ile Ile Val Asp Ile Glu Lys Arg Ile Leu 500 505 510 Asp Val Gln Val Pro Glu Glu Glu Trp Glu Lys Arg Lys Ala Asn Trp 515 520 525 Lys Gly Phe Glu Pro Lys Val Lys Thr Gly Tyr Leu Ala Arg Tyr Ser 530 535 540 Lys Leu Val Thr Ser Ala Asn Thr Gly Gly Ile Met Lys Ile 545 550 555 <210>191 <211>1647 <212>DNA <213>乳酸乳球菌 <400>191 atgtatacag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60 tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccacaaggat 120 atgaaatggg tcggaaatgc taatgaatta aatgcttcat atatggctga tggctatgct 180 cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240 aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300 acatcaaaag ttcaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360 aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420 aatgcaaccg ttgaaattga ccgagtactt tctgcactat taaaagaaag aaaacctgtc 480 tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540 aaaaaggaaa actcaacttc aaatacaagt gaccaagaaa ttttgaacaa aattcaagaa 600 agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagttttggc 660 ttagaaaaaa cagtcactca atttatttca aagacaaaac tacctattac gacattaaac 720 tttggtaaaa gttcagttga tgaagccctc ccttcatttt taggaatcta taatggtaca 780 ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatctt gatgcttgga 840 gttaaactca cagactcttc aacaggagcc ttcactcatc atttaaatga aaataaaatg 900 atttcactga atatagatga aggaaaaata tttaacgaaa gaatccaaaa ttttgatttt 960 gaatccctca tctcctctct cttagaccta agcgaaatag aatacaaagg aaaatatatc 1020 gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080 caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140 ttctttggcg cttcatcaat tttcttaaaa tcaaagagtc attttattgg tcaaccctta 1200 tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260 agcagacacc ttttatttat tggtgatggt tcacttcaac ttacagtgca agaattagga 1320 ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380 gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440 tcaaaattac cagaatcgtt tggagcaaca gaagatcgag tagtctcaaa aatcgttaga 1500 actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560 tggattgagt taattttggc aaaagaaggt gcaccaaaag tactgaaaaa aatgggcaaa 1620 ctatttgctg aacaaaataa atcataa 1647 <210>192 <211>1644 <212>DNA <213>乳酸乳球菌 <400>192 atgtatacag taggagatta cctgttagac cgattacacg agttgggaat tgaagaaatt 60 tttggagttc ctggtgacta taacttacaa tttttagatc aaattatttc acgcgaagat 120 atgaaatgga ttggaaatgc taatgaatta aatgcttctt atatggctga tggttatgct 180 cgtactaaaa aagctgccgc atttctcacc acatttggag tcggcgaatt gagtgcgatc 240 aatggactgg caggaagtta tgccgaaaat ttaccagtag tagaaattgt tggttcacca 300 acttcaaaag tacaaaatga cggaaaattt gtccatcata cactagcaga tggtgatttt 360 aaacacttta tgaagatgca tgaacctgtt acagcagcgc ggactttact gacagcagaa 420 aatgccacat atgaaattga ccgagtactt tctcaattac taaaagaaag aaaaccagtc 480 tatattaact taccagtcga tgttgctgca gcaaaagcag agaagcctgc attatcttta 540 gaaaaagaaa gctctacaac aaatacaact gaacaagtga ttttgagtaa gattgaagaa 600 agtttgaaaa atgcccaaaa accagtagtg attgcaggac acgaagtaat tagttttggt 660 ttagaaaaaa cggtaactca gtttgtttca gaaacaaaac taccgattac gacactaaat 720 tttggtaaaa gtgctgttga tgaatctttg ccctcatttt taggaatata taacgggaaa 780 ctttcagaaa tcagtcttaa aaattttgtg gagtccgcag actttatcct aatgcttgga 840 gtgaagctta cggactcctc aacaggtgca ttcacacatc atttagatga aaataaaatg 900 atttcactaa acatagatga aggaataatt ttcaataaag tggtagaaga ttttgatttt 960 agagcagtgg tttcttcttt atcagaatta aaaggaatag aatatgaagg acaatatatt 1020 gataagcaat atgaagaatt tattccatca agtgctccct tatcacaaga ccgtctatgg 1080 caggcagttg aaagtttgac tcaaagcaat gaaacaatcg ttgctgaaca aggaacctca 1140 ttttttggag cttcaacaat tttcttaaaa tcaaatagtc gttttattgg acaaccttta 1200 tggggttcta ttggatatac ttttccagcg gctttaggaa gccaaattgc ggataaagag 1260 agcagacacc ttttatttat tggtgatggt tcacttcaac ttaccgtaca agaattagga 1320 ctatcaatca gagaaaaact caatccaatt tgttttatca taaataatga tggttataca 1380 gttgaaagag aaatccacgg acctactcaa agttataacg acattccaat gtggaattac 1440 tcgaaattac cagaaacatt tggagcaaca gaagatcgtg tagtatcaaa aattgttaga 1500 acagagaatg aatttgtgtc tgtcatgaaa gaagcccaag cagatgtcaa tagaatgtat 1560 tggatagaac tagttttgga aaaagaagat gcgccaaaat tactgaaaaa aatgggtaaa 1620 ttatttgctg agcaaaataa atag 1644 <210>193 <211>547 <212>PRT <213>乳酸乳球菌 <400>193 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Ile 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Asp Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140 Glu Ile Asp Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln 180 185 190 Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro 195 200 205 Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu 325 330 335 Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala 340 345 350 Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Thr Ile Phe Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly TyrThr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys Leu Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Thr Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys 515 520 525 Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys 545 <210>194 <211>1653 <212>DNA <213>鼠伤寒沙门氏菌 <400>194 ttatcccccg ttgcgggctt ccagcgcccg ggtcacggta cgcagtaatt ccggcagatc 60 ggcttttggc aacatcactt caataaatga cagacgttgt gggcgcgcca accgttcgag 120 gacctctgcc agttggatag cctgcgtcac ccgccagcac tccgcctgtt gcgccgcgtt 180 tagcgccggt ggtatctgcg tccagttcca gctcgcgatg tcgttatacc gctgggccgc 240 gccgtgaatg gcgcgctcta cggtatagcc gtcattgttg agcagcagga tgaccggcgc 300 ctgcccgtcg cgtaacatcg agcccatctc ctgaatcgtg agctgcgccg cgccatcgcc 360 gataatcaga atcacccgcc gatcgggaca ggcggtttgc gcgccaaacg cggcgggcaa 420 ggaatagccg atagaccccc acagcggctg taacacaact tccgcgccgt caggaagcga 480 cagcgcggca gcgccaaaag ctgctgtccc ctggtcgaca aggataatat ctccgggttt 540 gagatactgc tgtaaggttt gccagaagct ttcctgggtc agttctcctt tatcaatccg 600 cactggctgt ccggcggaac gcgtcggcgg cggcgcaaaa gcgcattcca ggcacagttc 660 gcgcagcgta gacaccgcct gcgccatcgg gaggttgaac caggtttcgc cgatgcgcga 720 cgcgtaaggc tgaatctcca gcgtgcgttc cgccggtaat tgttgggtaa atccggccgt 780 aagggtatcg acaaaacggg tgccgacgca gataacccta tcggcgtcct ctatggcctg 840 acgcacttct ttgctgctgg cgccagcgct ataggtgcca acgaagttcg ggtgctgttc 900 atcaaaaagc cccttcccca tcagtagtgt cgcatgagcg atgggcgttt ccgccatcca 960 gcgctgcaac agtggtcgta aaccaaaacg cccggcaaga aagtcggcca atagcgcaat 1020 gcgccgactg ttcatcaggc actgacgggc gtgataacga aaggccgtct ccacgccgct 1080 ttgcgcttca tgcacgggca acgccagcgc ctgcgtaggt gggatggccg tttttttcgc 1140 cacatcggcg ggcaacatga tgtatcctgg cctgcgtgcg gcaagcattt cacccaacac 1200 gcggtcaatc tcgaaacagg cgttctgttc atctaatatt gcgctggcag cggatatcgc 1260 ctgactcatg cgataaaaat gacgaaaatc gccgtcaccg agggtatggt gcatcaattc 1320 gccacgctgc tgcgcagcgc tacagggcgc gccgacgata tgcaagaccg ggacatattc 1380 cgcgtaactg cccgcgatac cgttaatagc gctaagttct cccacgccaa aggtggtgag 1440 tagcgctcca gcgcccgaca tgcgcgcata gccgtccgcg gcataagcgg cgttcagctc 1500 attggcgcat cccacccaac gcagggtcgg gtggtcaatc acatggtcaa gaaactgcaa 1560 gttataatcg cccggtacgc caaaaagatg gccaatgccg catcctgcca gtctgtccag 1620 caaatagtcg gccacggtat aggggttttg cat 1653 <210>195 <211>550 <212>PRT <213>鼠伤寒沙门氏菌 <400>195 Met Gln Asn Pro Tyr Thr Val Ala Asp Tyr Leu Leu Asp Arg Leu Ala 1 5 10 15 Gly Cys Gly Ile Gly His Leu Phe Gly Val Pro Gly Asp Tyr Asn Leu 20 25 30 Gln Phe Leu Asp His Val Ile Asp His Pro Thr Leu Arg Trp Val Gly 35 40 45 Cys Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg 50 55 60 Met Ser Gly Ala Gly Ala Leu Leu Thr Thr Phe Gly Val Gly Glu Leu 65 70 75 80 Ser Ala Ile Asn Gly Ile Ala Gly Ser Tyr Ala Glu Tyr Val Pro Val 85 90 95 Leu His Ile Val Gly Ala Pro Cys Ser Ala Ala Gln Gln Arg Gly Glu 100 105 110 Leu Met His His Thr Leu Gly Asp Gly Asp Phe Arg His Phe Tyr Arg 115 120 125 Met Ser Gln Ala Ile Ser Ala Ala Ser Ala Ile Leu Asp Glu Gln Asn 130 135 140 Ala Cys Phe Glu Ile Asp Arg Val Leu Gly Glu Met Leu Ala Ala Arg 145 150 155 160 Arg Pro Gly Tyr Ile Met Leu Pro Ala Asp Val Ala Lys Lys Thr Ala 165 170 175 Ile Pro Pro Thr Gln Ala Leu Ala Leu Pro Val His Glu Ala Gln Ser 180 185 190 Gly Val Glu Thr Ala Phe Arg Tyr His Ala Arg Gln Cys Leu Met Asn 195 200 205 Ser Arg Arg Ile Ala Leu Leu Ala Asp Phe Leu Ala Gly Arg Phe Gly 210 215 220 Leu Arg Pro Leu Leu Gln Arg Trp Met Ala Glu Thr Pro Ile Ala His 225 230 235 240 Ala Thr Leu Leu Met Gly Lys Gly Leu Phe Asp Glu Gln His Pro Asn 245 250 255 Phe Val Gly Thr Tyr Ser Ala Gly Ala Ser Ser Lys Glu Val Arg Gln 260 265 270 Ala Ile Glu Asp Ala Asp Arg Val Ile Cys Val Gly Thr Arg Phe Val 275 280 285 Asp Thr Leu Thr Ala Gly Phe Thr Gln Gln Leu Pro Ala Glu Arg Thr 290 295 300 Leu Glu Ile Gln Pro Tyr Ala Ser Arg Ile Gly Glu Thr Trp Phe Asn 305 310 315 320 Leu Pro Met Ala Gln Ala Val Ser Thr Leu Arg Glu Leu Cys Leu Glu 325 330 335 Cys Ala Phe Ala Pro Pro Pro Thr Arg Ser Ala Gly Gln Pro Val Arg 340 345 350 Ile Asp Lys Gly Glu Leu Thr Gln Glu Ser Phe Trp Gln Thr Leu Gln 355 360 365 Gln Tyr Leu Lys Pro Gly Asp Ile Ile Leu Val Asp Gln Gly Thr Ala 370 375 380 Ala Phe Gly Ala Ala Ala Leu Ser Leu Pro Asp Gly Ala Glu Val Val 385 390 395 400 Leu Gln Pro Leu Trp Gly Ser Ile Gly Tyr Ser Leu Pro Ala Ala Phe 405 410 415 Gly Ala Gln Thr Ala Cys Pro Asp Arg Arg Val Ile Leu Ile Ile Gly 420 425 430 Asp Gly Ala Ala Gln Leu Thr Ile Gln Glu Met Gly Ser Met Leu Arg 435 440 445 Asp Gly Gln Ala Pro Val Ile Leu Leu Leu Asn Asn Asp Gly Tyr Thr 450 455 460 Val Glu Arg Ala Ile His Gly Ala Ala Gln Arg Tyr Asn Asp Ile Ala 465 470 475 480 Ser Trp Asn Trp Thr Gln Ile Pro Pro Ala Leu Asn Ala Ala Gln Gln 485 490 495 Ala Glu Cys Trp Arg Val Thr Gln Ala Ile Gln Leu Ala Glu Val Leu 500 505 510 Glu Arg Leu Ala Arg Pro Gln Arg Leu Ser Phe Ile Glu Val Met Leu 515 520 525 Pro Lys Ala Asp Leu Pro Glu Leu Leu Arg Thr Val Thr Arg Ala Leu 530 535 540 Glu Ala Arg Asn Gly Gly 545 550 <210>196 <211>1665 <212>DNA <213>丙醇丁酮梭菌 <400>196 ttgaagagtg aatacacaat tggaagatat ttgttagacc gtttatcaga gttgggtatt 60 cggcatatct ttggtgtacc tggagattac aatctatcct ttttagacta tataatggag 120 tacaaaggga tagattgggt tggaaattgc aatgaattga atgctgggta tgctgctgat 180 ggatatgcaa gaataaatgg aattggagcc atacttacaa catttggtgt tggagaatta 240 agtgccatta acgcaattgc tggggcatac gctgagcaag ttccagttgt taaaattaca 300 ggtatcccca cagcaaaagt tagggacaat ggattatatg tacaccacac attaggtgac 360 ggaaggtttg atcacttttt tgaaatgttt agagaagtaa cagttgctga ggcattacta 420 agcgaagaaa atgcagcaca agaaattgat cgtgttctta tttcatgctg gagacaaaaa 480 cgtcctgttc ttataaattt accgattgat gtatatgata aaccaattaa caaaccatta 540 aagccattac tcgattatac tatttcaagt aacaaagagg ctgcatgtga atttgttaca 600 gaaatagtac ctataataaa tagggcaaaa aagcctgtta ttcttgcaga ttatggagta 660 tatcgttacc aagttcaaca tgtgcttaaa aacttggccg aaaaaaccgg atttcctgtg 720 gctacactaa gtatgggaaa aggtgttttc aatgaagcac accctcaatt tattggtgtt 780 tataatggtg atgtaagttc tccttattta aggcagcgag ttgatgaagc agactgcatt 840 attagcgttg gtgtaaaatt gacggattca accacagggg gattttctca tggattttct 900 aaaaggaatg taattcacat tgatcctttt tcaataaagg caaaaggtaa aaaatatgca 960 cctattacga tgaaagatgc tttaacagaa ttaacaagta aaattgagca tagaaacttt 1020 gaggatttag atataaagcc ttacaaatca gataatcaaa agtattttgc aaaagagaag 1080 ccaattacac aaaaacgttt ttttgagcgt attgctcact ttataaaaga aaaagatgta 1140 ttattagcag aacagggtac atgctttttt ggtgcgtcaa ccatacaact acccaaagat 1200 gcaactttta ttggtcaacc tttatgggga tctattggat acacacttcc tgctttatta 1260 ggttcacaat tagctgatca aaaaaggcgt aatattcttt taattgggga tggtgcattt 1320 caaatgacag cacaagaaat ttcaacaatg cttcgtttac aaatcaaacc tattattttt 1380 ttaattaata acgatggtta tacaattgaa cgtgctattc atggtagaga acaagtatat 1440 aacaatattc aaatgtggcg atatcataat gttccaaagg ttttaggtcc taaagaatgc 1500 agcttaacct ttaaagtaca aagtgaaact gaacttgaaa aggctctttt agtggcagat 1560 aaggattgtg aacatttgat ttttatagaa gttgttatgg atcgttatga taaacccgag 1620 cctttagaac gtctttcgaa acgttttgca aatcaaaata attag 1665 <210>197 <211>554 <212>PRT <213>丙醇丁酮梭菌 <400>197 Met Lys Ser Glu Tyr Thr Ile Gly Arg Tyr Leu Leu Asp Arg Leu Ser 1 5 10 15 Glu Leu Gly Ile Arg His Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu 20 25 30 Ser Phe Leu Asp Tyr Ile Met Glu Tyr Lys Gly Ile Asp Trp Val Gly 35 40 45 Asn Cys Asn Glu Leu Asn Ala Gly Tyr Ala Ala Asp Gly Tyr Ala Arg 50 55 60 Ile Asn Gly Ile Gly Ala Ile Leu Thr Thr Phe Gly Val Gly Glu Leu 65 70 75 80 Ser Ala Ile Asn Ala Ile Ala Gly Ala Tyr Ala Glu Gln Val Pro Val 85 90 95 Val Lys Ile Thr Gly Ile Pro Thr Ala Lys Val Arg Asp Asn Gly Leu 100 105 110 Tyr Val His His Thr Leu Gly Asp Gly Arg Phe Asp His Phe Phe Glu 115 120 125 Met Phe Arg Glu Val Thr Val Ala Glu Ala Leu Leu Ser Glu Glu Asn 130 135 140 Ala Ala Gln Glu Ile Asp Arg Val Leu Ile Ser Cys Trp Arg Gln Lys 145 150 155 160 Arg Pro Val Leu Ile Asn Leu Pro Ile Asp Val Tyr Asp Lys Pro Ile 165 170 175 Asn Lys Pro Leu Lys Pro Leu Leu Asp Tyr Thr Ile Ser Ser Asn Lys 180 185 190 Glu Ala Ala Cys Glu Phe Val Thr Glu Ile Val Pro Ile Ile Asn Arg 195 200 205 Ala Lys Lys Pro Val Ile Leu Ala Asp Tyr Gly Val Tyr Arg Tyr Gln 210 215 220 Val Gln His Val Leu Lys Asn Leu Ala Glu Lys Thr Gly Phe Pro Val 225 230 235 240 Ala Thr Leu Ser Met Gly Lys Gly Val Phe Asn Glu Ala His Pro Gln 245 250 255 Phe Ile Gly Val Tyr Asn Gly Asp Val Ser Ser Pro Tyr Leu Arg Gln 260 265 270 Arg Val Asp Glu Ala Asp Cys Ile Ile Ser Val Gly Val Lys Leu Thr 275 280 285 Asp Ser Thr Thr Gly Gly Phe Ser His Gly Phe Ser Lys Arg Asn Val 290 295 300 Ile His Ile Asp Pro Phe Ser Ile Lys Ala Lys Gly Lys Lys Tyr Ala 305 310 315 320 Pro Ile Thr Met Lys Asp Ala Leu Thr Glu Leu Thr Ser Lys Ile Glu 325 330 335 His Arg Asn Phe Glu Asp Leu Asp Ile Lys Pro Tyr Lys Ser Asp Asn 340 345 350 Gln Lys Tyr Phe Ala Lys Glu Lys Pro Ile Thr Gln Lys Arg Phe Phe 355 360 365 Glu Arg Ile Ala His Phe Ile Lys Glu Lys Asp Val Leu Leu Ala Glu 370 375 380 Gln Gly Thr Cys Phe Phe Gly Ala Ser Thr Ile Gln Leu Pro Lys Asp 385 390 395 400 Ala Thr Phe Ile Gly Gln Pro Leu Trp Gly Ser Ile Gly Tyr Thr Leu 405 410 415 Pro Ala Leu Leu Gly Ser Gln Leu Ala Asp Gln Lys Arg Arg Asn Ile 420 425 430 Leu Leu Ile Gly Asp Gly Ala Phe Gln Met Thr Ala Gln Glu Ile Ser 435 440 445 Thr Met Leu Arg Leu Gln Ile Lys Pro Ile Ile Phe Leu Ile Asn Asn 450 455 460 Asp Gly Tyr Thr Ile Glu Arg Ala Ile His Gly Arg Glu Gln Val Tyr 465 470 475 480 Asn Asn Ile Gln Met Trp Arg Tyr His Asn Val Pro Lys Val Leu Gly 485 490 495 Pro Lys Glu Cys Ser Leu Thr Phe Lys Val Gln Ser Glu Thr Glu Leu 500 505 510 Glu Lys Ala Leu Leu Val Ala Asp Lys Asp Cys Glu His Leu Ile Phe 515 520 525 Ile Glu Val Val Met Asp Arg Tyr Asp Lys Pro Glu Pro Leu Glu Arg 530 535 540 Leu Ser Lys Arg Phe Ala Asn Gln Asn Asn 545 550 <210>198 <211>939 <212>DNA <213>酿酒酵母 <400>198 atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60 ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120 gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180 gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240 aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300 ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360 agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420 gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480 ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540 gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600 ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660 ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720 aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780 gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840 cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900 gttgatatga agtggggatc cttcccaatt ttccaatga 939 <210>199 <211>312 <212>PRT <213>酿酒酵母 <400>199 Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr 1 5 10 15 Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp 20 25 30 Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg 35 40 45 His Ile Asp Ala Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg 50 55 60 Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr 65 70 75 80 Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys 85 90 95 Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His 100 105 110 Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu 115 120 125 Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu 130 135 140 Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr 145 150 155 160 Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile 165 170 175 Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn 180 185 190 Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe 195 200 205 Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser 210 215 220 Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Ile Asp Met Ala Lys 225 230 235 240 Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln 245 250 255 Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val 260 265 270 Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile 275 280 285 Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys 290 295 300 Trp Gly Ser Phe Pro Ile Phe Gln 305 310 <210>200 <211>1083 <212>DNA <213>酿酒酵母 <400>200 ctagtctgaa aattctttgt cgtagccgac taaggtaaat ctatatctaa