具体实施方式

本发明对酿酒酵母BY4742(MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0)菌株内在的代谢途径进行了改变，如图1所示，可以从葡萄糖出发，通过酶系催化实现β-丙氨酸的生物合成。

在该代谢途径中，酿酒酵母菌可以摄入葡萄糖作为碳源，代谢生成丙酮酸和琥珀酰-CoA，其中丙酮酸可在乳酸脱氢酶的催化下生成乳酸；琥珀酰-CoA先后经甲基丙二酰-CoA变构酶和甲基丙二酰-CoA脱羧酶催化生成丙酰-CoA；丙酰-CoA和乳酸经丙酸CoA转移酶催化发生转酰基反应，生成乳酰基-CoA；进一步经乳酰基-CoA脱水酶、硫酯裂解酶催化生成丙烯酸；丙烯酸可在无机铵存在的条件下，经氨基裂合酶催化生成β-丙氨酸。

在本文中，为了描述简便，有时会将某种酶比如硫酯裂解酶tesB与其编码基因(DNA)名称混用，本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如，对于TesB，用于描述硫酯裂解酶的功能或类别时，指的是蛋白质；在作为一种基因描述时，指的是编码该酶的基因。

应理解，本发明中整合入酿酒酵母基因组的MMUT基因、YgfG基因、Pct基因、lcdA基因、lcdB基因、TesB基因和AspB基因还可以分别单独地克隆在一个质粒上，然后分别地、或者同时地转入同一个酿酒酵母菌感受态细胞中；也可以将两个以上基因相组合地克隆在一个质粒上；也可以像图2所示的那样，将这7种基因形成基