cgtcaccctt 60 ttccatcctt tcgaaggctt catggacgcc ggcttcacca acaggtaatg tttccaccca 120 aattttgata tctttttcag agactaattt caagagttgg ttcaattctt tgatggaacc 180 taaagcactg taagaaatgg agacagcctt taagccatat ggctttagcg ataacatttc 240 gtgttgttct ggtatagaga ttgagacaat tctaccacca accttcatag cctttggcat 300 aatgttgaag tcaatgtcgg taagggagga agcacagact acaatcaggt cgaaggtgtc 360 aaagtacttt tcaccccaat caccttcttc taatgtagca atgtagtgat cggcgcccat 420 cttcattgca tcttctcttt ttctcgaaga acgagaaata acatacgtct ctgcccccat 480 ggctttggaa atcaatgtac ccatactgcc gataccacca agaccaacta taccaacttt 540 tttacctgga ccgcaaccgt tacgaaccaa tggagagtac acagtcaaac caccacataa 600 tagtggagca gccaaatgtg atggaatatt ctctgggata ggcaccacaa aatgttcatg 660 aactctgacg tagtttgcat agccaccctg cgacacatag ccgtcttcat aaggctgact 720 gtatgtggta acaaacttgg tgcagtatgg ttcattatca ttcttacaac ggtcacattc 780 caagcatgaa aagacttgag cacctacacc aacacgttga ccgactttca acccactgtt 840 tgacttgggc cctagcttga caactttacc aacgatttca tgaccaacga ctagcggcat 900 cttcatattg ccccaatgac cagctgcaca atgaatatca ctaccgcaga caccacatgc 960 ttcgatctta atgtcaatgt catgatcgta aaatggtttt gggtcatact ttgtcttctt 1020 tgggtttttc caatcttcgt gtgattgaat agcgatacct tcaaatttct caggataaga 1080 cat 1083 <210>201 <211>360 <212>PRT <213>酿酒酵母 <400>201 Met Ser Tyr Pro Glu Lys Phe Glu Gly Ile Ala Ile Gln Ser His Glu 1 5 10 15 Asp Trp Lys Asn Pro Lys Lys Thr Lys Tyr Asp Pro Lys Pro Phe Tyr 20 25 30 Asp His Asp Ile Asp Ile Lys Ile Glu Ala Cys Gly Val Cys Gly Ser 35 40 45 Asp Ile His Cys Ala Ala Gly His Trp Gly Asn Met Lys Met Pro Leu 50 55 60 Val Val Gly His Glu Ile Val Gly Lys Val Val Lys Leu Gly Pro Lys 65 70 75 80 Ser Asn Ser Gly Leu Lys Val Gly Gln Arg Val Gly Val Gly Ala Gln 85 90 95 Val Phe Ser Cys Leu Glu Cys Asp Arg Cys Lys Asn Asp Asn Glu Pro 100 105 110 Tyr Cys Thr Lys Phe Val Thr Thr Tyr Ser Gln Pro Tyr Glu Asp Gly 115 120 125 Tyr Val Ser Gln Gly Gly Tyr Ala Asn Tyr Val Arg Val His Glu His 130 135 140 Phe Val Val Pro Ile Pro Glu Asn Ile Pro Ser His Leu Ala Ala Pro 145 150 155 160 Leu Leu Cys Gly Gly Leu Thr Val Tyr Ser Pro Leu Val Arg Asn Gly 165 170 175 Cys Gly Pro Gly Lys Lys Val Gly Ile Val Gly Leu Gly Gly Ile Gly 180 185 190 Ser Met Gly Thr Leu Ile Ser Lys Ala Met Gly Ala Glu Thr Tyr Val 195 200 205 Ile Ser Arg Ser Ser Arg Lys Arg Glu Asp Ala Met Lys Met Gly Ala 210 215 220 Asp His Tyr Ile Ala Thr Leu Glu Glu Gly Asp Trp Gly Glu Lys Tyr 225 230 235 240 Phe Asp Thr Phe Asp Leu Ile Val Val Cys Ala Ser Ser Leu Thr Asp 245 250 255 Ile Asp Phe Asn Ile Met Pro Lys Ala Met Lys Val Gly Gly Arg Ile 260 265 270 Val Ser Ile Ser Ile Pro Glu Gln His Glu Met Leu Ser Leu Lys Pro 275 280 285 Tyr Gly Leu Lys Ala Val Ser Ile Ser Tyr Ser Ala Leu Gly Ser Ile 290 295 300 Lys Glu Leu Asn Gln Leu Leu Lys Leu Val Ser Glu Lys Asp Ile Lys 305 310 315 320 Ile Trp Val Glu Thr Leu Pro Val Gly Glu Ala Gly Val His Glu Ala 325 330 335 Phe Glu Arg Met Glu Lys Gly Asp Val Arg Tyr Arg Phe Thr Leu Val 340 345 350 Gly Tyr Asp Lys Glu Phe Ser Asp 355 360 <210>202 <211>1170 <212>DNA <213>丙醇丁酮梭菌 <400>202 ttaataagat tttttaaata tctcaagaac atcctctgca tttattggtc ttaaacttcc 60 tattgttcct ccagaatttc taacagcttg ctttgccatt agttctagtt tatcttttcc 120 tattccaact tctctaagct ttgaaggaat acccaatgaa ttaaagtatt ctctcgtatt 180 tttaatagcc tctcgtgcta tttcatagtt atctttgttc ttgtctattc cccaaacatt 240 tattccataa gaaacaaatt tatgaagtgt atcgtcattt agaatatatt ccatccaatt 300 aggtgttaaa attgcaagtc ctacaccatg tgttatatca taatatgcac ttaactcgtg 360 ttccatagga tgacaactcc attttctatc cttaccaagt gataatagac catttatagc 420 taaacttgaa gcccacatca aattagctct agcctcgtaa tcatcagtct tctccattgc 480 tatttttcca tactttatac atgttcttaa gattgcttct gctataccgt cctgcacata 540 agcaccttca acaccactaa agtaagattc aaaggtgtga ctcataatgt cagctgttcc 600 cgctgctgtt tgatttttag gtactgtaaa agtatatgta ggatctaaca ctgaaaattt 660 aggtctcata tcatcatgtc ctactccaag cttttcatta gtctccatat ttgaaattac 720 tgcaatttga tccatttcag accctgttgc tgaaagagta agtatacttg caattggaag 780 aactttagtt attttagatg gatctttaac catgtcccat gtatcgccat cataataaac 840 tccagctgca attaccttag aacagtctat tgcacttcct ccccctattg ctaatactaa 900 atccacatta ttttctctac atatttctat gccttttttt actgttgtta tcctaggatt 960 tggctctact cctgaaagtt catagaaagc tatattgttt tcttttaata tagctgttgc 1020 tctatcatat ataccgttcc tttttatact tcctccgcca taaactataa gcactcttga 1080 gccatatttc ttaatttctt ctccaattac gtctattttt ccttttccaa aaaaaacttt 1140 agttggtatt gaataatcaa aacttagcat 1170 <210>203 <211>389 <212>PRT <213>丙醇丁酮梭菌 <400>203 Met Leu Ser Phe Asp Tyr Ser Ile Pro Thr Lys Val Phe Phe Gly Lys 1 5 10 15 Gly Lys Ile Asp Val Ile Gly Glu Glu Ile Lys Lys Tyr Gly Ser Arg 20 25 30 Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr 35 40 45 Asp Arg Ala Thr Ala Ile Leu Lys Glu Asn Asn Ile Ala Phe Tyr Glu 50 55 60 Leu Ser Gly Val Glu Pro Asn Pro Arg Ile Thr Thr Val Lys Lys Gly 65 70 75 80 Ile Glu Ile Cys Arg Glu Asn Asn Val Asp Leu Val Leu Ala Ile Gly 85 90 95 Gly Gly Ser Ala Ile Asp Cys Ser Lys Val Ile Ala Ala Gly Val Tyr 100 105 110 Tyr Asp Gly Asp Thr Trp Asp Met Val Lys Asp Pro Ser Lys Ile Thr 115 120 125 Lys Val Leu Pro Ile Ala Ser Ile Leu Thr Leu Ser Ala Thr Gly Ser 130 135 140 Glu Met Asp Gln Ile Ala Val Ile Ser Asn Met Glu Thr Asn Glu Lys 145 150 155 160 Leu Gly Val Gly His Asp Asp Met Arg Pro Lys Phe Ser Val Leu Asp 165 170 175 Pro Thr Tyr Thr Phe Thr Val Pro Lys Asn Gln Thr Ala Ala Gly Thr 180 185 190 Ala Asp Ile Met Ser His Thr Phe Glu Ser Tyr Phe Ser Gly Val Glu 195 200 205 Gly Ala Tyr Val Gln Asp Gly Ile Ala Glu Ala Ile Leu Arg Thr Cys 210 215 220 Ile Lys Tyr Gly Lys Ile Ala Met Glu Lys Thr Asp Asp Tyr Glu Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu 245 250 255 Ser Leu Gly Lys Asp Arg Lys Trp Ser Cys His Pro Met Glu His Glu 260 265 270 Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu 275 280 285 Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asp Asp Thr Leu His Lys 290 295 300 Phe Val Ser Tyr Gly Ile Asn Val Trp Gly Ile Asp Lys Asn Lys Asp 305 310 315 320 Asn Tyr Glu Ile Ala Arg Glu Ala Ile Lys Asn Thr Arg Glu Tyr Phe 325 330 335 Asn Ser Leu Gly Ile Pro Ser Lys Leu Arg Glu Val Gly Ile Gly Lys 340 345 350 Asp Lys Leu Glu Leu Met Ala Lys Gln Ala Val Arg Asn Ser Gly Gly 355 360 365 Thr Ile Gly Ser Leu Arg Pro Ile Asn Ala Glu Asp Val Leu Glu Ile 370 375 380 Phe Lys Lys Ser Tyr 385 <210>204 <211>390 <212>PRT <213>丙醇丁酮梭菌 <400>204 Met Val Asp Phe Glu Tyr Ser Ile Pro Thr Arg Ile Phe Phe Gly Lys 1 5 10 15 Asp Lys Ile Asn Val Leu Gly Arg Glu Leu Lys Lys Tyr Gly Ser Lys 20 25 30 Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr 35 40 45 Asp Lys Ala Val Ser Ile Leu Glu Lys Asn Ser Ile Lys Phe Tyr Glu 50 55 60 Leu Ala Gly Val Glu Pro Asn Pro Arg Val Thr Thr Val Glu Lys Gly 65 70 75 80 Val Lys Ile Cys Arg Glu Asn Gly Val Glu Val Val Leu Ala Ile Gly 85 90 95 Gly Gly Ser Ala Ile Asp Cys Ala Lys Val Ile Ala Ala Ala Cys Glu 100 105 110 Tyr Asp Gly Asn Pro Trp Asp Ile Val Leu Asp Gly Ser Lys Ile Lys 115 120 125 Arg Val Leu Pro Ile Ala Ser Ile Leu Thr Ile Ala Ala Thr Gly Ser 130 135 140 Glu Met Asp Thr Trp Ala Val Ile Asn Asn Met Asp Thr Asn Glu Lys 145 150 155 160 Leu Ile Ala Ala His Pro Asp Met Ala Pro Lys Phe Ser Ile Leu Asp 165 170 175 Pro Thr Tyr Thr Tyr Thr Val Pro Thr Asn Gln Thr Ala Ala Gly Thr 180 185 190 Ala Asp Ile Met Ser His Ile Phe Glu Val Tyr Phe Ser Asn Thr Lys 195 200 205 Thr Ala Tyr Leu Gln Asp Arg Met Ala Glu Ala Leu Leu Arg Thr Cys 210 215 220 Ile Lys Tyr Gly Gly Ile Ala Leu Glu Lys Pro Asp Asp Tyr Glu Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu 245 250 255 Thr Tyr Gly Lys Asp Thr Asn Trp Ser Val His Leu Met Glu His Glu 260 265 270 Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu 275 280 285 Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asn Asp Thr Val Tyr Lys 290 295 300 Phe Val Glu Tyr Gly Val Asn Val Trp Gly Ile Asp Lys Glu Lys Asn 305 310 315 320 His Tyr Asp Ile Ala His Gln Ala Ile Gln Lys Thr Arg Asp Tyr Phe 325 330 335 Val Asn Val Leu Gly Leu Pro Ser Arg Leu Arg Asp Val Gly Ile Glu 340 345 350 Glu Glu Lys Leu Asp Ile Met Ala Lys Glu Ser Val Lys Leu Thr Gly 355 360 365 Gly Thr Ile Gly Asn Leu Arg Pro Val Asn Ala Ser Glu Val Leu Gln 370 375 380 Ile Phe Lys Lys Ser Val 385 390 <210>205 <211>993 <212>DNA <213>枯草芽孢杆菌 <400>205 atgagtacaa accgacatca agcactaggg ctgactgatc aggaagccgt tgatatgtat 60 agaaccatgc tgttagcaag aaaaatcgat gaaagaatgt ggctgttaaa ccgttctggc 120 aaaattccat ttgtaatctc ttgtcaagga caggaagcag cacaggtagg agcggctttc 180 gcacttgacc gtgaaatgga ttatgtattg ccgtactaca gagacatggg tgtcgtgctc 240 gcgtttggca tgacagcaaa ggacttaatg atgtccgggt ttgcaaaagc agcagatccg 300 aactcaggag gccgccagat gccgggacat ttcggacaaa agaaaaaccg cattgtgacg 360 ggatcatctc cggttacaac gcaagtgccg cacgcagtcg gtattgcgct tgcgggacgt 420 atggagaaaa aggatatcgc agcctttgtt acattcgggg aagggtcttc aaaccaaggc 480 gatttccatg aaggggcaaa ctttgccgct gtccataagc tgccggttat tttcatgtgt 540 gaaaacaaca aatacgcaat ctcagtgcct tacgataagc aagtcgcatg tgagaacatt 600 tccgaccgtg ccataggcta tgggatgcct ggcgtaactg tgaatggaaa tgatccgctg 660 gaagtttatc aagcggttaa agaagcacgc gaaagggcac gcagaggaga aggcccgaca 720 ttaattgaaa cgatttctta ccgccttaca ccacattcca gtgatgacga tgacagcagc 780 tacagaggcc gtgaagaagt agaggaagcg aaaaaaagtg atcccctgct tacttatcaa 840 gcttacttaa aggaaacagg cctgctgtcc gatgagatag aacaaaccat gctggatgaa 900 attatggcaa tcgtaaatga agcgacggat gaagcggaga acgccccata tgcagctcct 960 gagtcagcgc ttgattatgt ttatgcgaag tag 993 <210>206 <211>330 <212>PRT <213>枯草芽孢杆菌 <400>206 Met Ser Thr Asn Arg His Gln Ala Leu Gly Leu Thr Asp Gln Glu Ala 1 5 10 15 Val Asp Met Tyr Arg Thr Met Leu Leu Ala Arg Lys Ile Asp Glu Arg 20 25 30 Met Trp Leu Leu Asn Arg Ser Gly Lys Ile Pro Phe Val Ile Ser Cys 35 40 45 Gln Gly Gln Glu Ala Ala Gln Val Gly Ala Ala Phe Ala Leu Asp Arg 50 55 60 Glu Met Asp Tyr Val Leu Pro Tyr Tyr Arg Asp Met Gly Val Val Leu 65 70 75 80 Ala Phe Gly Met Thr Ala Lys Asp Leu Met Met Ser Gly Phe Ala Lys 85 90 95 Ala Ala Asp Pro Asn Ser Gly Gly Arg Gln Met Pro Gly His Phe Gly 100 105 110 Gln Lys Lys Asn Arg Ile Val Thr Gly Ser Ser Pro Val Thr Thr Gln 115 120 125 Val Pro His Ala Val Gly Ile Ala Leu Ala Gly Arg Met Glu Lys Lys 130 135 140 Asp Ile Ala Ala Phe Val Thr Phe Gly Glu Gly Ser Ser Asn Gln Gly 145 150 155 160 Asp Phe His Glu Gly Ala Asn Phe Ala Ala Val His Lys Leu Pro Val 165 170 175 Ile Phe Met Cys Glu Asn Asn Lys Tyr Ala Ile Ser Val Pro Tyr Asp 180 185 190 Lys Gln Val Ala Cys Glu Asn Ile Ser Asp Arg Ala Ile Gly Tyr Gly 195 200 205 Met Pro Gly Val Thr Val Asn Gly Asn Asp Pro Leu Glu Val Tyr Gln 210 215 220 Ala Val Lys Glu Ala Arg Glu Arg Ala Arg Arg Gly Glu Gly Pro Thr 225 230 235 240 Leu Ile Glu Thr Ile Ser Tyr Arg Leu Thr Pro His Ser Ser Asp Asp 245 250 255 Asp Asp Ser Ser Tyr Arg Gly Arg Glu Glu Val Glu Glu Ala Lys Lys 260 265 270 Ser Asp Pro Leu Leu Thr Tyr Gln Ala Tyr Leu Lys Glu Thr Gly Leu 275 280 285 Leu Ser Asp Glu Ile Glu Gln Thr Met Leu Asp Glu Ile Met Ala Ile 290 295 300 Val Asn Glu Ala Thr Asp Glu Ala Glu Asn Ala Pro Tyr Ala Ala Pro 305 310 315 320 Glu Ser Ala Leu Asp Tyr Val Tyr Ala Lys 325 330 <210>207 <211>984 <212>DNA <213>枯草芽孢杆菌 <400>207 atgtcagtaa tgtcatatat tgatgcaatc aatttggcga tgaaagaaga aatggaacga 60 gattctcgcg ttttcgtcct tggggaagat gtaggaagaa aaggcggtgt gtttaaagcg 120 acagcgggac tctatgaaca atttggggaa gagcgcgtta tggatacgcc gcttgctgaa 180 tctgcaatcg caggagtcgg tatcggagcg gcaatgtacg gaatgagacc gattgctgaa 240 atgcagtttg ctgatttcat tatgccggca gtcaaccaaa ttatttctga agcggctaaa 300 atccgctacc gcagcaacaa tgactggagc tgtccgattg tcgtcagagc gccatacggc 360 ggaggcgtgc acggagccct gtatcattct caatcagtcg aagcaatttt cgccaaccag 420 cccggactga aaattgtcat gccatcaaca ccatatgacg cgaaagggct cttaaaagcc 480 gcagttcgtg acgaagaccc cgtgctgttt tttgagcaca agcgggcata ccgtctgata 540 aagggcgagg ttccggctga tgattatgtc ctgccaatcg gcaaggcgga cgtaaaaagg 600 gaaggcgacg acatcacagt gatcacatac ggcctgtgtg tccacttcgc cttacaagct 660 gcagaacgtc tcgaaaaaga tggcatttca gcgcatgtgg tggatttaag aacagtttac 720 ccgcttgata aagaagccat catcgaagct gcgtccaaaa ctggaaaggt tcttttggtc 780 acagaagata caaaagaagg cagcatcatg agcgaagtag ccgcaattat atccgagcat 840 tgtctgttcg acttagacgc gccgatcaaa cggcttgcag gtcctgatat tccggctatg 900 ccttatgcgc cgacaatgga aaaatacttt atggtcaacc ctgataaagt ggaagcggcg 960 atgagagaat tagcggagtt ttaa 984 <210>208 <211>327 <212>PRT <213>枯草芽孢杆菌 <400>208 Met Ser Val Met Ser Tyr Ile Asp Ala Ile Asn Leu Ala Met Lys Glu 1 5 10 15 Glu Met Glu Arg Asp Ser Arg Val Phe Val Leu Gly Glu Asp Val Gly 20 25 30 Arg Lys Gly Gly Val Phe Lys Ala Thr Ala Gly Leu Tyr Glu Gln Phe 35 40 45 Gly Glu Glu Arg Val Met Asp Thr Pro Leu Ala Glu Ser Ala Ile Ala 50 55 60 Gly Val Gly Ile Gly Ala Ala Met Tyr Gly Met Arg Pro Ile Ala Glu 65 70 75 80 Met Gln Phe Ala Asp Phe Ile Met Pro Ala Val Asn Gln Ile Ile Ser 85 90 95 Glu Ala Ala Lys Ile Arg Tyr Arg Ser Asn Asn Asp Trp Ser Cys Pro 100 105 110 Ile Val Val Arg Ala Pro Tyr Gly Gly Gly Val His Gly Ala Leu Tyr 115 120 125 His Ser Gln Ser Val Glu Ala Ile Phe Ala Asn Gln Pro Gly Leu Lys 130 135 140 Ile Val Met Pro Ser Thr Pro Tyr Asp Ala Lys Gly Leu Leu Lys Ala 145 150 155 160 Ala Val Arg Asp Glu Asp Pro Val Leu Phe Phe Glu His Lys Arg Ala 165 170 175 Tyr Arg Leu Ile Lys Gly Glu Val Pro Ala Asp Asp Tyr Val Leu Pro 180 185 190 Ile Gly Lys Ala Asp Val Lys Arg Glu Gly Asp Asp Ile Thr Val Ile 195 200 205 Thr Tyr Gly Leu Cys Val His Phe Ala Leu Gln Ala Ala Glu Arg Leu 210 215 220 Glu Lys Asp Gly Ile Ser Ala His Val Val Asp Leu Arg Thr Val Tyr 225 230 235 240 Pro Leu Asp Lys Glu Ala Ile Ile Glu Ala Ala Ser Lys Thr Gly Lys 245 250 255 Val Leu Leu Val Thr Glu Asp Thr Lys Glu Gly Ser Ile Met Ser Glu 260 265 270 Val Ala Ala Ile Ile Ser Glu His Cys Leu Phe Asp Leu Asp Ala Pro 275 280 285 Ile Lys Arg Leu Ala Gly Pro Asp Ile Pro Ala Met ProTyr Ala Pro 290 295 300 Thr Met Glu Lys Tyr Phe Met Val Asn Pro Asp Lys Val Glu Ala Ala 305 310 315 320 Met Arg Glu Leu Ala Glu Phe 325 <210>209 <211>1275 <212>DNA <213>枯草芽孢杆菌 <400>209 atggcaattg aacaaatgac gatgccgcag cttggagaaa gcgtaacaga ggggacgatc 60 agcaaatggc ttgtcgcccc cggtgataaa gtgaacaaat acgatccgat cgcggaagtc 120 atgacagata aggtaaatgc agaggttccg tcttctttta ctggtacgat aacagagctt 180 gtgggagaag aaggccaaac cctgcaagtc ggagaaatga tttgcaaaat tgaaacagaa 240 ggcgcgaatc cggctgaaca aaaacaagaa cagccagcag catcagaagc cgctgagaac 300 cctgttgcaa aaagtgctgg agcagccgat cagcccaata aaaagcgcta ctcgccagct 360 gttctccgtt tggccggaga gcacggcatt gacctcgatc aagtgacagg aactggtgcc 420 ggcgggcgca tcacacgaaa agatattcag cgcttaattg aaacaggcgg cgtgcaagaa 480 cagaatcctg aggagctgaa aacagcagct cctgcaccga agtctgcatc aaaacctgag 540 ccaaaagaag agacgtcata tcctgcgtct gcagccggtg ataaagaaat ccctgtcaca 600 ggtgtaagaa aagcaattgc ttccaatatg aagcgaagca aaacagaaat tccgcatgct 660 tggacgatga tggaagtcga cgtcacaaat atggttgcat atcgcaacag tataaaagat 720 tcttttaaga agacagaagg ctttaattta acgttcttcg ccttttttgt aaaagcggtc 780 gctcaggcgt taaaagaatt cccgcaaatg aatagcatgt gggcggggga caaaattatt 840 cagaaaaagg atatcaatat ttcaattgca gttgccacag aggattcttt atttgttccg 900 gtgattaaaa acgctgatga aaaaacaatt aaaggcattg cgaaagacat taccggccta 960 gctaaaaaag taagagacgg aaaactcact gcagatgaca tgcagggagg cacgtttacc 1020 gtcaacaaca caggttcgtt cgggtctgtt cagtcgatgg gcattatcaa ctaccctcag 1080 gctgcgattc ttcaagtaga atccatcgtc aaacgcccgg ttgtcatgga caatggcatg 1140 attgctgtca gagacatggt taatctgtgc ctgtcattag atcacagagt gcttgacggt 1200 ctcgtgtgcg gacgattcct cggacgagtg aaacaaattt tagaatcgat tgacgagaag 1260 acatctgttt actaa 1275 <210>210 <211>424 <212>PRT <213>枯草芽孢杆菌 <400>210 Met Ala Ile Glu Gln Met Thr Met Pro Gln Leu Gly Glu Ser Val Thr 1 5 10 15 Glu Gly Thr Ile Ser Lys Trp Leu Val Ala Pro Gly Asp Lys Val Asn 20 25 30 Lys Tyr Asp Pro Ile Ala Glu Val Met Thr Asp Lys Val Asn Ala Glu 35 40 45 Val Pro Ser Ser Phe Thr Gly Thr Ile Thr Glu Leu Val Gly Glu Glu 50 55 60 Gly Gln Thr Leu Gln Val Gly Glu Met Ile Cys Lys Ile Glu Thr Glu 65 70 75 80 Gly Ala Asn Pro Ala Glu Gln Lys Gln Glu Gln Pro Ala Ala Ser Glu 85 90 95 Ala Ala Glu Asn Pro Val Ala Lys Ser Ala Gly Ala Ala Asp Gln Pro 100 105 110 Asn Lys Lys Arg Tyr Ser Pro Ala Val Leu Arg Leu Ala Gly Glu His 115 120 125 Gly Ile Asp Leu Asp Gln Val Thr Gly Thr Gly Ala Gly Gly Arg Ile 130 135 140 Thr Arg Lys Asp Ile Gln Arg Leu Ile Glu Thr Gly Gly Val Gln Glu 145 150 155 160 Gln Asn Pro Glu Glu Leu Lys Thr Ala Ala Pro Ala Pro Lys Ser Ala 165 170 175 Ser Lys Pro Glu Pro Lys Glu Glu Thr Ser Tyr Pro Ala Ser Ala Ala 180 185 190 Gly Asp Lys Glu Ile Pro Val Thr Gly Val Arg Lys Ala Ile Ala Ser 195 200 205 Asn Met Lys Arg Ser Lys Thr Glu Ile Pro His Ala Trp Thr Met Met 210 215 220 Glu Val Asp Val Thr Asn Met Val Ala Tyr Arg Asn Ser Ile Lys Asp 225 230 235 240 Ser Phe Lys Lys Thr Glu Gly Phe Asn Leu Thr Phe Phe Ala Phe Phe 245 250 255 Val Lys Ala Val Ala Gln Ala Leu Lys Glu Phe Pro Gln Met Asn Ser 260 265 270 Met Trp Ala Gly Asp Lys Ile Ile Gln Lys Lys Asp Ile Asn Ile Ser 275 280 285 Ile Ala Val Ala Thr Glu Asp Ser Leu Phe Val Pro Val Ile Lys Asn 290 295 300 Ala Asp Glu Lys Thr Ile Lys Gly Ile Ala Lys Asp Ile Thr Gly Leu 305 310 315 320 Ala Lys Lys Val Arg Asp Gly Lys Leu Thr Ala Asp Asp Met Gln Gly 325 330 335 Gly Thr Phe Thr Val Asn Asn Thr Gly Ser Phe Gly Ser Val Gln Ser 340 345 350 Met Gly Ile Ile Asn Tyr Pro Gln Ala Ala Ile Leu Gln Val Glu Ser 355 360 365 Ile Val Lys Arg Pro Val Val Met Asp Asn Gly Met Ile Ala Val Arg 370 375 380 Asp Met Val Asn Leu Cys Leu Ser Leu Asp His Arg Val Leu Asp Gly 385 390 395 400 Leu Val Cys Gly Arg Phe Leu Gly Arg Val Lys Gln Ile Leu Glu Ser 405 410 415 Ile Asp Glu Lys Thr Ser Val Tyr 420 <210>211 <211>1374 <212>DNA <213>枯草芽孢杆菌 <400>211 atggcaactg agtatgacgt agtcattctg ggcggcggta ccggcggtta tgttgcggcc 60 atcagagccg ctcagctcgg cttaaaaaca gccgttgtgg aaaaggaaaa actcggggga 120 acatgtctgc ataaaggctg tatcccgagt aaagcgctgc ttagaagcgc agaggtatac 180 cggacagctc gtgaagccga tcaattcgga gtggaaacgg ctggcgtgtc cctcaacttt 240 gaaaaagtgc agcagcgtaa gcaagccgtt gttgataagc ttgcagcggg tgtaaatcat 300 ttaatgaaaa aaggaaaaat tgacgtgtac accggatatg gacgtatcct tggaccgtca 360 atcttctctc cgctgccggg aacaatttct gttgagcggg gaaatggcga agaaaatgac 420 atgctgatcc cgaaacaagt gatcattgca acaggatcaa gaccgagaat gcttccgggt 480 cttgaagtgg acggtaagtc tgtactgact tcagatgagg cgctccaaat ggaggagctg 540 ccacagtcaa tcatcattgt cggcggaggg gttatcggta tcgaatgggc gtctatgctt 600 catgattttg gcgttaaggt aacggttatt gaatacgcgg atcgcatatt gccgactgaa 660 gatctagaga tttcaaaaga aatggaaagt cttcttaaga aaaaaggcat ccagttcata 720 acaggggcaa aagtgctgcc tgacacaatg acaaaaacat cagacgatat cagcatacaa 780 gcggaaaaag acggagaaac cgttacctat tctgctgaga aaatgcttgt ttccatcggc 840 agacaggcaa atatcgaagg catcggccta gagaacaccg atattgttac tgaaaatggc 900 atgatttcag tcaatgaaag ctgccaaacg aaggaatctc atatttatgc aatcggagac 960 gtaatcggtg gcctgcagtt agctcacgtt gcttcacatg agggaattat tgctgttgag 1020 cattttgcag gtctcaatcc gcatccgctt gatccgacgc ttgtgccgaa gtgcatttac 1080 tcaagccctg aagctgccag tgtcggctta accgaagacg aagcaaaggc gaacgggcat 1140 aatgtcaaaa tcggcaagtt cccatttatg gcgattggaa aagcgcttgt atacggtgaa 1200 agcgacggtt ttgtcaaaat cgtggctgac cgagatacag atgatattct cggcgttcat 1260 atgattggcc cgcatgtcac cgacatgatt tctgaagcgg gtcttgccaa agtgctggac 1320 gcaacaccgt gggaggtcgg gcaaacgatt tcacccgcat ccaacgcttt ctga 1374 <210>212 <211>457 <212>PRT <213>枯草芽孢杆菌 <400>212 Met Ala Thr Glu Tyr Asp Val Val Ile Leu Gly Gly Gly Thr Gly Gly 1 5 10 15 Tyr Val Ala Ala Ile Arg Ala Ala Gln Leu Gly Leu Lys Thr Ala Val 20 25 30 Val Glu Lys Glu Lys Leu Gly Gly Thr Cys Leu His Lys Gly Cys Ile 35 40 45 Pro Ser Lys Ala Leu Leu Arg Ser Ala Glu Val Tyr Arg Thr Ala Arg 50 55 60 Glu Ala Asp Gln Phe Gly Val Glu Thr Ala Gly Val Ser Leu Asn Phe 65 70 75 80 Glu Lys Val Gln Gln Arg Lys Gln Ala Val Val Asp Lys Leu Ala Ala 85 90 95 Gly Val Asn His Leu Met Lys Lys Gly Lys Ile Asp Val Tyr Thr Gly 100 105 110 Tyr Gly Arg Ile Leu Gly Pro Ser Ile Phe Ser Pro Leu Pro Gly Thr 115 120 125 Ile Ser Val Glu Arg Gly Asn Gly Glu Glu Asn Asp Met Leu Ile Pro 130 135 140 Lys Gln Val Ile Ile Ala Thr Gly Ser Arg Pro Arg Met Leu Pro Gly 145 150 155c160 Leu Glu Val Asp Gly Lys Ser Val Leu Thr Ser Asp Glu Ala Leu Gln 165 170 175 Met Glu Glu Leu Pro Gln Ser Ile Ile Ile Val Gly Gly Gly Val Ile 180 185 190 Gly Ile Glu Trp Ala Ser Met Leu His Asp Phe Gly Val Lys Val Thr 195 200 205 Val Ile Glu Tyr Ala Asp Arg Ile Leu Pro Thr Glu Asp Leu Glu Ile 210 215 220 Ser Lys Glu Met Glu Ser Leu Leu Lys Lys Lys Gly Ile Gln Phe Ile 225 230 235 240 Thr Gly Ala Lys Val Leu Pro Asp Thr Met Thr Lys Thr Ser Asp Asp 245 250 255 Ile Ser Ile Gln Ala Glu Lys Asp Gly Glu Thr Val Thr Tyr Ser Ala 260 265 270 Glu Lys Met Leu Val Ser Ile Gly Arg Gln Ala Asn Ile Glu Gly Ile 275 280 285 Gly Leu Glu Asn Thr Asp Ile Val Thr Glu Asn Gly Met Ile Ser Val 290 295 300 Asn Glu Ser Cys Gln Thr Lys Glu Ser His Ile Tyr Ala Ile Gly Asp 305 310 315 320 Val Ile Gly Gly Leu Gln Leu Ala His Val Ala Ser His Glu Gly Ile 325 330 335 Ile Ala Val Glu His Phe Ala Gly Leu Asn Pro His Pro Leu Asp Pro 340 345 350 Thr Leu Val Pro Lys Cys Ile Tyr Ser Ser Pro Glu Ala Ala Ser Val 355 360 365 Gly Leu Thr Glu Asp Glu Ala Lys Ala Asn Gly His Asn Val Lys Ile 370 375 380 Gly Lys Phe Pro Phe Met Ala Ile Gly Lys Ala Leu Val Tyr Gly Glu 385 390 395 400 Ser Asp Gly Phe Val Lys Ile Val Ala Asp Arg Asp Thr Asp Asp Ile 405 410 415 Leu Gly Val His Met Ile Gly Pro His Val Thr Asp Met Ile Ser Glu 420 425 430 Ala Gly Leu Ala Lys Val Leu Asp Ala Thr Pro Trp Glu Val Gly Gln 435 440 445 Thr Ile Ser Pro Ala Ser Asn Ala Phe 450 455 <210>213 <211>1233 <212>DNA <213>恶臭假单胞菌 <400>213 atgaacgagt acgcccccct gcgtttgcat gtgcccgagc ccaccggccg gccaggctgc 60 cagaccgatt tttcctacct gcgcctgaac gatgcaggtc aagcccgtaa accccctgtc 120 gatgtcgacg ctgccgacac cgccgacctg tcctacagcc tggtccgcgt gctcgacgag 180 caaggcgacg cccaaggccc gtgggctgaa gacatcgacc cgcagatcct gcgccaaggc 240 atgcgcgcca tgctcaagac gcggatcttc gacagccgca tggtggttgc ccagcgccag 300 aagaagatgt ccttctacat gcagagcctg ggcgaagaag ccatcggcag cggccaggcg 360 ctggcgctta accgcaccga catgtgcttc cccacctacc gtcagcaaag catcctgatg 420 gcccgcgacg tgtcgctggt ggagatgatc tgccagttgc tgtccaacga acgcgacccc 480 ctcaagggcc gccagctgcc gatcatgtac tcggtacgcg aggccggctt cttcaccatc 540 agcggcaacc tggcgaccca gttcgtgcag gcggtcggct gggccatggc ctcggcgatc 600 aagggcgata ccaagattgc ctcggcctgg atcggcgacg gcgccactgc cgaatcggac 660 ttccacaccg ccctcacctt tgcccacgtt taccgcgccc cggtgatcct caacgtggtc 720 aacaaccagt gggccatctc aaccttccag gccatcgccg gtggcgagtc gaccaccttc 780 gccggccgtg gcgtgggctg cggcatcgct tcgctgcggg tggacggcaa cgacttcgtc 840 gccgtttacg ccgcttcgcg ctgggctgcc gaacgtgccc gccgtggttt gggcccgagc 900 ctgatcgagt gggtcaccta ccgtgccggc ccgcactcga cctcggacga cccgtccaag 960 taccgccctg ccgatgactg gagccacttc ccgctgggtg acccgatcgc ccgcctgaag 1020 cagcacctga tcaagatcgg ccactggtcc gaagaagaac accaggccac cacggccgag 1080 ttcgaagcgg ccgtgattgc tgcgcaaaaa gaagccgagc agtacggcac cctggccaac 1140 ggtcacatcc cgagcgccgc ctcgatgttc gaggacgtgt acaaggagat gcccgaccac 1200 ctgcgccgcc aacgccagga actgggggtt tga 1233 <210>214 <211>410 <212>PRT <213>恶臭假单胞菌 <400>214 Met Asn Glu Tyr Ala Pro Leu Arg Leu His Val Pro Glu Pro Thr Gly 1 5 10 15 Arg Pro Gly Cys Gln Thr Asp Phe Ser Tyr Leu Arg Leu Asn Asp Ala 20 25 30 Gly Gln Ala Arg Lys Pro Pro Val Asp Val Asp Ala Ala Asp Thr Ala 35 40 45 Asp Leu Ser Tyr Ser Leu Val Arg Val Leu Asp Glu Gln Gly Asp Ala 50 55 60 Gln Gly Pro Trp Ala Glu Asp Ile Asp Pro Gln Ile Leu Arg Gln Gly 65 70 75 80 Met Arg Ala Met Leu Lys Thr Arg Ile Phe Asp Ser Arg Met Val Val 85 90 95 Ala Gln Arg Gln Lys Lys Met Ser Phe Tyr Met Gln Ser Leu Gly Glu 100 105 110 Glu Ala Ile Gly Ser Gly Gln Ala Leu Ala Leu Asn Arg Thr Asp Met 115 120 125 Cys Phe Pro Thr Tyr Arg Gln Gln Ser Ile Leu Met Ala Arg Asp Val 130 135 140 Ser Leu Val Glu Met Ile Cys Gln Leu Leu Ser Asn Glu Arg Asp Pro 145 150 155 160 Leu Lys Gly Arg Gln Leu Pro Ile Met Tyr Ser Val Arg Glu Ala Gly 165 170 175 Phe Phe Thr Ile Ser Gly Asn Leu Ala Thr Gln Phe Val Gln Ala Val 180 185 190 Gly Trp Ala Met Ala Ser Ala Ile Lys Gly Asp Thr Lys Ile Ala Ser 195 200 205 Ala Trp Ile Gly Asp Gly Ala Thr Ala Glu Ser Asp Phe His Thr Ala 210 215 220 Leu Thr Phe Ala His Val Tyr Arg Ala Pro Val Ile Leu Asn Val Val 225 230 235 240 Asn Asn Gln Trp Ala Ile Ser Thr Phe Gln Ala Ile Ala Gly Gly Glu 245 250 255 Ser Thr Thr Phe Ala Gly Arg Gly Val Gly Cys Gly Ile Ala Ser Leu 260 265 270 Arg Val Asp Gly Asn Asp Phe Val Ala Val Tyr Ala Ala Ser Arg Trp 275 280 285 Ala Ala Glu Arg Ala Arg Arg Gly Leu Gly Pro Ser Leu Ile Glu Trp 290 295 300 Val Thr Tyr Arg Ala Gly Pro His Ser Thr Ser Asp Asp Pro Ser Lys 305 310 315 320 Tyr Arg Pro Ala Asp Asp Trp Ser His Phe Pro Leu Gly Asp Pro Ile 325 330 335 Ala Arg Leu Lys Gln His Leu Ile Lys Ile Gly His Trp Ser Glu Glu 340 345 350 Glu His Gln Ala Thr Thr Ala Glu Phe Glu Ala Ala Val Ile Ala Ala 355 360 365 Gln Lys Glu Ala Glu Gln Tyr Gly Thr Leu Ala Asn Gly His Ile Pro 370 375 380 Ser Ala Ala Ser Met Phe Glu Asp Val Tyr Lys Glu Met Pro Asp His 385 390 395 400 Leu Arg Arg Gln Arg Gln Glu Leu Gly Val 405 410 <210>215 <211>1059 <212>DNA <213>恶臭假单胞菌 <400>215 atgaacgacc acaacaacag catcaacccg gaaaccgcca tggccaccac taccatgacc 60 atgatccagg ccctgcgctc ggccatggat gtcatgcttg agcgcgacga caatgtggtg 120 gtgtacggcc aggacgtcgg ctacttcggc ggcgtgttcc gctgcaccga aggcctgcag 180 accaagtacg gcaagtcccg cgtgttcgac gcgcccatct ctgaaagcgg catcgtcggc 240 accgccgtgg gcatgggtgc ctacggcctg cgcccggtgg tggaaatcca gttcgctgac 300 tacttctacc cggcctccga ccagatcgtt tctgaaatgg cccgcctgcg ctaccgttcg 360 gccggcgagt tcatcgcccc gctgaccctg cgtatgccct gcggtggcgg tatctatggc 420 ggccagacac acagccagag cccggaagcg atgttcactc aggtgtgcgg cctgcgcacc 480 gtaatgccat ccaacccgta cgacgccaaa ggcctgctga ttgcctcgat cgaatgcgac 540 gacccggtga tcttcctgga gcccaagcgc ctgtacaacg gcccgttcga cggccaccat 600 gaccgcccgg ttacgccgtg gtcgaaacac ccgcacagcg ccgtgcccga tggctactac 660 accgtgccac tggacaaggc cgccatcacc cgccccggca atgacgtgag cgtgctcacc 720 tatggcacca ccgtgtacgt ggcccaggtg gccgccgaag aaagtggcgt ggatgccgaa 780 gtgatcgacc tgcgcagcct gtggccgcta gacctggaca ccatcgtcga gtcggtgaaa 840 aagaccggcc gttgcgtggt agtacacgag gccacccgta cttgtggctt tggcgcagaa 900 ctggtgtcgc tggtgcagga gcactgcttc caccacctgg aggcgccgat cgagcgcgtc 960 accggttggg acacccccta ccctcacgcg caggaatggg cttacttccc agggccttcg 1020 cgggtaggtg cggcattgaa aaaggtcatg gaggtctga 1059 <210>216 <211>352 <212>PRT <213>恶臭假单胞菌 <400>216 Met Asn Asp His Asn Asn Ser Ile Asn Pro Glu Thr Ala Met Ala Thr 1 5 10 15 Thr Thr Met Thr Met Ile Gln Ala Leu Arg Ser Ala Met Asp Val Met 20 25 30 Leu Glu Arg Asp Asp Asn Val Val Val Tyr Gly Gln Asp Val Gly Tyr 35 40 45 Phe Gly Gly Val Phe Arg Cys Thr Glu Gly Leu Gln Thr Lys Tyr Gly 50 55 60 Lys Ser Arg Val Phe Asp Ala Pro Ile Ser Glu Ser Gly Ile Val Gly 65 70 75 80 Thr Ala Val Gly Met Gly Ala Tyr Gly Leu Arg Pro Val Val Glu Ile 85 90 95 Gln Phe Ala Asp Tyr Phe Tyr Pro Ala Ser Asp Gln Ile Val Ser Glu 100 105 110 Met Ala Arg Leu Arg Tyr Arg Ser Ala Gly Glu Phe Ile Ala Pro Leu 115 120 125 Thr Leu Arg Met Pro Cys Gly Gly Gly Ile Tyr Gly Gly Gln Thr His 130 135 140 Ser Gln Ser Pro Glu Ala Met Phe Thr Gln Val Cys Gly Leu Arg Thr 145 150 155 160 Val Met Pro Ser Asn Pro Tyr Asp Ala Lys Gly Leu Leu Ile Ala Ser 165 170 175 Ile Glu Cys Asp Asp Pro Val Ile Phe Leu Glu Pro Lys Arg Leu Tyr 180 185 190 Asn Gly Pro Phe Asp Gly His His Asp Arg Pro Val Thr Pro Trp Ser 195 200 205 Lys His Pro His Ser Ala Val Pro Asp Gly Tyr Tyr Thr Val Pro Leu 210 215 220 Asp Lys Ala Ala Ile Thr Arg Pro Gly Asn Asp Val Ser Val Leu Thr 225 230 235 240 Tyr Gly Thr Thr Val Tyr Val Ala Gln Val Ala Ala Glu Glu Ser Gly 245 250 255 Val Asp Ala Glu Val Ile Asp Leu Arg Ser Leu Trp Pro Leu Asp Leu 260 265 270 Asp Thr Ile Val Glu Ser Val Lys Lys Thr Gly Arg Cys Val Val Val 275 280 285 His Glu Ala Thr Arg Thr Cys Gly Phe Gly Ala Glu Leu Val Ser Leu 290 295 300 Val Gln Glu His Cys Phe His His Leu Glu Ala Pro Ile Glu Arg Val 305 310 315 320 Thr Gly Trp Asp Thr Pro Tyr Pro His Ala Gln Glu Trp Ala Tyr Phe 325 330 335 Pro Gly Pro Ser Arg Val Gly Ala Ala Leu Lys Lys Val Met Glu Val 340 345 350 <210>217 <211>1272 <212>DNA <213>恶臭假单胞菌 <400>217 atgggcacgc acgtcatcaa gatgccggac attggcgaag gcatcgcgca ggtcgaattg 60 gtggaatggt tcgtcaaggt gggcgacatc atcgccgagg accaagtggt agccgacgtc 120 atgaccgaca aggccaccgt ggaaatcccg tcgccggtca gcggcaaggt gctggccctg 180 ggtggccagc caggtgaagt gatggcggtc ggcagtgagc tgatccgcat cgaagtggaa 240 ggcagcggca accatgtgga tgtgccgcaa gccaagccgg ccgaagtgcc tgcggcaccg 300 gtagccgcta aacctgaacc acagaaagac gttaaaccgg cggcgtacca ggcgtcagcc 360 agccacgagg cagcgcccat cgtgccgcgc cagccgggcg acaagccgct ggcctcgccg 420 gcggtgcgca aacgcgccct cgatgccggc atcgaattgc gttatgtgca cggcagcggc 480 ccggccgggc gcatcctgca cgaagacctc gacgcgttca tgagcaaacc gcaaagcgct 540 gccgggcaaa cccccaatgg ctatgccagg cgcaccgaca gcgagcaggt gccggtgatc 600 ggcctgcgcc gcaagatcgc ccagcgcatg caggacgcca agcgccgggt cgcgcacttc 660 agctatgtgg aagaaatcga cgtcaccgcc ctggaagccc tgcgccagca gctcaacagc 720 aagcacggcg acagccgcgg caagctgaca ctgctgccgt tcctggtgcg cgccctggtc 780 gtggcactgc gtgacttccc gcagataaac gccacctacg atgacgaagc gcagatcatc 840 acccgccatg gcgcggtgca tgtgggcatc gccacccaag gtgacaacgg cctgatggta 900 cccgtgctgc gccacgccga agcgggcagc ctgtgggcca atgccggtga gatttcacgc 960 ctggccaacg ctgcgcgcaa caacaaggcc agccgcgaag agctgtccgg ttcgaccatt 1020 accctgacca gcctcggcgc cctgggcggc atcgtcagca cgccggtggt caacaccccg 1080 gaagtggcga tcgtcggtgt caaccgcatg gttgagcggc ccgtggtgat cgacggccag 1140 atcgtcgtgc gcaagatgat gaacctgtcc agctcgttcg accaccgcgt ggtcgatggc 1200 atggacgccg ccctgttcat ccaggccgtg cgtggcctgc tcgaacaacc cgcctgcctg 1260 ttcgtggagt ga 1272 <210>218 <211>423 <212>PRT <213>恶臭假单胞菌 <400>218 Met Gly Thr His Val Ile Lys Met Pro Asp Ile Gly Glu Gly Ile Ala 1 5 10 15 Gln Val Glu Leu Val Glu Trp Phe Val Lys Val Gly Asp Ile Ile Ala 20 25 30 Glu Asp Gln Val Val Ala Asp Val Met Thr Asp Lys Ala Thr Val Glu 35 40 45 Ile Pro Ser Pro Val Ser Gly Lys Val Leu Ala Leu Gly Gly Gln Pro 50 55 60 Gly Glu Val Met Ala Val Gly Ser Glu Leu Ile Arg Ile Glu Val Glu 65 70 75 80 Gly Ser Gly Asn His Val Asp Val Pro Gln Ala Lys Pro Ala Glu Val 85 90 95 Pro Ala Ala Pro Val Ala Ala Lys Pro Glu Pro Gln Lys Asp Val Lys 100 105 110 Pro Ala Ala Tyr Gln Ala Ser Ala Ser His Glu Ala Ala Pro Ile Val 115 120 125 Pro Arg Gln Pro Gly Asp Lys Pro Leu Ala Ser Pro Ala Val Arg Lys 130 135 140 Arg Ala Leu Asp Ala Gly Ile Glu Leu Arg Tyr Val His Gly Ser Gly 145 150 155 160 Pro Ala Gly Arg Ile Leu His Glu Asp Leu Asp Ala Phe Met Ser Lys 165 170 175 Pro Gln Ser Ala Ala Gly Gln Thr Pro Asn Gly Tyr Ala Arg Arg Thr 180 185 190 Asp Ser Glu Gln Val Pro Val Ile Gly Leu Arg Arg Lys Ile Ala Gln 195 200 205 Arg Met Gln Asp Ala Lys Arg Arg Val Ala His Phe Ser Tyr Val Glu 210 215 220 Glu Ile Asp Val Thr Ala Leu Glu Ala Leu Arg Gln Gln Leu Asn Ser 225 230 235 240 Lys His Gly Asp Ser Arg Gly Lys Leu Thr Leu Leu Pro Phe Leu Val 245 250 255 Arg Ala Leu Val Val Ala Leu Arg Asp Phe Pro Gln Ile Asn Ala Thr 260 265 270 Tyr Asp Asp Glu Ala Gln Ile Ile Thr Arg His Gly Ala Val His Val 275 280 285 Gly Ile Ala Thr Gln Gly Asp Asn Gly Leu Met Val Pro Val Leu Arg 290 295 300 His Ala Glu Ala Gly Ser Leu Trp Ala Asn Ala Gly Glu Ile Ser Arg 305 310 315 320 Leu Ala Asn Ala Ala Arg Asn Asn Lys Ala Ser Arg Glu Glu Leu Ser 325 330 335 Gly Ser Thr Ile Thr Leu Thr Ser Leu Gly Ala Leu Gly Gly Ile Val 340 345 350 Ser Thr Pro Val Val Asn Thr Pro Glu Val Ala Ile Val Gly Val Asn 355 360 365 Arg Met Val Glu Arg Pro Val Val Ile Asp Gly Gln Ile Val Val Arg 370 375 380 Lys Met Met Asn Leu Ser Ser Ser Phe Asp His Arg Val Val Asp Gly 385 390 395 400 Met Asp Ala Ala Leu Phe Ile Gln Ala Val Arg Gly Leu Leu Glu Gln 405 410 415 Pro Ala Cys Leu Phe Val Glu 420 <210>219 <211>1380 <212>DNA <213>恶臭假单胞菌 <400>219 atgcaacaga ctatccagac aaccctgttg atcatcggcg gcggccctgg cggctatgtg 60 gcggccatcc gcgccgggca actgggcatc cctaccgtgc tggtggaagg ccaggcgctg 120 ggcggtacct gcctgaacat cggctgcatt ccgtccaagg cgctgatcca tgtggccgag 180 cagttccacc aggcctcgcg ctttaccgaa ccctcgccgc tgggcatcag cgtggcttcg 240 ccacgcctgg acatcggcca gagcgtggcc tggaaagacg gcatcgtcga tcgcctgacc 300 actggtgtcg ccgccctgct gaaaaagcac ggggtgaagg tggtgcacgg ctgggccaag 360 gtgcttgatg gcaagcaggt cgaggtggat ggccagcgca tccagtgcga gcacctgttg 420 ctggccacgg gctccagcag tgtcgaactg ccgatgctgc cgttgggtgg gccggtgatt 480 tcctcgaccg aggccctggc accgaaagcc ctgccgcaac acctggtggt ggtgggcggt 540 ggctacatcg gcctggagct gggtatcgcc taccgcaagc tcggcgcgca ggtcagcgtg 600 gtggaagcgc gcgagcgcat cctgccgact tacgacagcg aactgaccgc cccggtggcc 660 gagtcgctga aaaagctggg tatcgccctg caccttggcc acagcgtcga aggttacgaa 720 aatggctgcc tgctggccaa cgatggcaag ggcggacaac tgcgcctgga agccgaccgg 780 gtgctggtgg ccgtgggccg ccgcccacgc accaagggct tcaacctgga atgcctggac 840 ctgaagatga atggtgccgc gattgccatc gacgagcgct gccagaccag catgcacaac 900 gtctgggcca tcggcgacgt ggccggcgaa ccgatgctgg cgcaccgggc catggcccag 960 ggcgagatgg tggccgagat catcgccggc aaggcacgcc gcttcgaacc cgctgcgata 1020 gccgccgtgt gcttcaccga cccggaagtg gtcgtggtcg gcaagacgcc ggaacaggcc 1080 agtcagcaag gcctggactg catcgtcgcg cagttcccgt tcgccgccaa cggccgggcc 1140 atgagcctgg agtcgaaaag cggtttcgtg cgcgtggtcg cgcggcgtga caaccacctg 1200 atcctgggct ggcaagcggt tggcgtggcg gtttccgagc tgtccacggc gtttgcccag 1260 tcgctggaga tgggtgcctg cctggaggat gtggccggta ccatccatgc ccacccgacc 1320 ctgggtgaag cggtacagga agcggcactg cgtgccctgg gccacgccct gcatatctga 1380 <210>220 <211>459 <212>PRT <213>恶臭假单胞菌 <400>220 Met Gln Gln Thr Ile Gln Thr Thr Leu Leu Ile Ile Gly Gly Gly Pro 1 5 10 15 Gly Gly Tyr Val Ala Ala Ile Arg Ala Gly Gln Leu Gly Ile Pro Thr 20 25 30 Val Leu Val Glu Gly Gln Ala Leu Gly Gly Thr Cys Leu Asn Ile Gly 35 40 45 Cys Ile Pro Ser Lys Ala Leu Ile His Val Ala Glu Gln Phe His Gln 50 55 60 Ala Ser Arg Phe Thr Glu Pro Ser Pro Leu Gly Ile Ser Val Ala Ser 65 70 75 80 Pro Arg Leu Asp Ile Gly Gln Ser Val Ala Trp Lys Asp Gly Ile Val 85 90 95 Asp Arg Leu Thr Thr Gly Val Ala Ala Leu Leu Lys Lys His Gly Val 100 105 110 Lys Val Val His Gly Trp Ala Lys Val Leu Asp Gly Lys Gln Val Glu 115 120 125 Val Asp Gly Gln Arg Ile Gln Cys Glu His Leu Leu Leu Ala Thr Gly 130 135 140 Ser Ser Ser Val Glu Leu Pro Met Leu Pro Leu Gly Gly Pro Val Ile 145 150 155 160 Ser Ser Thr Glu Ala Leu Ala Pro Lys Ala Leu Pro Gln His Leu Val 165 170 175 Val Val Gly Gly Gly Tyr Ile Gly Leu Glu Leu Gly Ile Ala Tyr Arg 180 185 190 Lys Leu Gly Ala Gln Val Ser Val Val Glu Ala Arg Glu Arg Ile Leu 195 200 205 Pro Thr Tyr Asp Ser Glu Leu Thr Ala Pro Val Ala Glu Ser Leu Lys 210 215 220 Lys Leu Gly Ile Ala Leu His Leu Gly His Ser Val Glu Gly Tyr Glu 225 230 235 240 Asn Gly Cys Leu Leu Ala Asn Asp Gly Lys Gly Gly Gln Leu Arg Leu 245 250 255 Glu Ala Asp Arg Val Leu Val Ala Val Gly Arg Arg Pro Arg Thr Lys 260 265 270 Gly Phe Asn Leu Glu Cys Leu Asp Leu Lys Met Asn Gly Ala Ala Ile 275 280 285 Ala Ile Asp Glu Arg Cys Gln Thr Ser Met His Asn Val Trp Ala Ile 290 295 300 Gly Asp Val Ala Gly Glu Pro Met Leu Ala His Arg Ala Met Ala Gln 305 310 315 320 Gly Glu Met Val Ala Glu Ile Ile Ala Gly Lys Ala Arg Arg Phe Glu 325 330 335 Pro Ala Ala Ile Ala Ala Val Cys Phe Thr Asp Pro Glu Val Val Val 340 345 350 Val Gly Lys Thr Pro Glu Gln Ala Ser Gln Gln Gly Leu Asp Cys Ile 355 360 365 Val Ala Gln Phe Pro Phe Ala Ala Asn Gly Arg Ala Met Ser Leu Glu 370 375 380 Ser Lys Ser Gly Phe Val Arg Val Val Ala Arg Arg Asp Asn His Leu 385 390 395 400 Ile Leu Gly Trp Gln Ala Val Gly Val Ala Val Ser Glu Leu Ser Thr 405 410 415 Ala Phe Ala Gln Ser Leu Glu Met Gly Ala Cys Leu Glu Asp Val Ala 420 425 430 Gly Thr Ile His Ala His Pro Thr Leu Gly Glu Ala Val Gln Glu Ala 435 440 445 Ala Leu Arg Ala Leu Gly His Ala Leu His Ile 450 455 <210>221 <211>1407 <212>DNA <213>拜氏梭菌 <400>221 atgaataaag acacactaat acctacaact aaagatttaa aattaaaaac aaatgttgaa 60 aacattaatt taaagaacta caaggataat tcttcatgtt tcggagtatt cgaaaatgtt 120 gaaaatgcta taaacagcgc tgtacacgcg caaaagatat tatcccttca ttatacaaaa 180 gaacaaagag aaaaaatcat aactgagata agaaaggccg cattagaaaa taaagaggtt 240 ttagctacca tgattctgga agaaacacat atgggaaggt atgaagataa aatattaaag 300 catgaattag tagctaaata tactcctggt acagaagatt taactactac tgcttggtca 360 ggtgataatg gtcttacagt tgtagaaatg tctccatatg gcgttatagg tgcaataact 420 ccttctacga atccaactga aactgtaata tgtaatagca tcggcatgat agctgctgga 480 aatgctgtag tatttaacgg acacccaggc gctaaaaaat gtgttgcttt tgctattgaa 540 atgataaata aagcaattat ttcatgtggc ggtcctgaga atttagtaac aactataaaa 600 aatccaacta tggaatccct agatgcaatt attaagcatc ctttaataaa acttctttgc 660 ggaactggag gtccaggaat ggtaaaaacc ctcttaaatt ctggcaagaa agctataggt 720 gctggtgctg gaaatccacc agttattgta gatgataccg ctgatataga aaaggctggt 780 aagagtatca ttgaaggctg ttcttttgat aataatttac cttgtattgc agaaaaagaa 840 gtatttgttt ttgagaatgt tgcagatgat ttaatatcta acatgctaaa aaataatgct 900 gtaattataa atgaagatca agtatcaaaa ttaatagatt tagtattaca aaaaaataat 960 gaaactcaag aatactttat aaacaaaaaa tgggtaggaa aagatgcaaa attattctca 1020 gatgaaatag atgttgagtc tccttcaaat attaaatgca tagtctgcga agtaaatgca 1080 aatcatccat ttgtcatgac agaactcatg atgccaatat taccaattgt aagagttaaa 1140 gatatagatg aagctgttaa atatacaaag atagcagaac aaaatagaaa acatagtgcc 1200 tatatttatt ctaaaaatat agacaaccta aatagatttg aaagagaaat tgatactact 1260 atttttgtaa agaatgctaa atcttttgct ggtgttggtt atgaagctga aggatttaca 1320 actttcacta ttgctggatc tactggtgaa ggcataacct ctgcaagaaa ttttacaaga 1380 caaagaagat gtgtacttgc cggctaa 1407 <210>222 <211>468 <212>PRT <213>拜氏梭菌 <400>222 Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Leu Lys 1 5 10 15 Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser 20 25 30 Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Asn Ser Ala Val 35 40 45 His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu 50 55 60 Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val 65 70 75 80 Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp 85 90 95 Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu 100 105 110 Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val 115 120 125 Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn 130 135 140 Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly 145 150 155 160 Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala 165 170 175 Phe Ala Ile Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro 180 185 190 Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp 195 200 205 Ala Ile Ile Lys His Pro Leu Ile Lys Leu Leu Cys Gly Thr Gly Gly 210 215 220 Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly 225 230 235 240 Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile 245 250 255 Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn 260 265 270 Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala 275 280 285 Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn 290 295 300 Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn 305 310 315 320 Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala 325 330 335 Lys Leu Phe Ser Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Ile Lys 340 345 350 Cys Ile Val Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu 355 360 365 Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu 370 375 380 Ala Val Lys Tyr Thr Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala 385 390 395 400 Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu 405 410 415 Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val 420 425 430 Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr 435 440 445 Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys 450 455 460 Val Leu Ala Gly 465 <210>223 <211>2589 <212>DNA <213>丙醇丁酮梭菌 <400>223 atgaaagtca caacagtaaa ggaattagat gaaaaactca aggtaattaa agaagctcaa 60 aaaaaattct cttgttactc gcaagaaatg gttgatgaaa tctttagaaa tgcagcaatg 120 gcagcaatcg acgcaaggat agagctagca aaagcagctg ttttggaaac cggtatgggc 180 ttagttgaag acaaggttat aaaaaatcat tttgcaggcg aatacatcta taacaaatat 240 aaggatgaaa aaacctgcgg tataattgaa cgaaatgaac cctacggaat tacaaaaata 300 gcagaaccta taggagttgt agctgctata atccctgtaa caaaccccac atcaacaaca 360 atatttaaat ccttaatatc ccttaaaact agaaatggaa ttttcttttc gcctcaccca 420 agggcaaaaa aatccacaat actagcagct aaaacaatac ttgatgcagc cgttaagagt 480 ggtgccccgg aaaatataat aggttggata gatgaacctt caattgaact aactcaatat 540 ttaatgcaaa aagcagatat aacccttgca actggtggtc cctcactagt taaatctgct 600 tattcttccg gaaaaccagc aataggtgtt ggtccgggta acaccccagt aataattgat 660 gaatctgctc atataaaaat ggcagtaagt tcaattatat tatccaaaac ctatgataat 720 ggtgttatat gtgcttctga acaatctgta atagtcttaa aatccatata taacaaggta 780 aaagatgagt tccaagaaag aggagcttat ataataaaga aaaacgaatt ggataaagtc 840 cgtgaagtga tttttaaaga tggatccgta aaccctaaaa tagtcggaca gtcagcttat 900 actatagcag ctatggctgg cataaaagta cctaaaacca caagaatatt aataggagaa 960 gttacctcct taggtgaaga agaacctttt gcccacgaaa aactatctcc tgttttggct 1020 atgtatgagg ctgacaattt tgatgatgct ttaaaaaaag cagtaactct aataaactta 1080 ggaggcctcg gccatacctc aggaatatat gcagatgaaa taaaagcacg agataaaata 1140 gatagattta gtagtgccat gaaaaccgta agaacctttg taaatatccc aacctcacaa 1200 ggtgcaagtg gagatctata taattttaga ataccacctt ctttcacgct tggctgcgga 1260 ttttggggag gaaattctgt ttccgagaat gttggtccaa aacatctttt gaatattaaa 1320 accgtagctg aaaggagaga aaacatgctt tggtttagag ttccacataa agtatatttt 1380 aagttcggtt gtcttcaatt tgctttaaaa gatttaaaag atctaaagaa aaaaagagcc 1440 tttatagtta ctgatagtga cccctataat ttaaactatg ttgattcaat aataaaaata 1500 cttgagcacc tagatattga ttttaaagta tttaataagg ttggaagaga agctgatctt 1560 aaaaccataa aaaaagcaac tgaagaaatg tcctccttta tgccagacac tataatagct 1620 ttaggtggta cccctgaaat gagctctgca aagctaatgt gggtactata tgaacatcca 1680 gaagtaaaat ttgaagatct tgcaataaaa tttatggaca taagaaagag aatatatact 1740 ttcccaaaac tcggtaaaaa ggctatgtta gttgcaatta caacttctgc tggttccggt 1800 tctgaggtta ctccttttgc tttagtaact gacaataaca ctggaaataa gtacatgtta 1860 gcagattatg aaatgacacc aaatatggca attgtagatg cagaacttat gatgaaaatg 1920 ccaaagggat taaccgctta ttcaggtata gatgcactag taaatagtat agaagcatac 1980 acatccgtat atgcttcaga atacacaaac ggactagcac tagaggcaat acgattaata 2040 tttaaatatt tgcctgaggc ttacaaaaac ggaagaacca atgaaaaagc aagagagaaa 2100 atggctcacg cttcaactat ggcaggtatg gcatccgcta atgcatttct aggtctatgt 2160 cattccatgg caataaaatt aagttcagaa cacaatattc ctagtggcat tgccaatgca 2220 ttactaatag aagaagtaat aaaatttaac gcagttgata atcctgtaaa acaagcccct 2280 tgcccacaat ataagtatcc aaacaccata tttagatatg ctcgaattgc agattatata 2340 aagcttggag gaaatactga tgaggaaaag gtagatctct taattaacaa aatacatgaa 2400 ctaaaaaaag ctttaaatat accaacttca ataaaggatg caggtgtttt ggaggaaaac 2460 ttctattcct cccttgatag aatatctgaa cttgcactag atgatcaatg cacaggcgct 2520 aatcctagat ttcctcttac aagtgagata aaagaaatgt atataaattg ttttaaaaaa 2580 caaccttaa 2589 <210>224 <211>862 <212>PRT <213>丙醇丁酮梭菌 <400>224 Met Lys Val Thr Thr Val Lys Glu Leu Asp Glu Lys Leu Lys Val Ile 1 5 10 15 Lys Glu Ala Gln Lys Lys Phe Ser Cys Tyr Ser Gln Glu Met Val Asp 20 25 30 Glu Ile Phe Arg Asn Ala Ala Met Ala Ala Ile Asp Ala Arg Ile Glu 35 40 45 Leu Ala Lys Ala Ala Val Leu Glu Thr Gly Met Gly Leu Val Glu Asp 50 55 60 Lys Val Ile Lys Asn His Phe Ala Gly Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asp Glu Lys Thr Cys Gly Ile Ile Glu Arg Asn Glu Pro Tyr Gly 85 90 95 Ile Thr Lys Ile Ala Glu Pro Ile Gly Val Val Ala Ala Ile Ile Pro 100 105 110 Val Thr Asn Pro Thr Ser Thr Thr Ile Phe Lys Ser Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Gly Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys 130 135 140 Ser Thr Ile Leu Ala Ala Lys Thr Ile Leu Asp Ala Ala Val Lys Ser 145 150 155 160 Gly Ala Pro Glu Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu 165 170 175 Leu Thr Gln Tyr Leu Met Gln Lys Ala Asp Ile Thr Leu Ala Thr Gly 180 185 190 Gly Pro Ser Leu Val Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205 Gly Val Gly Pro Gly Asn Thr Pro Val Ile Ile Asp Glu Ser Ala His 210 215 220 Ile Lys Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Val Ile Val Leu Lys Ser Ile 245 250 255 Tyr Asn Lys Val Lys Asp Glu Phe Gln Glu Arg Gly Ala Tyr Ile Ile 260 265 270 Lys Lys Asn Glu Leu Asp Lys Val Arg Glu Val Ile Phe Lys Asp Gly 275 280 285 Ser Val Asn Pro Lys Ile Val Gly Gln Ser Ala Tyr Thr Ile Ala Ala 290 295 300 Met Ala Gly Ile Lys Val Pro Lys Thr Thr Arg Ile Leu Ile Gly Glu 305 310 315 320 Val Thr Ser Leu Gly Glu Glu Glu Pro Phe Ala His Glu Lys Leu Ser 325 330 335 Pro Val Leu Ala Met Tyr Glu Ala Asp Asn Phe Asp Asp Ala Leu Lys 340 345 350 Lys Ala Val Thr Leu Ile Asn Leu Gly Gly Leu Gly His Thr Ser Gly 355 360 365 Ile Tyr Ala Asp Glu Ile Lys Ala Arg Asp Lys Ile Asp Arg Phe Ser 370 375 380 Ser Ala Met Lys Thr Val Arg Thr Phe Val Asn Ile Pro Thr Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Arg Ile Pro Pro Ser Phe Thr 405 410 415 Leu Gly Cys Gly Phe Trp Gly Gly Asn Ser Val Ser Glu Asn Val Gly 420 425 430 Pro Lys His Leu Leu Asn Ile Lys Thr Val Ala Glu Arg Arg Glu Asn 435 440 445 Met Leu Trp Phe Arg Val Pro His Lys Val Tyr Phe Lys Phe Gly Cys 450 455 460 Leu Gln Phe Ala Leu Lys Asp Leu Lys Asp Leu Lys Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Ser Asp Pro Tyr Asn Leu Asn Tyr Val Asp Ser 485 490 495 Ile Ile Lys Ile Leu Glu His Leu Asp Ile Asp Phe Lys Val Phe Asn 500 505 510 Lys Val Gly Arg Glu Ala Asp Leu Lys Thr Ile Lys Lys Ala Thr Glu 515 520 525 Glu Met Ser Ser Phe Met Pro Asp Thr Ile Ile Ala Leu Gly Gly Thr 530 535 540 Pro Glu Met Ser Ser Ala Lys Leu Met Trp Val Leu Tyr Glu His Pro 545 550 555 560 Glu Val Lys Phe Glu Asp Leu Ala Ile Lys Phe Met Asp Ile Arg Lys 565 570 575 Arg Ile Tyr Thr Phe Pro Lys Leu Gly Lys Lys Ala Met Leu Val Ala 580 585 590 Ile Thr Thr Ser Ala Gly Ser Gly Ser Glu Val Thr Pro Phe Ala Leu 595 600 605 Val Thr Asp Asn Asn Thr Gly Asn Lys Tyr Met Leu Ala Asp Tyr Glu 610 615 620 Met Thr Pro Asn Met Ala Ile Val Asp Ala Glu Leu Met Met Lys Met 625 630 635 640 Pro Lys Gly Leu Thr Ala Tyr Ser Gly Ile Asp Ala Leu Val Asn Ser 645 650 655 Ile Glu Ala Tyr Thr Ser Val Tyr Ala Ser Glu Tyr Thr Asn Gly Leu 660 665 670 Ala Leu Glu Ala Ile Arg Leu Ile Phe Lys Tyr Leu Pro Glu Ala Tyr 675 680 685 Lys Asn Gly Arg Thr Asn Glu Lys Ala Arg Glu Lys Met Ala His Ala 690 695 700 Ser Thr Met Ala Gly Met Ala Ser Ala Asn Ala Phe Leu Gly Leu Cys 705 710 715 720 His Ser Met Ala Ile Lys Leu Ser Ser Glu His Asn Ile Pro Ser Gly 725 730 735 Ile Ala Asn Ala Leu Leu Ile Glu Glu Val Ile Lys Phe Asn Ala Val 740 745 750 Asp Asn Pro Val Lys Gln Ala Pro Cys Pro Gln Tyr Lys Tyr Pro Asn 755 760 765 Thr Ile Phe Arg Tyr Ala Arg Ile Ala Asp Tyr Ile Lys Leu Gly Gly 770 775 780 Asn Thr Asp Glu Glu Lys Val Asp Leu Leu Ile Asn Lys Ile His Glu 785 790 795 800 Leu Lys Lys Ala Leu Asn Ile Pro Thr Ser Ile Lys Asp Ala Gly Val 805 810 815 Leu Glu Glu Asn Phe Tyr Ser Ser Leu Asp Arg Ile Ser Glu Leu Ala 820 825 830 Leu Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Phe Pro Leu Thr Ser 835 840 845 Glu Ile Lys Glu Met Tyr Ile Asn Cys Phe Lys Lys Gln Pro 850 855 860 <210>225 <211>2577 <212>DNA <213>丙醇丁酮梭菌 <400>225 atgaaagtta caaatcaaaa agaactaaaa caaaagctaa atgaattgag agaagcgcaa 60 aagaagtttg caacctatac tcaagagcaa gttgataaaa tttttaaaca atgtgccata 120 gccgcagcta aagaaagaat aaacttagct aaattagcag tagaagaaac aggaataggt 180 cttgtagaag ataaaattat aaaaaatcat tttgcagcag aatatatata caataaatat 240 aaaaatgaaa aaacttgtgg cataatagac catgacgatt ctttaggcat aacaaaggtt 300 gctgaaccaa ttggaattgt tgcagccata gttcctacta ctaatccaac ttccacagca 360 attttcaaat cattaatttc tttaaaaaca agaaacgcaa tattcttttc accacatcca 420 cgtgcaaaaa aatctacaat tgctgcagca aaattaattt tagatgcagc tgttaaagca 480 ggagcaccta aaaatataat aggctggata gatgagccat caatagaact ttctcaagat 540 ttgatgagtg aagctgatat aatattagca acaggaggtc cttcaatggt taaagcggcc 600 tattcatctg gaaaacctgc aattggtgtt ggagcaggaa atacaccagc aataatagat 660 gagagtgcag atatagatat ggcagtaagc tccataattt tatcaaagac ttatgacaat 720 ggagtaatat gcgcttctga acaatcaata ttagttatga attcaatata cgaaaaagtt 780 aaagaggaat ttgtaaaacg aggatcatat atactcaatc aaaatgaaat agctaaaata 840 aaagaaacta tgtttaaaaa tggagctatt aatgctgaca tagttggaaa atctgcttat 900 ataattgcta aaatggcagg aattgaagtt cctcaaacta caaagatact tataggcgaa 960 gtacaatctg ttgaaaaaag cgagctgttc tcacatgaaa aactatcacc agtacttgca 1020 atgtataaag ttaaggattt tgatgaagct ctaaaaaagg cacaaaggct aatagaatta 1080 ggtggaagtg gacacacgtc atctttatat atagattcac aaaacaataa ggataaagtt 1140 aaagaatttg gattagcaat gaaaacttca aggacattta ttaacatgcc ttcttcacag 1200 ggagcaagcg gagatttata caattttgcg atagcaccat catttactct tggatgcggc 1260 acttggggag gaaactctgt atcgcaaaat gtagagccta aacatttatt aaatattaaa 1320 agtgttgctg aaagaaggga aaatatgctt tggtttaaag tgccacaaaa aatatatttt 1380 aaatatggat gtcttagatt tgcattaaaa gaattaaaag atatgaataa gaaaagagcc 1440 tttatagtaa cagataaaga tctttttaaa cttggatatg ttaataaaat aacaaaggta 1500 ctagatgaga tagatattaa atacagtata tttacagata ttaaatctga tccaactatt 1560 gattcagtaa aaaaaggtgc taaagaaatg cttaactttg aacctgatac tataatctct 1620 attggtggtg gatcgccaat ggatgcagca aaggttatgc acttgttata tgaatatcca 1680 gaagcagaaa ttgaaaatct agctataaac tttatggata taagaaagag aatatgcaat 1740 ttccctaaat taggtacaaa ggcgatttca gtagctattc ctacaactgc tggtaccggt 1800 tcagaggcaa caccttttgc agttataact aatgatgaaa caggaatgaa atacccttta 1860 acttcttatg aattgacccc aaacatggca ataatagata ctgaattaat gttaaatatg 1920 cctagaaaat taacagcagc aactggaata gatgcattag ttcatgctat agaagcatat 1980 gtttcggtta tggctacgga ttatactgat gaattagcct taagagcaat aaaaatgata 2040 tttaaatatt tgcctagagc ctataaaaat gggactaacg acattgaagc aagagaaaaa 2100 atggcacatg cctctaatat tgcggggatg gcatttgcaa atgctttctt aggtgtatgc 2160 cattcaatgg ctcataaact tggggcaatg catcacgttc cacatggaat tgcttgtgct 2220 gtattaatag aagaagttat taaatataac gctacagact gtccaacaaa gcaaacagca 2280 ttccctcaat ataaatctcc taatgctaag agaaaatatg ctgaaattgc agagtatttg 2340 aatttaaagg gtactagcga taccgaaaag gtaacagcct taatagaagc tatttcaaag 2400 ttaaagatag atttgagtat tccacaaaat ataagtgccg ctggaataaa taaaaaagat 2460 ttttataata cgctagataa aatgtcagag cttgcttttg atgaccaatg tacaacagct 2520 aatcctaggt atccacttat aagtgaactt aaggatatct atataaaatc attttaa 2577 <210>226 <211>800 <212>PRT <213>丙醇丁酮梭菌 <400>226 Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu 1 5 10 15 Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp 20 25 30 Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn 35 40 45 Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp 50 55 60 Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly 85 90 95 Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro 100 105 110 Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys 130 135 140 Ser Thr Ile Ala Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala 145 150 155 160 Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu 165 170 175 Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly 180 185 190 Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205 Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp 210 215 220 Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile 245 250 255 Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu 260 265 270 Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly 275 280 285 Ala Ile Asn Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys 290 295 300 Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu 305 310 315 320 Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser 325 330 335 Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys 340 345 350 Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser 355 360 365 Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly 370 375 380 Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr 405 410 415 Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu 420 425 430 Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn 435 440 445 Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys 450 455 460 Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys 485 490 495 Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr 500 505 510 Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys 515 520 525 Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly 530 535 540 Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro 545 550 555 560 Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys 565 570 575 Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala 580 585 590 Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val 595 600 605 Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu 610 615 620 Leu Thr Pro Asn Met Ala Ile Ile Asp Thr Glu Leu Met Leu Asn Met 625 630 635 640 Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala 645 650 655 Ile Glu Ala Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu 660 665 670 Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr 675 680 685 Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala 690 695 700 Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys 705 710 715 720 His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly 725 730 735 Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr 740 745 750 Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn 755 760 765 Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly 770 775 780 Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys 785 790 795 800 <210>227 <211>924 <212>DNA <213>恶臭假单胞菌 <400>227 atgagcaaga aactcaaggc ggccatcata ggccccggca atatcggtac cgatctggtg 60 atgaagatgc tccgttccga gtggattgag ccggtgtgga tggtcggcat cgaccccaac 120 tccgacggcc tcaaacgcgc ccgcgatttc ggcatgaaga ccacagccga aggcgtcgac 180 ggcctgctcc cgcacgtgct ggacgacgac atccgcatcg ccttcgacgc cacctcggcc 240 tatgtgcatg ccgagaatag ccgcaagctc aacgcgcttg gcgtgctgat ggtcgacctg 300 accccggcgg ccatcggccc ctactgcgtg ccgccggtca acctcaagca gcatgtcggc 360 cgcctggaaa tgaacgtcaa catggtcacc tgcggcggcc aggccaccat ccccatggtc 420 gccgcggtgt cccgcgtgca gccggtggcc tacgccgaga tcgtcgccac cgtctcctcg 480 cgctcggtcg gcccgggcac gcgcaagaac atcgacgagt tcacccgcac caccgccggc 540 gccatcgagc aggtcggcgg cgccagggaa ggcaaggcga tcatcgtcat caacccggcc 600 gagccgccgc tgatgatgcg cgacaccatc cactgcctga ccgacagcga gccggaccag 660 gctgcgatca ccgcttcggt tcacgcgatg atcgccgagg tgcagaaata cgtgcccggc 720 taccgcctga agaacggccc ggtgttcgac ggcaaccgcg tgtcgatctt catggaagtc 780 gaaggcctgg gcgactacct gcccaagtac gccggcaacc tcgacatcat gaccgccgcc 840 gcgctgcgta ccggcgagat gttcgccgag gaaatcgccg ccggcaccat tcaactgccg 900 cgtcgcgaca tcgcgctggc ttga 924 <210>228 <211>307 <212>PRT <213>恶臭假单胞菌 <400>228 Met Ser Lys Lys Leu Lys Ala Ala Ile Ile Gly Pro Gly Asn Ile Gly 1 5 10 15 Thr Asp Leu Val Met Lys Met Leu Arg Ser Glu Trp Ile Glu Pro Val 20 25 30 Trp Met Val Gly Ile Asp Pro Asn Ser Asp Gly Leu Lys Arg Ala Arg 35 40 45 Asp Phe Gly Met Lys Thr Thr Ala Glu Gly Val Asp Gly Leu Leu Pro 50 55 60 His Val Leu Asp Asp Asp Ile Arg Ile Ala Phe Asp Ala Thr Ser Ala 65 70 75 80 Tyr Val His Ala Glu Asn Ser Arg Lys Leu Asn Ala Leu Gly Val Leu 85 90 95 Met Val Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val Pro Pro 100 105 110 Val Asn Leu Lys Gln His Val Gly Arg Leu Glu Met Asn Val Asn Met 115 120 125 Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val Ser 130 135 140 Arg Val Gln Pro Val Ala Tyr Ala Glu Ile Val Ala Thr Val Ser Ser 145 150 155 160 Arg Ser Val Gly Pro Gly Thr Arg Lys Asn Ile Asp Glu Phe Thr Arg 165 170 175 Thr Thr Ala Gly Ala Ile Glu Gln Val Gly Gly Ala Arg Glu Gly Lys 180 185 190 Ala Ile Ile Val Ile Asn Pro Ala Glu Pro Pro Leu Met Met Arg Asp 195 200 205 Thr Ile His Cys Leu Thr Asp Ser Glu Pro Asp Gln Ala Ala Ile Thr 210 215 220 Ala Ser Val His Ala Met Ile Ala Glu Val Gln Lys Tyr Val Pro Gly 225 230 235 240 Tyr Arg Leu Lys Asn Gly Pro Val Phe Asp Gly Asn Arg Val Ser Ile 245 250 255 Phe Met Glu Val Glu Gly Leu Gly Asp Tyr Leu Pro Lys Tyr Ala Gly 260 265 270 Asn Leu Asp Ile Met Thr Ala Ala Ala Leu Arg Thr Gly Glu Met Phe 275 280 285 Ala Glu Glu Ile Ala Ala Gly Thr Ile Gln Leu Pro Arg Arg Asp Ile 290 295 300 Ala Leu Ala 305 <210>229 <211>924 <212>DNA <213>嗜热栖热菌 <400>229 atgtccgaaa gggttaaggt agccatcctg ggctccggca acatcgggac ggacctgatg 60 tacaagctcc tgaagaaccc gggccacatg gagcttgtgg cggtggtggg gatagacccc 120 aagtccgagg gcctggcccg ggcgcgggcc ttagggttag aggcgagcca cgaagggatc 180 gcctacatcc tggagaggcc ggagatcaag atcgtctttg acgccaccag cgccaaggcc 240 cacgtgcgcc acgccaagct cctgagggag gcggggaaga tcgccataga cctcacgccg 300 gcggcccggg gcccttacgt ggtgcccccg gtgaacctga aggaacacct ggacaaggac 360 aacgtgaacc tcatcacctg cggggggcag gccaccatcc ccctggtcta cgcggtgcac 420 cgggtggccc ccgtgctcta cgcggagatg gtctccacgg tggcctcccg ctccgcgggc 480 cccggcaccc ggcagaacat cgacgagttc accttcacca ccgcccgggg cctggaggcc 540 atcggggggg ccaagaaggg gaaggccatc atcatcctga acccggcgga accccccatc 600 ctcatgacca acaccgtgcg ctgcatcccc gaggacgagg gctttgaccg ggaggccgtg 660 gtggcgagcg tccgggccat ggagcgggag gtccaggcct acgtgcccgg ctaccgcctg 720 aaggcggacc cggtgtttga gaggcttccc accccctggg gggagcgcac cgtggtctcc 780 atgctcctgg aggtggaggg ggcgggggac tatttgccca aatacgccgg caacctggac 840 atcatgacgg cttctgcccg gagggtgggg gaggtcttcg cccagcacct cctggggaag 900 cccgtggagg aggtggtggc gtga 924 <210>230 <211>307 <212>PRT <213>嗜热栖热菌 <400>230 Met Ser Glu Arg Val Lys Val Ala Ile Leu Gly Ser Gly Asn Ile Gly 1 5 10 15 Thr Asp Leu Met Tyr Lys Leu Leu Lys Asn Pro Gly His Met Glu Leu 20 25 30 Val Ala Val Val Gly Ile Asp Pro Lys Ser Glu Gly Leu Ala Arg Ala 35 40 45 Arg Ala Leu Gly Leu Glu Ala Ser His Glu Gly Ile Ala Tyr Ile Leu 50 55 60 Glu Arg Pro Glu Ile Lys Ile Val Phe Asp Ala Thr Ser Ala Lys Ala 65 70 75 80 His Val Arg His Ala Lys Leu Leu Arg Glu Ala Gly Lys Ile Ala Ile 85 90 95 Asp Leu Thr Pro Ala Ala Arg Gly Pro Tyr Val Val Pro Pro Val Asn 100 105 110 Leu Lys Glu His Leu Asp Lys Asp Asn Val Asn Leu Ile Thr Cys Gly 115 120 125 Gly Gln Ala Thr Ile Pro Leu Val Tyr Ala Val His Arg Val Ala Pro 130 135 140 Val Leu Tyr Ala Glu Met Val Ser Thr Val Ala Ser Arg Ser Ala Gly 145 150 155 160 Pro Gly Thr Arg Gln Asn Ile Asp Glu Phe Thr Phe Thr Thr Ala Arg 165 170 175 Gly Leu Glu Ala Ile Gly Gly Ala Lys Lys Gly Lys Ala Ile Ile Ile 180 185 190 Leu Asn Pro Ala Glu Pro Pro Ile Leu Met Thr Asn Thr Val Arg Cys 195 200 205 Ile Pro Glu Asp Glu Gly Phe Asp Arg Glu Ala Val Val Ala Ser Val 210 215 220 Arg Ala Met Glu Arg Glu Val Gln Ala Tyr Val Pro Gly Tyr Arg Leu 225 230 235 240 Lys Ala Asp Pro Val Phe Glu Arg Leu Pro Thr Pro Trp Gly Glu Arg 245 250 255 Thr Val Val Ser Met Leu Leu Glu Val Glu Gly Ala Gly Asp Tyr Leu 260 265 270 Pro Lys Tyr Ala Gly Asn Leu Asp Ile Met Thr Ala Ser Ala Arg Arg 275 280 285 Val Gly Glu Val Phe Ala Gln His Leu Leu Gly Lys Pro Val Glu Glu 290 295 300 Val Val Ala 305 <210>231 <211>1254 <212>DNA <213>大肠杆菌 <400>231 atgacattct ccctttttgg tgacaaattt acccgccact ccggcattac gctgttgatg 60 gaagatctga acgacggttt acgcacgcct ggcgcgatta tgctcggcgg cggtaatccg 120 gcgcagatcc cggaaatgca ggactacttc cagacgctac tgaccgacat gctggaaagt 180 ggcaaagcga ctgatgcact gtgtaactac gacggtccac aggggaaaac ggagctactc 240 acactgcttg ccggaatgct gcgcgagaag ttgggttggg atatcgaacc acagaatatt 300 gcactaacaa acggcagcca gagcgcgttt ttctacttat ttaacctgtt tgccggacgc 360 cgtgccgatg gtcgggtcaa aaaagtgctg ttcccgcttg caccggaata cattggctat 420 gctgacgccg gactggaaga agatctgttt gtctctgcgc gtccgaatat tgaactgctg 480 ccggaaggcc agtttaaata ccacgtcgat tttgagcatc tgcatattgg cgaagaaacc 540 gggatgattt gcgtctcccg gccgacgaat ccaacaggca atgtgattac tgacgaagag 600 ttgctgaagc ttgacgcgct ggcgaatcaa cacggcattc cgctggtgat tgataacgct 660 tatggcgtcc cgttcccggg tatcatcttc agtgaagcgc gcccgctatg gaatccgaat 720 atcgtgctgt gcatgagtct ttccaagctg ggtctacctg gctcccgctg cggcattatc 780 atcgccaatg aaaaaatcat caccgccatc accaatatga acggcattat cagcctggca 840 cctggcggta ttggtccggc gatgatgtgt gaaatgatta agcgtaacga tctgctgcgc 900 ctgtctgaaa cagtcatcaa accgttttac taccagcgtg ttcaggaaac tatcgccatc 960 attcgccgct atttaccgga aaatcgctgc ctgattcata aaccggaagg agccattttc 1020 ctctggctat ggtttaagga tttgcccatt acgaccaagc agctctatca gcgcctgaaa 1080 gcacgcggcg tgctgatggt gccggggcac aacttcttcc cagggctgga taaaccgtgg 1140 ccgcatacgc atcaatgtat gcgcatgaac tacgtaccag agccggagaa aattgaggcg 1200 ggggtgaaga ttctggcgga agagatagaa agagcctggg ctgaaagtca ctaa 1254 <210>232 <211>417 <212>PRT <213>大肠杆菌 <400>232 Met Thr Phe Ser Leu Phe Gly Asp Lys Phe Thr Arg His Ser Gly Ile 1 5 10 15 Thr Leu Leu Met Glu Asp Leu Asn Asp Gly Leu Arg Thr Pro Gly Ala 20 25 30 Ile Met Leu Gly Gly Gly Asn Pro Ala Gln Ile Pro Glu Met Gln Asp 35 40 45 Tyr Phe Gln Thr Leu Leu Thr Asp Met Leu Glu Ser Gly Lys Ala Thr 50 55 60 Asp Ala Leu Cys Asn Tyr Asp Gly Pro Gln Gly Lys Thr Glu Leu Leu 65 70 75 80 Thr Leu Leu Ala Gly Met Leu Arg Glu Lys Leu Gly Trp Asp Ile Glu 85 90 95 Pro Gln Asn Ile Ala Leu Thr Asn Gly Ser Gln Ser Ala Phe Phe Tyr 100 105 110 Leu Phe Asn Leu Phe Ala Gly Arg Arg Ala Asp Gly Arg Val Lys Lys 115 120 125 Val Leu Phe Pro Leu Ala Pro Glu Tyr Ile Gly Tyr Ala Asp Ala Gly 130 135 140 Leu Glu Glu Asp Leu Phe Val Ser Ala Arg Pro Asn Ile Glu Leu Leu 145 150 155 160 Pro Glu Gly Gln Phe Lys Tyr His Val Asp Phe Glu His Leu His Ile 165 170 175 Gly Glu Glu Thr Gly Met Ile Cys Val Ser Arg Pro Thr Asn Pro Thr 180 185 190 Gly Asn Val Ile Thr Asp Glu Glu Leu Leu Lys Leu Asp Ala Leu Ala 195 200 205 Asn Gln His Gly Ile Pro Leu Val Ile Asp Asn Ala Tyr Gly Val Pro 210 215 220 Phe Pro Gly Ile Ile Phe Ser Glu Ala Arg Pro Leu Trp Asn Pro Asn 225 230 235 240 Ile Val Leu Cys Met Ser Leu Ser Lys Leu Gly Leu Pro Gly Ser Arg 245 250 255 Cys Gly Ile Ile Ile Ala Asn Glu Lys Ile Ile Thr Ala Ile Thr Asn 260 265 270 Met Asn Gly Ile Ile Ser Leu Ala Pro Gly Gly Ile Gly Pro Ala Met 275 280 285 Met Cys Glu Met Ile Lys Arg Asn Asp Leu Leu Arg Leu Ser Glu Thr 290 295 300 Val Ile Lys Pro Phe Tyr Tyr Gln Arg Val Gln Glu Thr Ile Ala Ile 305 310 315 320 Ile Arg Arg Tyr Leu Pro Glu Asn Arg Cys Leu Ile His Lys Pro Glu 325 330 335 Gly Ala Ile Phe Leu Trp Leu Trp Phe Lys Asp Leu Pro Ile Thr Thr 340 345 350 Lys Gln Leu Tyr Gln Arg Leu Lys Ala Arg Gly Val Leu Met Val Pro 355 360 365 Gly His Asn Phe Phe Pro Gly Leu Asp Lys Pro Trp Pro His Thr His 370 375 380 Gln Gys Met Arg Met Asn Tyr Val Pro Glu Pro Glu Lys Ile Glu Ala 385 390 395 400 Gly Val Lys Ile Leu Ala Glu Glu Ile Glu Arg Ala Trp Ala Glu Ser 405 410 415 His <210>233 <211>1278 <212>DNA <213>地衣芽孢杆菌 <400>233 ttataagtat tcaacctgtt tctcatatac acccttcgca attttagcta aaacatcgat 60 tccccttata atatcttcat ccgccgcggt taggctgatt cgtatacact ggtgtgaatg 120 cgccaggcgc cgggattgac ggtgaaagaa agatgatccg ggaacgataa tgactccatc 180 cgctttcata tactcataca gcgctgcatc ggtcaccggc aggtcttcaa accacagcca 240 tccgaaaagc gatccttccc cttgatgcag ataccatttg atgtcttcag gcatcttgca 300 taaaagcgtt tccttgagca gcatgaattt attgcggtaa tatggcctga cttcattcag 360 cgacacgtcg gcgaggcgcc cgtcattcaa tactgatgca gccatatact gccccagcct 420 tgaagaatgg atcgccgcat tcgactgaaa agcttccatt gcctgaatat accgggacgg 480 cccgatggcg attccgatcc tttcgccagg caggccggct tttgaaaggc tcatacagtg 540 aatgatctgc tcgttgaaaa tcggttccat gtcgataaag tgaatcgccg gaaaaggcgg 600 agcatatgcg gaatcaatga acagcggaac attcgcttct cggcatgcgt ctgaaatgaa 660 tgctacatct tctttaggca agatgtttcc gcaaggattg ttcgggcgcg atagcaagac 720 agcaccgatg cgcatcctct ctaaaaaccc cttacggtcg agctcatatc gaaacgtatg 780 atcatccaat ttcgatatga gcggagggat cccctcaatc atctcccgct ccagtgccgc 840 cccgctgtat cccgaatagt caggcagcat cgggatcaag gcttttttca tcacagatcc 900 gcttcccatt ccgcaaaacg aattgatcgc cagaaaaaac agctgctggc ttccggctgt 960 aatcaacacg ttctcttttc gaatgccggc gctataccgc tctgaaaaga agcggacaac 1020 acttgcaatc agttcatcgg ttccatagct cgatccgtat tggccgatca ccgaagaaaa 1080 cctgtcatcg tcaaggagat cggcaagagc cgacttccac atggctgaca cgccgggcaa 1140 aatcatcgga ttgcccgcac ttaaattaat gtatgaccgt tcaccgccgg ccaggacttc 1200 ctgaatatcg ctcatcacag ccctgacccc tgttttctca atcattttct ctccgatttt 1260 gcttaatggc ggcttcac 1278 <210>234 <211>425 <212>PRT <213>地衣芽孢杆菌 <400>234 Met Lys Pro Pro Leu Ser Lys Ile Gly Glu Lys Met Ile Glu Lys Thr 1 5 10 15 Gly Val Arg Ala Val Met Ser Asp Ile Gln Glu Val Leu Ala Gly Gly 20 25 30 Glu Arg Ser Tyr Ile Asn Leu Ser Ala Gly Asn Pro Met Ile Leu Pro 35 40 45 Gly Val Ser Ala Met Trp Lys Ser Ala Leu Ala Asp Leu Leu Asp Asp 50 55 60 Asp Arg Phe Ser Ser Val Ile Gly Gln Tyr Gly Ser Ser Tyr Gly Thr 65 70 75 80 Asp Glu Leu Ile Ala Ser Val Val Arg Phe Phe Ser Glu Arg Tyr Ser 85 90 95 Ala Gly Ile Arg Lys Glu Asn Val Leu Ile Thr Ala Gly Ser Gln Gln 100 105 110 Leu Phe Phe Leu Ala Ile Asn Ser Phe Cys Gly Met Gly Ser Gly Ser 115 120 125 Val Met Lys Lys Ala Leu Ile Pro Met Leu Pro Asp Tyr Ser Gly Tyr 130 135 140 Ser Gly Ala Ala Leu Glu Arg Glu Met Ile Glu Gly Ile Pro Pro Leu l45 150 155 160 Ile Ser Lys Leu Asp Asp His Thr Phe Arg Tyr Glu Leu Asp Arg Lys 165 170 175 Gly Phe Leu Glu Arg Met Arg Ile Gly Ala Val Leu Leu Ser Arg Pro 180 185 190 Asn Asn Pro Cys Gly Asn Ile Leu Pro Lys Glu Asp Val Ala Phe Ile 195 200 205 Ser Asp Ala Cys Arg Glu Ala Asn Val Pro Leu Phe Ile Asp Ser Ala 210 215 220 Tyr Ala Pro Pro Phe Pro Ala Ile His Phe Ile Asp Met Glu Pro Ile 225 230 235 240 Phe Asn Glu Gln Ile Ile His Cys Met Ser Leu Ser Lys Ala Gly Leu 245 250 255 Pro Gly Glu Arg Ile Gly Ile Ala Ile Gly Pro Ser Arg Tyr Ile Gln 260 265 270 Ala Met Glu Ala Phe Gln Ser Asn Ala Ala Ile His Ser Ser Arg Leu 275 280 285 Gly Gln Tyr Met Ala Ala Ser Val Leu Asn Asp Gly Arg Leu Ala Asp 290 295 300 Val Ser Leu Asn Glu Val Arg Pro Tyr Tyr Arg Asn Lys Phe Met Leu 305 310 315 320 Leu Lys Glu Thr Leu Leu Cys Lys Met Pro Glu Asp Ile Lys Trp Tyr 325 330 335 Leu His Gln Gly Glu Gly Ser Leu Phe Gly Trp Leu Trp Phe Glu Asp 340 345 350 Leu Pro Val Thr Asp Ala Ala Leu Tyr Glu Tyr Met Lys Ala Asp Gly 355 360 365 Val Ile Ile Val Pro Gly Ser Ser Phe Phe His Arg Gln Ser Arg Arg 370 375 380 Leu Ala His Ser His Gln Cys Ile Arg Ile Ser Leu Thr Ala Ala Asp 385 390 395 400 Glu Asp Ile Ile Arg Gly Ile Asp Val Leu Ala Lys Ile Ala Lys Gly 405 410 415 Val Tyr Glu Lys Gln Val Glu Tyr Leu 420 425 <210>235 <211>930 <212>DNA <213>大肠杆菌 <400>235 atgaccacga agaaagctga ttacatttgg ttcaatgggg agatggttcg ctgggaagac 60 gcgaaggtgc atgtgatgtc gcacgcgctg cactatggca cttcggtttt tgaaggcatc 120 cgttgctacg actcgcacaa aggaccggtt gtattccgcc atcgtgagca tatgcagcgt 180 ctgcatgact ccgccaaaat ctatcgcttc ccggtttcgc agagcattga tgagctgatg 240 gaagcttgtc gtgacgtgat ccgcaaaaac aatctcacca gcgcctatat ccgtccgctg 300 atcttcgtcg gtgatgttgg catgggagta aacccgccag cgggatactc aaccgacgtg 360 attatcgctg ctttcccgtg gggagcgtat ctgggcgcag aagcgctgga gcaggggatc 420 gatgcgatgg tttcctcctg gaaccgcgca gcaccaaaca ccatcccgac ggcggcaaaa 480 gccggtggta actacctctc ttccctgctg gtgggtagcg aagcgcgccg ccacggttat 540 caggaaggta tcgcgctgga tgtgaacggt tatatctctg aaggcgcagg cgaaaacctg 600 tttgaagtga aagatggtgt gctgttcacc ccaccgttca cctcctccgc gctgccgggt 660 attacccgtg atgccatcat caaactggcg aaagagctgg gaattgaagt acgtgagcag 720 gtgctgtcgc gcgaatccct gtacctggcg gatgaagtgt ttatgtccgg tacggcggca 780 gaaatcacgc cagtgcgcag cgtagacggt attcaggttg gcgaaggccg ttgtggcccg 840 gttaccaaac gcattcagca agccttcttc ggcctcttca ctggcgaaac cgaagataaa 900 tggggctggt tagatcaagt taatcaataa 930 <210>236 <211>309 <212>PRT <213>大肠杆菌 <400>236 Met Thr Thr Lys Lys Ala Asp Tyr Ile Trp Phe Asn Gly Glu Met Val 1 5 10 15 Arg Trp Glu Asp Ala Lys Val His Val Met Ser His Ala Leu His Tyr 20 25 30 Gly Thr Ser Val Phe Glu Gly Ile Arg Cys Tyr Asp Ser His Lys Gly 35 40 45 Pro Val Val Phe Arg His Arg Glu His Met Gln Arg Leu His Asp Ser 50 55 60 Ala Lys Ile Tyr Arg Phe Pro Val Ser Gln Ser Ile Asp Glu Leu Met 65 70 75 80 Glu Ala Cys Arg Asp Val Ile Arg Lys Asn Asn Leu Thr Ser Ala Tyr 85 90 95 Ile Arg Pro Leu Ile Phe Val Gly Asp Val Gly Met Gly Val Asn Pro 100 105 110 Pro Ala Gly Tyr Ser Thr Asp Val Ile Ile Ala Ala Phe Pro Trp Gly 115 120 125 Ala Tyr Leu Gly Ala Glu Ala Leu Glu Gln Gly Ile Asp Ala Met Val 130 135 140 Ser Ser Trp Asn Arg Ala Ala Pro Asn Thr Ile Pro Thr Ala Ala Lys 145 150 155 160 Ala Gly Gly Asn Tyr Leu Ser Ser Leu Leu Val Gly Ser Glu Ala Arg 165 170 175 Arg His Gly Tyr Gln Glu Gly Ile Ala Leu Asp Val Asn Gly Tyr Ile 180 185 190 Ser Glu Gly Ala Gly Glu Asn Leu Phe Glu Val Lys Asp Gly Val Leu 195 200 205 Phe Thr Pro Pro Phe Thr Ser Ser Ala Leu Pro Gly Ile Thr Arg Asp 210 215 220 Ala Ile Ile Lys Leu Ala Lys Glu Leu Gly Ile Glu Val Arg Glu Gln 225 230 235 240 Val Leu Ser Arg Glu Ser Leu Tyr Leu Ala Asp Glu Val Phe Met Ser 245 250 255 Gly Thr Ala Ala Glu Ile Thr Pro Val Arg Ser Val Asp Gly Ile Gln 260 265 270 Val Gly Glu Gly Arg Cys Gly Pro Val Thr Lys Arg IIe Gln Gln Ala 275 280 285 Phe Phe Gly Leu Phe Thr Gly Glu Thr Glu Asp Lys Trp Gly Trp Leu 290 295 300 Asp Gln Val Asn Gln 305 <210>237 <211>1131 <212>DNA <213>酿酒酵母 <400>237 atgaccttgg cacccctaga cgcctccaaa gttaagataa ctaccacaca acatgcatct 60 aagccaaaac cgaacagtga gttagtgttt ggcaagagct tcacggacca catgttaact 120 gcggaatgga cagctgaaaa agggtggggt accccagaga ttaaacctta tcaaaatctg 180 tctttagacc cttccgcggt ggttttccat tatgcttttg agctattcga agggatgaag 240 gcttacagaa cggtggacaa caaaattaca atgtttcgtc cagatatgaa tatgaagcgc 300 atgaataagt ctgctcagag aatctgtttg ccaacgttcg acccagaaga gttgattacc 360 ctaattggga aactgatcca gcaagataag tgcttagttc ctgaaggaaa aggttactct 420 ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 480 agagccttgc tatatgtcat ttgctgccct gtgggtcctt attacaaaac tggatttaag 540 gcggtcagac tggaagccac tgattatgcc acaagagctt ggccaggagg ctgtggtgac 600 aagaaactag gtgcaaacta cgccccctgc gtcctgccac aattgcaagc tgcttcaagg 660 ggttaccaac aaaatttatg gctatttggt ccaaataaca acattactga agtcggcacc 720 atgaatgctt ttttcgtgtt taaagatagt aaaacgggca agaaggaact agttactgct 780 ccactagacg gtaccatttt ggaaggtgtt actagggatt ccattttaaa tcttgctaaa 840 gaaagactcg aaccaagtga atggaccatt agtgaacgct acttcactat aggcgaagtt 900 actgagagat ccaagaacgg tgaactactt gaagcctttg gttctggtac tgctgcgatt 960 gtttctccca ttaaggaaat cggctggaaa ggcgaacaaa ttaatattcc gttgttgccc 1020 ggcgaacaaa ccggtccatt ggccaaagaa gttgcacaat ggattaatgg aatccaatat 1080 ggcgagactg agcatggcaa ttggtcaagg gttgttactg atttgaactg a 1131 <210>238 <211>376 <212>PRT <213>酿酒酵母 <400>238 Met Thr Leu Ala Pro Leu Asp Ala Ser Lys Val Lys Ile Thr Thr Thr 1 5 l0 15 Gln His Ala Ser Lys Pro Lys Pro Asn Ser Glu Leu Val Phe Gly Lys 20 25 30 Ser Phe Thr Asp His Met Leu Thr Ala Glu Trp Thr Ala Glu Lys Gly 35 40 45 Trp Gly Thr Pro Glu Ile Lys Pro Tyr Gln Asn Leu Ser Leu Asp Pro 50 55 60 Ser Ala Val Val Phe His Tyr Ala Phe Glu Leu Phe Glu Gly Met Lys 65 70 75 80 Ala Tyr Arg Thr Val Asp Asn Lys Ile Thr Met Phe Arg Pro Asp Met 85 90 95 Asn Met Lys Arg Met Asn Lys Ser Ala Gln Arg Ile Cys Leu Pro Thr 100 105 110 Phe Asp Pro Glu Glu Leu Ile Thr Leu Ile Gly Lys Leu Ile Gln Gln 115 120 125 Asp Lys Cys Leu Val Pro Glu Gly Lys Gly Tyr Ser Leu Tyr Ile Arg 130 135 140 Pro Thr Leu Ile Gly Thr Thr Ala Gly Leu Gly Val Ser Thr Pro Asp 145 150 155 160 Arg Ala Leu Leu Tyr Val Ile Cys Cys Pro Val Gly Pro Tyr Tyr Lys 165 170 175 Thr Gly Phe Lys Ala Val Arg Leu Glu Ala Thr Asp Tyr Ala Thr Arg 180 185 190 Ala Trp Pro Gly Gly Cys Gly Asp Lys Lys Leu Gly Ala Asn Tyr Ala 195 200 205 Pro Cys Val Leu Pro Gln Leu Gln Ala Ala Ser Arg Gly Tyr Gln Gln 210 215 220 Asn Leu Trp Leu Phe Gly Pro Asn Asn Asn Ile Thr Glu Val Gly Thr 225 230 235 240 Met Asn Ala Phe Phe Val Phe Lys Asp Ser Lys Thr Gly Lys Lys Glu 245 250 255 Leu Val Thr Ala Pro Leu Asp Gly Thr Ile Leu Glu Gly Val Thr Arg 260 265 270 Asp Ser Ile Leu Asn Leu Ala Lys Glu Arg Leu Glu Pro Ser Glu Trp 275 280 285 Thr Ile Ser Glu Arg Tyr Phe Thr Ile Gly Glu Val Thr Glu Arg Ser 290 295 300 Lys Asn Gly Glu Leu Leu Glu Ala Phe Gly Ser Gly Thr Ala Ala Ile 305 310 3l5 320 Val Ser Pro Ile Lys Glu Ile Gly Trp Lys Gly Glu Gln Ile Asn Ile 325 330 335 Pro Leu Leu Pro Gly Glu Gln Thr Gly Pro Leu Ala Lys Glu Val Ala 340 345 350 Gln Trp Ile Asn Gly Ile Gln Tyr Gly Glu Thr Glu His Gly Asn Trp 355 360 365 Ser Arg Val Val Thr Asp Leu Asn 370 375 <210>239 <211>993 <212>DNA <213>嗜热碱甲烷杆菌 <400>239 tcagatgtag gtgagccatc cgaagctgtc ctctgtctct gccctgatta tcctgaagaa 60 ctcatcctgc agcagctttg taacgggacc ccttcgcccg gcacctatct ctataccatc 120 aactgatctg atgggtgtta tctctgcggc tgtacctgtg aagaaggcct catctgcgat 180 gtagagcatc tccctggtta tgggttcctc atgcacggta acaccctcgg tcctggctat 240 ctttattacg gagtcccttg ttatccccct cagaagggat gatgaaacag ggggggtgta 300 aatttcaccc tcactgacga ggaatatgtt ctccccgcta ccctcactta tgtagccatg 360 gtagtccagc attatggcct catcatagcc gtgtctcaca gcctccatct tggcaagctg 420 tgagttgagg tagttaccgc cggcctttgc catgttgggc attgtgtttg gtgccatcct 480 ccgccaggtt gaaacaccag catcgacacc aacctcaagg gcctctgcac ccagataggc 540 cccccattcc caggcagcca cagcgacgtc cactgggcag ttcaccgggt gaacacccat 600 ctcaccgtat cccctgaata ccacgggtct tatatagcac tcctcaagtc cgttctccct 660 gacggtctca actatggcat cacatatctg ctcctgggtg tagggtatgt ccatccggta 720 tatctttgca gaatcaaaaa ggcgtttaac atgctcccgc aaacggaaga tggctgaccc 780 cttactgttc ctgtagcacc ttattccctc aaagacagat gatccataat gcacaacatg 840 tgagagtacg tggacggtgg cttcttccca ttcaaccatt tcaccgttta accatatctt 900 tccactggct tcgcatgaca tgataataac ctcaggtgat ttactaggat aggttatggt 960 tggaggccta tataatgctc tccataaccg caa 993 <210>240 <211>330 <212>PRT <213>嗜热碱甲烷杆菌 <400>240 Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro Thr Ile Thr Tyr Pro 1 5 10 15 Ser Lys Ser Pro Glu Val Ile Ile Met Ser Cys Glu Ala Ser Gly Lys 20 25 30 Ile Trp Leu Asn Gly Glu Met Val Glu Trp Glu Glu Ala Thr Val His 35 40 45 Val Leu Ser His Val Val His Tyr Gly Ser Ser Val Phe Glu Gly Ile 50 55 60 Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala Ile Phe Arg Leu Arg Glu 65 70 75 80 His Val Lys Arg Leu Phe Asp Ser Ala Lys Ile Tyr Arg Met Asp Ile 85 90 95 Pro Tyr Thr Gln Glu Gln Ile Cys Asp Ala Ile Val Glu Thr Val Arg 100 105 110 Glu Asn Gly Leu Glu Glu Cys Tyr Ile Arg Pro Val Val Phe Arg Gly 115 120 125 Tyr Gly Glu Met Gly Val His Pro Val Asn Cys Pro Val Asp Val Ala 130 135 140 Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu Gly Ala Glu Ala Leu Glu 145 150 155 160 Val Gly Val Asp Ala Gly Val Ser Thr Trp Arg Arg Met Ala Pro Asn 165 170 175 Thr Met Pro Asn Met Ala Lys Ala Gly Gly Asn Tyr Leu Asn Ser Gln 180 185 190 Leu Ala Lys Met Glu Ala Val Arg His Gly Tyr Asp Glu Ala Ile Met 195 200 205 Leu Asp Tyr His Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe 210 215 220 Leu Val Ser Glu Gly Glu Ile Tyr Thr Pro Pro Val Ser Ser Ser Leu 225 230 235 240 Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys Ile Ala Arg Thr Glu 245 250 255 Gly Val Thr Val His Glu Glu Pro Ile Thr Arg Glu Met Leu Tyr Ile 260 265 270 Ala Asp Glu Ala Phe Phe Thr Gly Thr Ala Ala Glu Ile Thr Pro Ile 275 280 285 Arg Ser Val Asp Gly Ile Glu Ile Gly Ala Gly Arg Arg Gly Pro Val 290 295 300 Thr Lys Leu Leu Gln Asp Glu Phe Phe Arg Ile Ile Arg Ala Glu Thr 305 310 315 320 Glu Asp Ser Phe Gly Trp Leu Thr Tyr Ile 325 330 <210>241 <211>1095 <212>DNA <213>天蓝色链霉菌 <400>241 tcacggccgg ggacgggcct ccgccatccg ctgctcggcg atccggtcgg ccgccgcggc 60 cggcggaata ccgtcctcct tcgcacgtgc gaatatggcc agcgtggtgt cgtagatctt 120 cgaggccttc gccttgcacc ggtcgaagtc gaacccgtgc agctcgtcgg cgacctggat 180 gacaccgccg gcgttcacca catagtccgg cgcgtagagg atcccgcggt cggcgaggtc 240 cttctcgacg cccgggtggg cgagctggtt gttggccgcg ccgcacacca ccttggcggt 300 cagcaccggc acggtgtcgt cgttcagcgc gccgccgagc gcgcagggcg cgtagatgtc 360 caggttctcc acccggatca gcgcgtcggt gtcggcgacg gcgaccaccg acgggtgccg 420 ctccgtgatc ccgcgcacca cgtccttgcg cacgtccgtg acgacgacgt gggcgccctc 480 ggcgagcagg tgctcgacca ggtggtggcc gaccttgccg acgcccgcga tgccgacggt 540 gcggtcgcgc agcgtcgggt cgccccacag gtgctgggcg gcggcccgca tgccctggta 600 gacgccgaag gaggtgagca cggaggagtc gcccgcgccg ccgttctccg gggaacgccc 660 ggtcgtccag cggcactcgc gggccacgac gtccatgtcg gcgacgtagg tgccgacgtc 720 gcacgcggtg acgtagcggc cgcccagcga ggcgacgaac cggccgtagg cgaggagcag 780 ctcctcgctc ttgatctgct ccggatcgcc gatgatcacg gccttgccgc caccgtggtc 840 cagaccggcc atggcgttct tgtacgacat cccgcgggcg aggttcagcg cgtcggcgac 900 ggcctccgcc tcgctcgcgt acgggtagaa gcgggtaccg ccgagcgccg ggcccagggc 960 ggtggagtgg agggcgatca cggccttgag gccgctggca cggtcctggc agagcacgac 1020 ttgctcatgt cccccctgat ccgagtggaa cagggtgtgc agtacatcag caggtgcgcc 1080 gtttacgtcg gtcac 1095 <210>242 <211>364 <212>PRT <213>天蓝色链霉菌 <400>242 Met Thr Asp Val Asn Gly Ala Pro Ala Asp Val Leu His Thr Leu Phe 1 5 10 15 His Ser Asp Gln Gly Gly His Glu Gln Val Val Leu Cys Gln Asp Arg 20 25 30 Ala Ser Gly Leu Lys Ala Val Ile Ala Leu His Ser Thr Ala Leu Gly 35 40 45 Pro Ala Leu Gly Gly Thr Arg Phe Tyr Pro Tyr Ala Ser Glu Ala Glu 50 55 60 Ala Val Ala Asp Ala Leu Asn Leu Ala Arg Gly Met Ser Tyr Lys Asn 65 70 75 80 Ala Met Ala Gly Leu Asp His Gly Gly Gly Lys Ala Val Ile Ile Gly 85 90 95 Asp Pro Glu Gln Ile Lys Ser Glu Glu Leu Leu Leu Ala Tyr Gly Arg 100 105 110 Phe Val Ala Ser Leu Gly Gly Arg Tyr Val Thr Ala Cys Asp Val Gly 115 120 125 Thr Tyr Val Ala Asp Met Asp Val Val Ala Arg Glu Cys Arg Trp Thr 130 135 140 Thr Gly Arg Ser Pro Glu Asn Gly Gly Ala Gly Asp Ser Ser Val Leu 145 150 155 160 Thr Ser Phe Gly Val Tyr Gln Gly Met Arg Ala Ala Ala Gln His Leu 165 170 175 Trp Gly Asp Pro Thr Leu Arg Asp Arg Thr Val Gly Ile Ala Gly Val 180 185 190 Gly Lys Val Gly His His Leu Val Glu His Leu Leu Ala Glu Gly Ala 195 200 205 His Val Val Val Thr Asp Val Arg Lys Asp Val Val Arg Gly Ile Thr 210 215 220 Glu Arg His Pro Ser Val Val Ala Val Ala Asp Thr Asp Ala Leu Ile 225 230 235 240 Arg Val Glu Asn Leu Asp Ile Tyr Ala Pro Cys Ala Leu Gly Gly Ala 245 250 255 Leu Asn Asp Asp Thr Val Pro Val Leu Thr Ala Lys Val Val Cys Gly 260 265 270 Ala Ala Asn Asn Gln Leu Ala His Pro Gly Val Glu Lys Asp Leu Ala 275 280 285 Asp Arg Gly Ile Leu Tyr Ala Pro Asp Tyr Val Val Asn Ala Gly Gly 290 295 300 Val Ile Gln Val Ala Asp Glu Leu His Gly Phe Asp Phe Asp Arg Cys 305 310 315 320 Lys Ala Lys Ala Ser Lys Ile Tyr Asp Thr Thr Leu Ala Ile Phe Ala 325 330 335 Arg Ala Lys Glu Asp Gly Ile Pro Pro Ala Ala Ala Ala Asp Arg Ile 340 345 350 Ala Glu Gln Arg Met Ala Glu Ala Arg Pro Arg Pro 355 360 <210>243 <211>1095 <212>DNA <213>枯草芽孢杆菌 <400>243 atggaacttt ttaaatatat ggagaaatac gattacgaac aattggtatt ctgccaggat 60 gaacaatctg gattaaaagc gattatcgcc attcatgata caacgcttgg tccggcgctt 120 ggcggaacga gaatgtggac atatgaaaat gaagaagcgg caattgaaga tgcgctcaga 180 ttggcaagag gcatgaccta taagaacgcg gcggcaggct taaaccttgg cggcggaaaa 240 acagtcatta tcggcgatcc gcgcaaagac aaaaatgagg aaatgttccg cgcgtttggc 300 cgctatattc aaggactgaa tggcagatac atcacggctg aagatgtggg cacaacggtc 360 gaggatatgg atatcattca tgatgagaca gactatgtca cagggatttc tcctgctttc 420 ggctcttctg gaaatccgtc cccagtcaca gcgtacgggg tgtacagagg aatgaaggca 480 gcagctaaag ctgctttcgg aaccgattct cttgaaggaa aaaccattgc tgtacagggt 540 gttgggaacg tagcctataa cctttgccgc cacctgcatg aagaaggagc aaacttaatc 600 gttacggata tcaacaaaca atctgtacag cgtgcagttg aagattttgg cgcccgtgcg 660 gtagatcctg atgacattta ttcacaagac tgcgatattt atgcgccgtg tgcccttggt 720 gcgactatta acgacgacac cattaaacag ctgaaggcga aagtgatcgc aggtgcggct 780 aacaaccaat taaaagagac acgccatggt gatcaaattc acgaaatggg catcgtttat 840 gcaccggatt acgtgattaa cgcgggcggt gtcatcaacg tggcagatga gctttacggc 900 tataatgcag aacgtgcatt gaaaaaagtt gaaggcattt acggcaatat cgagcgtgta 960 cttgagattt ctcagcgtga cggcattcct gcatatttag cggctgaccg cttagcagag 1020 gaacggattg aacgcatgcg ccgctcaaga agccagtttt tgcaaaacgg ccacagtgta 1080 ttaagcagac gttaa 1095 <210>244 <211>364 <212>PRT <213>枯草芽孢杆菌 <400>244 Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser 195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg 355 360 <210>245 <211>1785 <212>DNA <213>生绿链霉菌 <400>245 gtgtcaactt cctccgcttc ttccgggccg gacctcccct tcgggcccga ggacacgcca 60 tggcagaagg ccttcagcag gctgcgggcg gtggatggcg tgccgcgcgt caccgcgccg 120 tccagtgatc cgcgtgaggt ctacatggac atcccggaga tccccttctc caaggtccag 180 atccccccgg acggaatgga cgagcagcag tacgcagagg ccgagagcct cttccgccgc 240 tacgtagacg cccagacccg caacttcgcg ggataccagg tcaccagcga cctcgactac 300 cagcacctca gtcactatct caaccggcat ctgaacaacg tcggcgatcc ctatgagtcc 360 agctcctaca cgctgaactc caaggtcctt gagcgagccg ttctcgacta cttcgcctcc 420 ctgtggaacg ccaagtggcc ccatgacgca agcgatccgg aaacgtactg gggttacgtg 480 ctgaccatgg gctccagcga aggcaacctg tacgggttgt ggaacgcacg ggactatctg 540 tcgggcaagc tgctgcggcg ccagcaccgg gaggccggcg gcgacaaggc ctcggtcgtc 600 tacacgcaag cgctgcgaca cgaagggcag agtccgcatg cctacgagcc ggtggcgttc 660 ttctcgcagg acacgcacta ctcgctcacg aaggccgtgc gggttctggg catcgacacc 720 ttccacagca tcggcagcag tcggtatccg gacgagaacc cgctgggccc cggcactccg 780 tggccgaccg aagtgccctc ggttgacggt gccatcgatg tcgacaaact cgcctcgttg 840 gtccgcttct tcgccagcaa gggctacccg atactggtca gcctcaacta cgggtcaacg 900 ttcaagggcg cctacgacga cgtcccggcc gtggcacagg ccgtgcggga catctgcacg 960 gaatacggtc tggatcggcg gcgggtatac cacgaccgca gtaaggacag tgacttcgac 1020 gagcgcagcg gcttctggat ccacatcgat gccgccctgg gggcgggcta cgctccctac 1080 ctgcagatgg cccgggatgc cggcatggtc gaggaggcgc cgcccgtttt cgacttccgg 1140 ctcccggagg tgcactcgct gaccatgagc ggccacaagt ggatgggaac accgtgggca 1200 tgcggtgtct acatgacacg gaccgggctg cagatgaccc cgccgaagtc gtccgagtac 1260 atcggggcgg ccgacaccac cttcgcgggc tcccgcaacg gcttctcgtc actgctgctg 1320 tgggactacc tgtcccggca ttcgtatgac gatctggtgc gcctggccgc cgactgcgac 1380 cggctggccg gctacgccca cgaccggttg ctgaccttgc aggacaaact cggcatggat 1440 ctgtgggtcg cccgcagccc gcagtccctc acggtgcgct tccgtcagcc atgtgcagac 1500 atcgtccgca agtactcgct gtcgtgtgag acggtctacg aagacaacga gcaacggacc 1560 tacgtacatc tctacgccgt tccccacctc actcgggaac tcgtggatga gctcgtgcgc 1620 gatctgcgcc agcccggagc cttcaccaac gctggtgcac tggaggggga ggcctgggcc 1680 ggggtgatcg atgccctcgg ccgcccggac cccgacggaa cctatgccgg cgccttgagc 1740 gctccggctt ccggcccccg ctccgaggac ggcggcggga gctga 1785 <210>246 <211>594 <212>PRT <213>生绿链霉菌 <400>246 Met Ser Thr Ser Ser Ala Ser Ser Gly Pro Asp Leu Pro Phe Gly Pro 1 5 10 15 Glu Asp Thr Pro Trp Gln Lys Ala Phe Ser Arg Leu Arg Ala Val Asp 20 25 30 Gly Val Pro Arg Val Thr Ala Pro Ser Ser Asp Pro Arg Glu Val Tyr 35 40 45 Met Asp Ile Pro Glu Ile Pro Phe Ser Lys Val Gln Ile Pro Pro Asp 50 55 60 Gly Met Asp Glu Gln Gln Tyr Ala Glu Ala Glu Ser Leu Phe Arg Arg 65 70 75 80 Tyr Val Asp Ala Gln Thr Arg Asn Phe Ala Gly Tyr Gln Val Thr Ser 85 90 95 Asp Leu Asp Tyr Gln His Leu Ser His Tyr Leu Asn Arg His Leu Asn 100 105 110 Asn Val Gly Asp Pro Tyr Glu Ser Ser Ser Tyr Thr Leu Asn Ser Lys 115 120 125 Val Leu Glu Arg Ala Val Leu Asp Tyr Phe Ala Ser Leu Trp Asn Ala 130 135 140 Lys Trp Pro His Asp Ala Ser Asp Pro Glu Thr Tyr Trp Gly Tyr Val 145 150 155 160 Leu Thr Met Gly Ser Ser Glu Gly Asn Leu Tyr Gly Leu Trp Asn Ala 165 170 175 Arg Asp Tyr Leu Ser Gly Lys Leu Leu Arg Arg Gln His Arg Glu Ala 180 185 190 Gly Gly Asp Lys Ala Ser Val Val Tyr Thr Gln Ala Leu Arg His Glu 195 200 205 Gly Gln Ser Pro His Ala Tyr Glu Pro Val Ala Phe Phe Ser Gln Asp 210 215 220 Thr His Tyr Ser Leu Thr Lys Ala Val Arg Val Leu Gly Ile Asp Thr 225 230 235 240 Phe His Ser Ile Gly Ser Ser Arg Tyr Pro Asp Glu Asn Pro Leu Gly 245 250 255 Pro Gly Thr Pro Trp Pro Thr Glu Val Pro Ser Val Asp Gly Ala Ile 260 265 270 Asp Val Asp Lys Leu Ala Ser Leu Val Arg Phe Phe Ala Ser Lys Gly 275 280 285 Tyr Pro Ile Leu Val Ser Leu Asn Tyr Gly Ser Thr Phe Lys Gly Ala 290 295 300 Tyr Asp Asp Val Pro Ala Val Ala Gln Ala Val Arg Asp Ile Cys Thr 305 310 315 320 Glu Tyr Gly Leu Asp Arg Arg Arg Val Tyr His Asp Arg Ser Lys Asp 325 330 335 Ser Asp Phe Asp Glu Arg Ser Gly Phe Trp Ile His Ile Asp Ala Ala 340 345 350 Leu Gly Ala Gly Tyr Ala Pro Tyr Leu Gln Met Ala Arg Asp Ala Gly 355 360 365 Met Val Glu Glu Ala Pro Pro Val Phe Asp Phe Arg Leu Pro Glu Val 370 375 380 His Ser Leu Thr Met Ser Gly His Lys Trp Met Gly Thr Pro Trp Ala 385 390 395 400 Cys Gly Val Tyr Met Thr Arg Thr Gly Leu Gln Met Thr Pro Pro Lys 405 410 415 Ser Ser Glu Tyr Ile Gly Ala Ala Asp Thr Thr Phe Ala Gly Ser Arg 420 425 430 Asn Gly Phe Ser Ser Leu Leu Leu Trp Asp Tyr Leu Ser Arg His Ser 435 440 445 Tyr Asp Asp Leu Val Arg Leu Ala Ala Asp Cys Asp Arg Leu Ala Gly 450 455 460 Tyr Ala His Asp Arg Leu Leu Thr Leu Gln Asp Lys Leu Gly Met Asp 465 470 475 480 Leu Trp Val Ala Arg Ser Pro Gln Ser Leu Thr Val Arg Phe Arg Gln 485 490 495 Pro Cys Ala Asp Ile Val Arg Lys Tyr Ser Leu Ser Cys Glu Thr Val 500 505 510 Tyr Glu Asp Asn Glu Gln Arg Thr Tyr Val His Leu Tyr Ala Val Pro 515 520 525 His Leu Thr Arg Glu Leu Val Asp Glu Leu Val Arg Asp Leu Arg Gln 530 535 540 Pro Gly Ala Phe Thr Asn Ala Gly Ala Leu Glu Gly Glu Ala Trp Ala 545 550 555 560 Gly Val Ile Asp Ala Leu Gly Arg Pro Asp Pro Asp Gly Thr Tyr Ala 565 570 575 Gly Ala Leu Ser Ala Pro Ala Ser Gly Pro Arg Ser Glu Asp Gly Gly 580 585 590 Gly Ser <210>247 <211>1323 <212>DNA <213>反硝化产碱菌 <400>247 atgagcgctg ccaaactgcc cgacctgtcc cacctctgga tgccctttac cgccaaccgg 60 cagttcaagg cgaacccccg cctgctggcc tcggccaagg gcatgtacta cacgtctttc 120 gacggccgcc agatcctgga cggcacggcc ggcctgtggt gcgtgaacgc cggccactgc 180 cgcgaagaaa tcgtctccgc catcgccagc caggccggcg tcatggacta cgcgccgggg 240 ttccagctcg gccacccgct ggccttcgag gccgccaccg ccgtggccgg cctgatgccg 300 cagggcctgg accgcgtgtt cttcaccaat tcgggctccg aatcggtgga caccgcgctg 360 aagatcgccc tggcctacca ccgcgcgcgc ggcgaggcgc agcgcacccg cctcatcggg 420 cgcgagcgcg gctaccacgg cgtgggcttc ggcggcattt ccgtgggcgg catctcgccc 480 aaccgcaaga ccttctccgg cgcgctgctg ccggccgtgg accacctgcc gcacacccac 540 agcctggaac acaacgcctt cacgcgcggc cagcccgagt ggggcgcgca cctggccgac 600 gagttggaac gcatcatcgc cctgcacgac gcctccacca tcgcggccgt gatcgtcgag 660 cccatggccg gctccaccgg cgtgctcgtc ccgcccaagg gctatctcga aaaactgcgc 720 gaaatcaccg cccgccacgg cattctgctg atcttcgacg aagtcatcac cgcgtacggc 780 cgcctgggcg aggccaccgc cgcggcctat ttcggcgtaa cgcccgacct catcaccatg 840 gccaagggcg tgagcaacgc cgccgttccg gccggcgccg tcgcggtgcg ccgcgaagtg 900 catgacgcca tcgtcaacgg accgcaaggc ggcatcgagt tcttccacgg ctacacctac 960 tcggcccacc cgctggccgc cgccgccgtg ctcgccacgc tggacatcta ccgccgcgaa 1020 gacctgttcg cccgcgcccg caagctgtcg gccgcgttcg aggaagccgc ccacagcctc 1080 aagggcgcgc cgcacgtcat cgacgtgcgc aacatcggcc tggtggccgg catcgagctg 1140 tcgccgcgcg aaggcgcccc gggcgcgcgc gccgccgaag ccttccagaa atgcttcgac 1200 accggcctca tggtgcgcta cacgggcgac atcctcgcgg tgtcgcctcc gctcatcgtc 1260 gacgaaaacc agatcggcca gatcttcgag ggcatcggca aggtgctcaa ggaagtggct 1320 tag 1323 <210>248 <211>440 <212>PRT <213>ω氨基酸:丙酮酸转氨酶 <400>248 Met Ser Ala Ala Lys Leu Pro Asp Leu Ser His Leu Trp Met Pro Phe 1 5 10 15 Thr Ala Asn Arg Gln Phe Lys Ala Asn Pro Arg Leu Leu Ala Ser Ala 20 25 30 Lys Gly Met Tyr Tyr Thr Ser Phe Asp Gly Arg Gln Ile Leu Asp Gly 35 40 45 Thr Ala Gly Leu Trp Cys Val Asn Ala Gly His Cys Arg Glu Glu Ile 50 55 60 Val Ser Ala Ile Ala Ser Gln Ala Gly Val Met Asp Tyr Ala Pro Gly 65 70 75 80 Phe Gln Leu Gly His Pro Leu Ala Phe Glu Ala Ala Thr Ala Val Ala 85 90 95 Gly Leu Met Pro Gln Gly Leu Asp Arg Val Phe Phe Thr Asn Ser Gly 100 105 110 Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr His Arg 115 120 125 Ala Arg Gly Glu Ala Gln Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly 130 135 140 Tyr His Gly Val Gly Phe Gly Gly Ile Ser Val Gly Gly Ile Ser Pro 145 150 155 160 Asn Arg Lys Thr Phe Ser Gly Ala Leu Leu Pro Ala Val Asp His Leu 165 170 175 Pro His Thr His Ser Leu Glu His Asn Ala Phe Thr Arg Gly Gln Pro 180 185 190 Glu Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Ile Ala Leu 195 200 205 His Asp Ala Ser Thr Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly 210 215 220 Ser Thr Gly Val Leu Val Pro Pro Lys Gly Tyr Leu Glu Lys Leu Arg 225 230 235 240 Glu Ile Thr Ala Arg His Gly Ile Leu Leu Ile Phe Asp Glu Val Ile 245 250 255 Thr Ala Tyr Gly Arg Leu Gly Glu Ala Thr Ala Ala Ala Tyr Phe Gly 260 265 270 Val Thr Pro Asp Leu Ile Thr Met Ala Lys Gly Val Ser Asn Ala Ala 275 280 285 Val Pro Ala Gly Ala Val Ala Val Arg Arg Glu Val His Asp Ala Ile 290 295 300 Val Asn Gly Pro Gln Gly Gly Ile Glu Phe Phe His Gly Tyr Thr Tyr 305 310 315 320 Ser Ala His Pro Leu Ala Ala Ala Ala Val Leu Ala Thr Leu Asp Ile 325 330 335 Tyr Arg Arg Glu Asp Leu Phe Ala Arg Ala Arg Lys Leu Ser Ala Ala 340 345 350 Phe Glu Glu Ala Ala His Ser Leu Lys Gly Ala Pro His Val Ile Asp 355 360 365 Val Arg Asn Ile Gly Leu Val Ala Gly Ile Glu Leu Ser Pro Arg Glu 370 375 380 Gly Ala Pro Gly Ala Arg Ala Ala Glu Ala Phe Gln Lys Cys Phs Asp 385 390 395 400 Thr Gly Leu Met Val Arg Tyr Thr Gly Asp Ile Leu Ala Val Ser Pro 405 410 415 Pro Leu Ile Val Asp Glu Asn Gln Ile Gly Gln Ile Phe Glu Gly Ile 420 425 430 Gly Lys Val Leu Lys Glu Val Ala 435 440 <210>249 <211>1332 <212>DNA <213>真养雷氏菌 <400>249 atggacgccg cgaagaccgt gattcccgat ctcgatgccc tgtggatgcc ctttaccgcg 60 aaccgccagt acaaggcggc gccgcgcctg ctggcctcgg ccagcggcat gtactacacc 120 acccacgacg gacgccagat cctcgacggt tgcgcgggcc tctggtgcgt agcggccggc 180 cactgccgca aggagattgc cgaggccgtg gcccgccagg ccgccacgct cgactacgcg 240 ccgccgttcc agatgggcca tccgctgtcg ttcgaagccg ccaccaaggt ggccgcgatc 300 atgccgcagg gactggaccg catcttcttc acgaattccg gttcggaatc ggtggacacc 360 gcgctgaaga ttgcgctggc ctaccaccgt gcgcgcggcg agggccagcg cacccgcttc 420 atcgggcgcg aacgcggtta ccacggcgtg ggctttggcg gcatggctgt cggtggcatc 480 gggccgaacc gcaaggcgtt ctcggccaac ctgatgccgg gcaccgacca tctgccggcg 540 acgctgaata tcgccgaagc ggcgttctcc aagggtcagc cgacatgggg cgcgcacctt 600 gccgacgaac tcgagcgcat cgtcgcgctg catgatccgt ccacgattgc cgccgtcatc 660 gtggaaccgc tggcgggctc cgccggggtg ctggtgccgc cggtcggcta cctcgacaag 720 ctgcgcgaga tcacgaccaa gcacggcatc ctgctgatct tcgacgaggt catcacggcc 780 tttggtcgcc tgggtaccgc caccgcggcg gaacgcttca aggtcacgcc ggacctgatc 840 accatggcca aggccatcaa caacgccgcc gtgccgatgg gtgccgtggc cgtgcgccgc 900 gaagtccatg acaccgtggt caactcggcc gcgccgggcg cgatcgaact cgcgcatggc 960 tacacctact cgggccaccc gctggccgcc gccgctgcca tcgccacgct ggacctgtat 1020 cagcgcgaga acctgttcgg ccgtgccgcg gagctgtcgc cggtgttcga agcggccgtt 1080 cacagcgtac gcagcgcgcc gcatgtgaag gacatccgca acctcggcat ggtggccggc 1140 atcgagctgg agccgcgtcc gggccagccc ggcgcacgcg cctacgaagc cttcctcaaa 1200 tgccttgagc gtggcgtgct ggtgcgctac accggcgata tcctcgcgtt ctcgccgccg 1260 ctgatcatca gcgaggcgca gattgccgag ctgttcgata cggtcaagca ggccttgcag 1320 gaagtgcagt aa 1332 <210>250 <211>443 <212>PRT <213>真养雷氏菌 <400>250 Met Asp Ala Ala Lys Thr Val Ile Pro Asp Leu Asp Ala Leu Trp Met 1 5 10 15 Pro Phe Thr Ala Asn Arg Gln Tyr Lys Ala Ala Pro Arg Leu Leu Ala 20 25 30 Ser Ala Ser Gly Met Tyr Tyr Thr Thr His Asp Gly Arg Gln Ile Leu 35 40 45 Asp Gly Cys Ala Gly Leu Trp Cys Val Ala Ala Gly His Cys Arg Lys 50 55 60 Glu Ile Ala Glu Ala Val Ala Arg Gln Ala Ala Thr Leu Asp Tyr Ala 65 70 75 80 Pro Pro Phe Gln Met Gly His Pro Leu Ser Phe Glu Ala Ala Thr Lys 85 90 95 Val Ala Ala Ile Met Pro Gln Gly Leu Asp Arg Ile Phe Phe Thr Asn 100 105 110 Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr 115 120 125 His Arg Ala Arg Gly Glu Gly Gln Arg Thr Arg Phe Ile Gly Arg Glu 130 135 140 Arg Gly Tyr His Gly Val Gly Phe Gly Gly Met Ala Val Gly Gly Ile 145 150 155 160 Gly Pro Asn Arg Lys Ala Phe Ser Ala Asn Leu Met Pro Gly Thr Asp 165 170 175 His Leu Pro Ala Thr Leu Asn Ile Ala Glu Ala Ala Phe Ser Lys Gly 180 185 190 Gln Pro Thr Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Val 195 200 205 Ala Leu His Asp Pro Ser Thr Ile Ala Ala Val Ile Val Glu Pro Leu 210 215 220 Ala Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly Tyr Leu Asp Lys 225 230 235 240 Leu Arg Glu Ile Thr Thr Lys His Gly Ile Leu Leu Ile Phe Asp Glu 245 250 255 Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Ala Thr Ala Ala Glu Arg 260 265 270 Phe Lys Val Thr Pro Asp Leu Ile Thr Met Ala Lys Ala Ile Asn Asn 275 280 285 Ala Ala Val Pro Met Gly Ala Val Ala Val Arg Arg Glu Val His Asp 290 295 300 Thr Val Val Asn Ser Ala Ala Pro Gly Ala Ile Glu Leu Ala His Gly 305 310 315 320 Tyr Thr Tyr Ser Gly His Pro Leu Ala Ala Ala Ala Ala Ile Ala Thr 325 330 335 Leu Asp Leu Tyr Gln Arg Glu Asn Leu Phe Gly Arg Ala Ala Glu Leu 340 345 350 Ser Pro Val Phe Glu Ala Ala Val His Ser Val Arg Ser Ala Pro His 355 360 365 Val Lys Asp Ile Arg Asn Leu Gly Met Val Ala Gly Ile Glu Leu Glu 370 375 380 Pro Arg Pro Gly Gln Pro Gly Ala Arg Ala Tyr Glu Ala Phe Leu Lys 385 390 395 400 Cys Leu Glu Arg Gly Val Leu Val Arg Tyr Thr Gly Asp Ile Leu Ala 405 410 415 Phe Ser Pro Pro Leu Ile Ile Ser Glu Ala Gln Ile Ala Glu Leu Phe 420 425 430 Asp Thr Val Lys Gln Ala Leu Gln Glu Val Gln 435 440 <210>251 <211>1341 <212>DNA <213>Shewanella oneidensis <400>251 atggccgact cacccaacaa cctcgctcac gaacatcctt cacttgaaca ctattggatg 60 ccttttaccg ccaatcgcca attcaaagcg agccctcgtt tactcgccca agctgaaggt 120 atgtattaca cagatatcaa tggcaacaag gtattagact ctacagcggg cttatggtgt 180 tgtaatgctg gccatggtcg ccgtgagatc agtgaagccg tcagcaaaca aattcggcag 240 atggattacg ctccctcctt ccaaatgggc catcccatcg cttttgaact ggccgaacgt 300 ttaaccgaac tcagcccaga aggactcaac aaagtattct ttaccaactc aggctctgag 360 tcggttgata ccgcgctaaa aatggctctt tgctaccata gagccaatgg ccaagcgtca 420 cgcacccgct ttattggccg tgaaatgggt taccatggcg taggatttgg tgggatctcg 480 gtgggtggtt taagcaataa ccgtaaagcc ttcagcggcc agctattgca aggcgtggat 540 cacctgcccc acaccttaga cattcaacat gccgccttta gtcgtggctt accgagcctc 600 ggtgctgaaa aagctgaggt attagaacaa ttagtcacac tccatggcgc cgaaaatatt 660 gccgccgtta ttgttgaacc catgtcaggt tctgcagggg taattttacc acctcaaggc 720 tacttaaaac gcttacgtga aatcactaaa aaacacggca tcttattgat tttcgatgaa 780 gtcattaccg catttggccg tgtaggtgca gcattcgcca gccaacgttg gggcgttatt 840 ccagacataa tcaccacggc taaagccatt aataatggcg ccatccccat gggcgcagtg 900 tttgtacagg attatatcca cgatacttgc atgcaagggc caaccgaact gattgaattt 960 ttccacggtt atacctattc gggccaccca gtcgccgcag cagcagcact cgccacgctc 1020 tccatctacc aaaacgagca actgtttgag cgcagttttg agcttgagcg gtatttcgaa 1080 gaagccgttc atagcctcaa agggttaccg aatgtgattg atattcgcaa caccggatta 1140 gtcgcgggtt tccagctagc accgaatagc caaggtgttg gtaaacgcgg atacagcgtg 1200 ttcgagcatt gtttccatca aggcacactc gtgcgggcaa cgggcgatat tatcgccatg 1260 tccccaccac tcattgttga gaaacatcag attgaccaaa tggtaaatag ccttagcgat 1320 gcaattcacg ccgttggatg a 1341 <210>252 <211>446 <212>PRT <213>β丙氨酸-丙酮酸转氨酶 <400>252 Met Ala Asp Ser Pro Asn Asn Leu Ala His Glu His Pro Ser Leu Glu 1 5 10 15 His Tyr Trp Met Pro Phe Thr Ala Asn Arg Gln Phe Lys Ala Ser Pro 20 25 30 Arg Leu Leu Ala Gln Ala Glu Gly Met Tyr Tyr Thr Asp Ile Asn Gly 35 40 45 Asn Lys Val Leu Asp Ser Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly 50 55 60 His Gly Arg Arg Glu Ile Ser Glu Ala Val Ser Lys Gln Ile Arg Gln 65 70 75 80 Met Asp Tyr Ala Pro Ser Phe Gln Met Gly His Pro Ile Ala Phe Glu 85 90 95 Leu Ala Glu Arg Leu Thr Glu Leu Ser Pro Glu Gly Leu Asn Lys Val 100 105 110 Phe Phe Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Met 115 120 125 Ala Leu Cys Tyr His Arg Ala Asn Gly Gln Ala Ser Arg Thr Arg Phe 130 135 140 Ile Gly Arg Glu Met Gly Tyr His Gly Val Gly Phe Gly Gly Ile Ser 145 150 155 160 Val Gly Gly Leu Ser Asn Asn Arg Lys Ala Phe Ser Gly Gln Leu Leu 165 170 175 Gln Gly Val Asp His Leu Pro His Thr Leu Asp Ile Gln His Ala Ala 180 185 190 Phe Ser Arg Gly Leu Pro Ser Leu Gly Ala Glu Lys Ala Glu Val Leu 195 200 205 Glu Gln Leu Val Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile 210 215 220 Val Glu Pro Met Ser Gly Ser Ala Gly Val Ile Leu Pro Pro Gln Gly 225 230 235 240 Tyr Leu Lys Arg Leu Arg Glu Ile Thr Lys Lys His Gly Ile Leu Leu 245 250 255 Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Val Gly Ala Ala Phe 260 265 270 Ala Ser Gln Arg Trp Gly Val Ile Pro Asp Ile Ile Thr Thr Ala Lys 275 280 285 Ala Ile Asn Asn Gly Ala Ile Pro Met Gly Ala Val Phe Val Gln Asp 290 295 300 Tyr Ile His Asp Thr Cys Met Gln Gly Pro Thr Glu Leu Ile Glu Phe 305 310 315 320 Phe His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Ala Ala 325 330 335 Leu Ala Thr Leu Ser Ile Tyr Gln Asn Glu Gln Leu Phe Glu Arg Ser 340 345 350 Phe Glu Leu Glu Arg Tyr Phe Glu Glu Ala Val His Ser Leu Lys Gly 355 360 365 Leu Pro Asn Val Ile Asp Ile Arg Asn Thr Gly Leu Val Ala Gly Phe 370 375 380 Gln Leu Ala Pro Asn Ser Gln Gly Val Gly Lys Arg Gly Tyr Ser Val 385 390 395 400 Phe Glu His Cys Phe His Gln Gly Thr Leu Val Arg Ala Thr Gly Asp 405 410 415 Ile Ile Ala Met Ser Pro Pro Leu Ile Val Glu Lys His Gln Ile Asp 420 425 430 Gln Met Val Asn Ser Leu Ser Asp Ala Ile His Ala Val Gly 435 440 445 <210>253 <211>1347 <212>DNA <213>恶臭假单胞菌 <400>253 atgaacatgc ccgaaactgg tcctgccggt atcgccagcc agctcaagct ggacgcccac 60 tggatgccct acaccgccaa ccgcaacttc cagcgcgacc cacgcctgat cgtggcggcc 120 gaaggcaact acctggtcga tgaccacggg cgcaagatct tcgacgccct gtccggcctg 180 tggacctgcg gcgcagggca cactcgcaag gaaatcgctg acgcggtgac ccgtcaactg 240 agtacgctgg actactcccc agcgttccag ttcggccacc cgctgtcgtt ccagctggcg 300 gaaaagatcg ccgagctggt tccgggcaat ctgaatcacg tcttctatac caactccggt 360 tccgagtgcg ccgataccgc actgaagatg gtgcgtgcct actggcgcct gaaaggccag 420 gcaaccaaga ccaagatcat cggccgtgcc cgtggttacc atggcgtgaa catcgccggt 480 accagcctgg gtggcgtcaa cggtaaccgc aagatgtttg gccagctgct ggacgtcgac 540 cacctgcctc acactgtatt gccggtgaac gccttctcga aaggcttgcc ggaagagggc 600 ggtatcgcgc tggctgacga aatgctcaag ctgatcgagc tgcacgatgc ctccaacatc 660 gcagcagtca tcgtcgagcc gctggccggt tcggccggtg tgctgccgcc gccaaagggt 720 tacctgaagc gcctgcgtga aatctgcacc cagcacaaca ttctgctgat cttcgacgaa 780 gtgatcacag gcttcggccg catgggcgcg atgaccggct cggaagcctt cggcgttacc 840 ccggacctga tgtgcatcgc caagcaggtg accaacggcg ccatcccgat gggcgcagtg 900 attgccagca gcgagatcta ccagaccttc atgaaccagc cgaccccgga atacgccgtg 960 gaattcccac acggctacac ctattcggcg cacccggtag cctgtgccgc cggtctcgcc 1020 gcgctggacc tgctgcagaa ggaaaacctg gtgcagtccg cggctgaact ggcgccgcat 1080 ttcgagaagc tgctgcacgg cgtgaagggc accaagaata tcgtcgatat ccgcaactac 1140 ggcctggccg gcgccatcca gatcgccgcc cgtgacggtg atgccatcgt tcgcccttac 1200 gaagcggcca tgaagctgtg gaaagcgggc ttctatgtac gctttggtgg cgacaccctg 1260 cagttcggcc caaccttcaa taccaagccg caggaactgg accgcttgtt cgatgctgtt 1320 ggcgaaaccc tgaacctgat cgactga 1347 <210>254 <211>448 <212>PRT <213>恶臭假单胞菌 <400>254 Met Asn Met Pro Glu Thr Gly Pro Ala Gly Ile Ala Ser Gln Leu Lys 1 5 10 15 Leu Asp Ala His Trp Met Pro Tyr Thr Ala Asn Arg Asn Phe Gln Arg 20 25 30 Asp Pro Arg Leu Ile Val Ala Ala Glu Gly Asn Tyr Leu Val Asp Asp 35 40 45 His Gly Arg Lys Ile Phe Asp Ala Leu Ser Gly Leu Trp Thr Cys Gly 50 55 60 Ala Gly His Thr Arg Lys Glu Ile Ala Asp Ala Val Thr Arg Gln Leu 65 70 75 80 Ser Thr Leu Asp Tyr Ser Pro Ala Phe Gln Phe Gly His Pro Leu Ser 85 90 95 Phe Gln Leu Ala Glu Lys Ile Ala Glu Leu Val Pro Gly Asn Leu Asn 100 105 110 His Val Phe Tyr Thr Asn Ser Gly Ser Glu Cys Ala Asp Thr Ala Leu 115 120 125 Lys Met Val Arg Ala Tyr Trp Arg Leu Lys Gly Gln Ala Thr Lys Thr 130 135 140 Lys Ile Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Ile Ala Gly l45 150 155 160 Thr Ser Leu Gly Gly Val Asn Gly Asn Arg Lys Met Phe Gly Gln Leu 165 170 175 Leu Asp Val Asp His Leu Pro His Thr Val Leu Pro Val Asn Ala Phe 180 185 190 Ser Lys Gly Leu Pro Glu Glu Gly Gly Ile Ala Leu Ala Asp Glu Met 195 200 205 Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile 210 215 220 Val Glu Pro Leu Ala Gly Ser Ala Gly Val Leu Pro Pro Pro Lys Gly 225 230 235 240 Tyr Leu Lys Arg Leu Arg Glu Ile Cys Thr Gln His Asn Ile Leu Leu 245 250 255 Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Met Gly Ala Met Thr 260 265 270 Gly Ser Glu Ala Phe Gly Val Thr Pro Asp Leu Met Cys Ile Ala Lys 275 280 285 Gln Val Thr Asn Gly Ala Ile Pro Met Gly Ala Val Ile Ala Ser Ser 290 295 300 Glu Ile Tyr Gln Thr Phe Met Asn Gln Pro Thr Pro Glu Tyr Ala Val 305 310 315 320 Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala 325 330 335 Ala Gly Leu Ala Ala Leu Asp Leu Leu Gln Lys Glu Asn Leu Val Gln 340 345 350 Ser Ala Ala Glu Leu Ala Pro His Phe Glu Lys Leu Leu His Gly Val 355 360 365 Lys Gly Thr Lys Asn Ile Val Asp Ile Arg Asn Tyr Gly Leu Ala Gly 370 375 380 Ala Ile Gln Ile Ala Ala Arg Asp Gly Asp Ala Ile Val Arg Pro Tyr 385 390 395 400 Glu Ala Ala Met Lys Leu Trp Lys Ala Gly Phe Tyr Val Arg Phe Gly 405 410 415 Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Thr Lys Pro Gln Glu 420 425 430 Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Thr Leu Asn Leu Ile Asp 435 440 445 <210>255 <211>1701 <212>DNA <213>肉桂地链霉菌 <400>255 atggacgctg acgcgatcga ggaaggccgc cgacgctggc aggcccgtta cgacaaggcc 60 cgcaagcgcg acgcggactt caccacgctc tccggggacc ccgtcgaccc cgtctacggc 120 ccccggcccg gggacacgta cgacgggttc gagcggatcg gctggccggg ggagtacccc 180 ttcacccgcg ggctctacgc caccgggtac cgcggccgca cctggaccat ccgccagttc 240 gccggcttcg gcaacgccga gcagacgaac gagcgctaca agatgatcct ggccaacggc 300 ggcggcggcc tctccgtcgc cttcgacatg ccgaccctca tgggccgcga ctccgacgac 360 ccgcgctcgc tcggcgaggt cggccactgc ggtgtcgcca tcgactccgc cgccgacatg 420 gaggtcctct tcaaggacat cccgctcggc gacgtcacga cgtccatgac catcagcggg 480 cccgccgtgc ccgtcttctg catgtacctc gtcgcggccg agcgccaggg cgtcgacccg 540 gccgtcctca acggcacgct gcagaccgac atcttcaagg agtacatcgc ccagaaggag 600 tggctcttcc agcccgagcc gcacctgcgc ctcatcggcg acctgatgga gcactgcgcg 660 cgcgacatcc ccgcgtacaa gccgctctcg gtctccggct accacatccg cgaggccggg 720 gcgacggccg cgcaggagct cgcgtacacc ctcgcggacg gcttcgggta cgtggaactg 780 ggcctctcgc gcggcctgga cgtggacgtc ttcgcgcccg gcctctcctt cttcttcgac 840 gcgcacgtcg acttcttcga ggagatcgcg aagttccgcg ccgcacgccg catctgggcg 900 cgctggctcc gggacgagta cggagcgaag accgagaagg cacagtggct gcgcttccac 960 acgcagaccg cgggggtctc gctcacggcc cagcagccgt acaacaacgt ggtgcggacg 1020 gcggtggagg ccctcgccgc ggtgctcggc ggcacgaact ccctgcacac caacgctctc 1080 gacgagaccc ttgccctccc cagcgagcag gccgcggaga tcgcgctgcg cacccagcag 1140 gtgctgatgg aggagaccgg cgtcgccaac gtcgcggacc cgctgggcgg ctcctggtac 1200 atcgagcagc tcaccgaccg catcgaggcc gacgccgaga agatcttcga gcagatcagg 1260 gagcgggggc ggcgggcctg ccccgacggg cagcacccga tcgggccgat cacctccggc 1320 atcctgcgcg gcatcgagga cggctggttc accggcgaga tcgccgagtc cgccttccag 1380 taccagcggt ccctggagaa gggcgacaag cgggtcgtcg gcgtcaactg cctcgaaggc 1440 tccgtcaccg gcgacctgga gatcctgcgc gtcagccacg aggtcgagcg cgagcaggtg 1500 cgggagcttg cggggcgcaa ggggcggcgt gacgatgcgc gggtgcgggc ctcgctcgac 1560 gcgatgctcg ccgctgcgcg ggacgggtcg aacatgattg cccccatgct ggaggcggtg 1620 cgggccgagg cgaccctcgg ggagatctgc ggggtgcttc gcgatgagtg gggggtctac 1680 gtggagccgc ccgggttctg a 1701 <210>256 <211>566 <212>PRT <213>肉桂地链霉菌 <400>256 Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Lys Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly 20 25 30 Asp Pro Val Asp Pro Val Tyr Gly Pro Arg Pro Gly Asp Thr Tyr Asp 35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly 50 55 60 Leu Tyr Ala Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile 85 90 95 Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr 100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly 115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe 130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln 165 170 175 Gly Val Asp Pro Ala Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe 180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His 195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Arg Asp Ile Pro 210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala 260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu 275 280 285 Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg 290 295 300 Asp Glu Tyr Gly Ala Lys Thr Glu Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr 340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser 355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu 370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr 385 390 395 400 Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe 405 410 415 Glu Gln Ile Arg Glu Arg Gly Arg Arg Ala Cys Pro Asp Gly Gln His 420 425 430 Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly 435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Arg Ser 450 455 460 Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Cys Leu Glu Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu 485 490 495 Arg Glu Gln Val Arg Glu Leu Ala Gly Arg Lys Gly Arg Arg Asp Asp 500 505 510 Ala Arg Val Arg Ala Ser Leu Asp Ala Met Leu Ala Ala Ala Arg Asp 515 520 525 Gly Ser Asn Met Ile Ala Pro Met Leu Glu Ala Val Arg Ala Glu Ala 530 535 540 Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr 545 550 555 560 Val Glu Pro Pro Gly Phe 565 <210>257 <211>411 <212>DNA <213>肉桂地链霉菌 <400>257 atgggtgtgg cagccgggcc gatccgcgtg gtggtcgcca agccggggct cgacgggcac 60 gatcgcgggg ccaaggtgat cgcgcgggcg ttgcgtgacg cgggtatgga ggtcatctac 120 accgggctgc accagacgcc cgagcaggtg gtggacaccg cgatccagga ggacgccgac 180 gcgatcggcc tctccatcct ctccggagcg cacaacacgc tgttcgcgcg cgtgttggag 240 ctcttgaagg agcgggacgc ggaggacatc aaggtgtttg gtggcggcat catcccggag 300 gcggacatcg cgccgctgaa ggagaagggc gtcgcggaga tcttcacgcc cggggccacc 360 accacgtcga tcgtggagtg ggttcggggg aacgtgcgac aggccgtctg a 411 <210>258 <211>136 <212>PRT <213>肉桂地链霉菌 <400>258 Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg 20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu 35 40 45 Gln Val Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu 50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Arg Val Leu Glu 65 70 75 80 Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala 100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Thr Ser Ile Val Glu Trp Val 115 120 125 Arg Gly Asn Val Arg Gln Ala Val 130 135 <210>259 <211>1701 <212>DNA <213>天蓝色链霉菌 <400>259 atggacgctc atgccataga ggagggccgc cttcgctggc aggcccggta cgacgcggcg 60 cgcaagcgcg acgcggactt caccacgctc tccggagacc ccgtggagcc ggtgtacggg 120 ccccgccccg gggacgagta cgagggcttc gagcggatcg gctggccggg cgagtacccc 180 ttcacccgcg gcctgtatcc gaccgggtac cgggggcgta cgtggaccat ccggcagttc 240 gccgggttcg gcaacgccga gcagaccaac gagcgctaca agatgatcct ccgcaacggc 300 ggcggcgggc tctcggtcgc cttcgacatg ccgaccctga tgggccgcga ctccgacgac 360 ccgcgctcgc tgggcgaggt cgggcactgc ggggtggcca tcgactcggc cgccgacatg 420 gaagtgctgt tcaaggacat cccgctcggg gacgtgacga cctccatgac gatcagcggg 480 cccgccgtgc ccgtgttctg catgtacctc gtcgccgccg agcgccaggg cgtcgacgca 540 tccgtgctca acggcacgct gcagaccgac atcttcaagg agtacatcgc ccagaaggag 600 tggctcttcc agcccgagcc ccacctccgg ctcatcggcg acctcatgga gtactgcgcg 660 gccggcatcc ccgcctacaa gccgctctcc gtctccggct accacatccg cgaggcgggc 720 gcgacggccg cgcaggagct ggcgtacacg ctcgccgacg gcttcggata cgtggagctg 780 ggcctcagcc gcgggctcga cgtggacgtc ttcgcgcccg gcctctcctt cttcttcgac 840 gcgcacctcg acttcttcga ggagatcgcc aagttccgcg cggcccgcag gatctgggcc 900 cgctggatgc gcgacgtgta cggcgcgcgg accgacaagg cccagtggct gcggttccac 960 acccagaccg ccggagtctc gctcaccgcg cagcagccgt acaacaacgt cgtacgcacc 1020 gcggtggagg cgctggcggc cgtgctcggc ggcaccaact ccctgcacac caacgcgctc 1080 gacgagaccc tcgccctgcc cagcgagcag gccgccgaga tcgccctgcg cacccagcag 1140 gtgctgatgg aggagaccgg cgtcgccaac gtcgccgacc cgctgggcgg ttcctggttc 1200 atcgagcagc tgaccgaccg catcgaggcc gacgccgaga agatcttcga gcagatcaag 1260 gagcgggggc tgcgcgccca ccccgacggg cagcaccccg tcggaccgat cacctccggc 1320 ctgctgcgcg gcatcgagga cggctggttc accggcgaga tcgccgagtc cgccttccgc 1380 taccagcagt ccttggagaa ggacgacaag aaggtggtcg gcgtcaacgt ccacaccggc 1440 tccgtcaccg gcgacctgga gatcctgcgg gtcagccacg aggtcgagcg cgagcaggtg 1500 cgggtcctgg gcgagcgcaa ggacgcccgg gacgacgccg ccgtgcgcgg cgccctggac 1560 gccatgctgg ccgcggcccg ctccggcggc aacatgatcg ggccgatgct ggacgcggtg 1620 cgcgcggagg cgacgctggg cgagatctgc ggtgtgctgc gcgacgagtg gggggtgtac 1680 acggaaccgg cggggttctg a 1701 <210>260 <211>566 <212>PRT <213>天蓝色链霉菌 <400>260 Met Asp Ala His Ala Ile Glu Glu Gly Arg Leu Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Ala Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly 20 25 30 Asp Pro Val Glu Pro Val Tyr Gly Pro Arg Pro Gly Asp Glu Tyr Glu 35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly 50 55 60 Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile 85 90 95 Leu Arg Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr 100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly 115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe 130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln 165 170 175 Gly Val Asp Ala Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe 180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His 195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu Tyr Cys Ala Ala Gly Ile Pro 210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala 260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Leu Asp Phe Phe Glu Glu 275 280 285 Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Met Arg 290 295 300 Asp Val Tyr Gly Ala Arg Thr Asp Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr 340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser 355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu 370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Phe 385 390 395 400 Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe 405 410 415 Glu Gln Ile Lys Glu Arg Gly Leu Arg Ala His Pro Asp Gly Gln His 420 425 430 Pro Val Gly Pro Ile Thr Ser Gly Leu Leu Arg Gly Ile Glu Asp Gly 435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Arg Tyr Gln Gln Ser 450 455 460 Leu Glu Lys Asp Asp Lys Lys Val Val Gly Val Asn Val His Thr Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu 485 490 495 Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Asp Ala Arg Asp Asp 500 505 510 Ala Ala Val Arg Gly Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Ser 515 520 525 Gly Gly Asn Met Ile Gly Pro Met Leu Asp Ala Val Arg Ala Glu Ala 530 535 540 Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr 545 550 555 560 Thr Glu Pro Ala Gly Phe 565 <210>261 <211>417 <212>DNA <213>天蓝色链霉菌 <400>261 atgggtgtgg cagccggtcc gatccgcgtg gtggtggcca agccggggct cgacggccac 60 gatcgcgggg ccaaggtgat cgcgagggcc ctgcgtgacg ccggtatgga ggtgatctac 120 accgggctcc accagacgcc cgagcagatc gtcgacaccg cgatccagga ggacgccgac 180 gcgatcgggc tgtccatcct ctccggtgcg cacaacacgc tcttcgccgc cgtgatcgag 240 ctgctccggg agcgggacgc cgcggacatc ctggtcttcg gcggcgggat catccccgag 300 gcggacatcg ccccgctgaa ggagaagggc gtcgcggaga tcttcacgcc cggcgccacc 360 acggcgtcca tcgtggactg ggtccgggcg aacgtgcggg agcccgcggg agcatag 417 <210>262 <211>138 <212>PRT <213>天蓝色链霉菌 <400>262 Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg 20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu 35 40 45 Gln Ile Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu 50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Glu 65 70 75 80 Leu Leu Arg Glu Arg Asp Ala Ala Asp Ile Leu Val Phe Gly Gly Gly 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala 100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Asp Trp Val 115 120 125 Arg Ala Asn Val Arg Glu Pro Ala Gly Ala 130 135 <210>263 <211>1701 <212>DNA <213>除虫链霉菌 <400>263 tcagaaaccg gcgggctccg tgtagacccc ccactcctcc cggaggacat cgcagatctc 60 gcccagcgtg gcctccgcgc ggaccgcgtc cagcatcggg gcgatcatgt tcgacccgtc 120 gcgcgcggcg gcgagcatcg cgtccagggc cgcggttacg gccgtgtcgt cgcgccccga 180 cttccgctcg cccagcaccc gcacctgctc gcgctccacc tcgtggctga cgcgcaggat 240 ctccaggtcg cccgtcacgg acccgtggtg gacgttgacg ccgacgaccc gcttgtcgcc 300 cttctccagc gcctgctggt actggaaggc cgactcggcg atctccccgg tgaaccagcc 360 gtcctcgatg ccgcgcagga tgccggaggt gatgggcccg atcgggtgcc gcccgtccgg 420 gtgggcccgc agcccgcgct ccctgatctg ttcgaagatc ttctcggcgt cggcctcgat 480 ccggtcggtc agctgctcca cgtaccagga accgcccagc ggatcggcca cgttggcgac 540 gcccgtctcc tccatcagca cctgctgggt gcgcagggcg atctcggccg cctgctcgga 600 cggcagggcg agggtctcgt cgagggcgtt ggtgtgcagc gagttcgtcc cgccgagcac 660 cgcggcgagg gcctccacgg ccgtccgtac gacgttgttg tacggctgct gcgcggtgag 720 cgagacgccc gcggtctggg tgtggaagcg cagccactgc gccttctccg acttcgcccc 780 gtacacgtcc cgcagccagc gcgcccagat gcgccgcgcc gcacggaact tggcgatctc 840 ctcgaagaag tcgacgtgcg cgtcgaagaa gaaggagagc ccgggcgcga acacgtccac 900 gtccaggccg cggctcagcc ccagctccac gtatccgaaa ccgtcggcga gggtgtacgc 960 cagctcctgg gcggccgtgg caccggcctc ccggatgtgg tacccggaga cggacagcgg 1020 cttgtacgcg gggatcttcg aggcgcagtg ctccatcagg tcgccgatga gccgcagatg 1080 gggctcgggc tggaagagcc actccttctg cgcgatgtac tccttgaaga tgtcggtctg 1140 gagggtgccg ttgaggacgg aggggtcgac gccctgccgc tcggccgcga ccaggtacat 1200 gcagaagacg ggcacggcgg gcccgctgat cgtcatcgac gtcgtcacgt cacccagcgg 1260 gatgtccttg aacaggacct ccatgtcggc cgccgagtcg atcgcgaccc cgcagtgccc 1320 gacctcgccg agcgcgcggc ggtcgtcgga gtcgcgcccc atgagcgtcg gcatgtcgaa 1380 ggccacggac agcccaccgc cgccgttggc gaggatcttc ttgtagcgct cgttggtctg 1440 ctcggcgttg ccgaacccgg cgaactgccg gatggtccag gtccggcccc ggtagccggt 1500 cggatacaga ccgcgcgtga aggggtactc acccggccag ccgatccgct cgaaaccctc 1560 gtacgcgtcc ccgggccggg gcccgtacgc cggctccacg ggatcgccgg agagcgtggt 1620 gaaatcggcc tcgcgcttgc gtgaggcgtc gtagcgggcc tgccagcgtc ggcggccttc 1680 ctcgatggcg tcagcgtcca t 1701 <210>264 <211>566 <212>PRT <213>除虫链霉菌 <400>264 Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Ala Ser Arg Lys Arg Glu Ala Asp Phe Thr Thr Leu Ser Gly 20 25 30 Asp Pro Val Glu Pro Ala Tyr Gly Pro Arg Pro Gly Asp Ala Tyr Glu 35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly 50 55 60 Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Lys Ile 85 90 95 Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr 100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Arg Arg Ala Leu Gly Glu Val Gly 115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe 130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln 165 170 175 Gly Val Asp Pro Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe 180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His 195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Ser Lys Ile Pro 210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp ValAsp Val Phe Ala 260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu 275 280 285 Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg 290 295 300 Asp Val Tyr Gly Ala Lys Ser Glu Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr 340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser 355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu 370 375 380 Glu Thr Gly Val Ala Agn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr 385 390 395 400 Val Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe 405 410 415 Glu Gln Ile Arg Glu Arg Gly Leu Arg Ala His Pro Asp Gly Arg His 420 425 430 Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly 435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Gln Ala 450 455 460 Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Val His His Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu 485 490 495 Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Ser Gly Arg Asp Asp 500 505 510 Thr Ala Val Thr Ala Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Asp 515 520 525 Gly Ser Asn Met Ile Ala Pro Met Leu Asp Ala Val Arg Ala Glu Ala 530 535 540 Thr Leu Gly Glu Ile Cys Asp Val Leu Arg Glu Glu Trp Gly Val Tyr 545 550 555 560 Thr Glu Pro Ala Gly Phe 565 <210>265 <211>417 <212>DNA <213>除虫链霉菌 <400>265 ctacgccccg gcaggctgcc gcacgttcgc ccgcacccac tccacgatcg acgccgtggt 60 cgcccccgga gtgaagatct ccgcgacacc cttctccttc agcggcgcga tgtccgcctc 120 ggggatgatg ccgccaccga acaccttgat gtcctcggca tcgcgctcct tgagcagatc 180 gatgaccgcc gcgaacaacg tgttgtgcgc cccggacagg atcgacagcc cgatcgcgtc 240 ggcgtcctcc tggatggccg tgcccacgat ctgctccggc gtctggtgca gccccgtgta 300 aatgacctcc ataccggcat cgcgcagcgc ccgcgcgatc accttggccc cgcgatcgtg 360 gccatcgagc cccggcttgg ccaccaccac gcggatcgga ccggctgcca cacccat 417 <210>266 <211>138 <212>PRT <213>除虫链霉菌 <400>266 Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg 20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu 35 40 45 Gln Ile Val Gly Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu 50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Asp 65 70 75 80 Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala 100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Glu Trp Val 115 120 125 Arg Ala Asn Val Arg Gln Pro Ala Gly Ala 130 135 <210>267 <211>2910 <212>DNA <213>金黄色葡萄球菌 <400>267 gatcaatttc ttttaagtaa tctaaatccc cattttttaa tttcttttta gcctctttaa 60 ataatcctga ataaactaat acctgtttac ctttaagtga tttataaaat gcatcaaaga 120 ctttttgatt tatttattaa ataatcacta tctttaccag aatacttagc catttcatat 180 aattctttat tattattttg tcttattttt tgaacttgaa cttgtgttat ttctgaaatg 240 cccgttacat cacgccataa atctaaccat tcttgttggc taatataata tcttttatct 300 gtgaaatacg atttatttac tgcaattaac acatgaaaat gaggattata atcatctctt 360 tttttattat atgtaatctc taacttacga acatatccct ttataacact acctactttt 420 tttctcttta taagttttct aaaagaatta ttataacgtt ttatttcatt ttctaattca 480 tcactcatta cattaggtgt agtcaaagtt aaaaagataa actccttttt ctcttgctgc 540 ttaatatatt gcatcatcaa agataaaccc aatgcatctt ttctagcttt tctccaagca 600 cagacaggac aaaatcgatt tttacaagaa ttagctttat ataatttctg tttttctaaa 660 gttttatcag ctacaaaaga cagaaatgta ttgcaatctt caactaaatc catttgattc 720 tctccaatat gacgtttaat aaatttctga aatacttgat ttctttgttt tttctcagta 780 tacttttcca tgttataaca cataaaaaca acttagtttt cacaaactat gacaataaaa 840 aaagttgctt tttccccttt ctatgtatgt tttttactag tcatttaaaa cgatacatta 900 ataggtacga aaaagcaact ttttttgcgc ttaaaaccag tcataccaat aacttaaggg 960 taactagcct cgccggcaat agttaccctt attatcaaga taagaaagaa aaggattttt 1020 cgctacgctc aaatccttta aaaaaacaca aaagaccaca ttttttaatg tggtctttat 1080 tcttcaacta aagcacccat tagttcaaca aacgaaaatt ggataaagtg ggatattttt 1140 aaaatatata tttatgttac agtaatattg acttttaaaa aaggattgat tctaatgaag 1200 aaagcagaca agtaagcctc ctaaattcac tttagataaa aatttaggag gcatatcaaa 1260 tgaactttaa taaaattgat ttagacaatt ggaagagaaa agagatattt aatcattatt 1320 tgaaccaaca aacgactttt agtataacca cagaaattga tattagtgtt ttataccgaa 1380 acataaaaca agaaggatat aaattttacc ctgcatttat tttcttagtg acaagggtga 1440 taaactcaaa tacagctttt agaactggtt acaatagcga cggagagtta ggttattggg 1500 ataagttaga gccactttat acaatttttg atggtgtatc taaaacattc tctggtattt 1560 ggactcctgt aaagaatgac ttcaaagagt tttatgattt atacctttct gatgtagaga 1620 aatataatgg ttcggggaaa ttgtttccca aaacacctat acctgaaaat gctttttctc 1680 tttctattat tccatggact tcatttactg ggtttaactt aaatatcaat aataatagta 1740 attaccttct acccattatt acagcaggaa aattcattaa taaaggtaat tcaatatatt 1800 taccgctatc tttacaggta catcattctg tttgtgatgg ttatcatgca ggattgttta 1860 tgaactctat tcaggaattg tcagataggc ctaatgactg gcttttataa tatgagataa 1920 tgccgactgt actttttaca gtcggttttc taatgtcact aacctgcccc gttagttgaa 1980 gaaggttttt atattacagc tccagatcca tatccttctt tttctgaacc gacttctcct 2040 ttttcgcttc tttattccaa ttgctttatt gacgttgagc ctcggaaccc ttaacaatcc 2100 caaaacttgt cgaatggtcg gcttaatagc tcacgctatg ccgacattcg tctgcaagtt 2160 tagttaaggg ttcttctcaa cgcacaataa attttctcgg cataaatgcg tggtctaatt 2220 tttattttta ataaccttga tagcaaaaaa tgccattcca atacaaaacc acatacctat 2280 aatcgataac cacataacag tcataaaacc actccttttt aacaaacttt atcacaagaa 2340 atatttaaat tttaaatgcc tttattttga attttaaggg gcattttaaa gatttagggg 2400 taaatcatat agttttatgc ctaaaaacct acagaagctt ttaaaaagca aatatgagcc 2460 aaataaatat attctaattc tacaaacaaa aatttgagca aattcagtgt cgatttttta 2520 agacactgcc cagttacatg caaattaaaa ttttcatgat tttttatagt tcctaacagg 2580 gttaaaattt gtataacgaa agtataatgt ttatataacg ttagtataat aaagcatttt 2640 aacattatac ttttgataat cgtttatcgt cgtcatcaca ataactttta aaatactcgt 2700 gcataattca cgctgacctc ccaataacta catggtgtta tcgggaggtc agctgttagc 2760 acttatattt tgttattgtt cttcctcgat ttcgtctatc attttgtgat taatttctct 2820 tttttcttgt tctgttaagt cataaagttc actagctaaa tactcttttt gtttccaaat 2880 ataaaaaatt tgatagatat attacggttg 2910 |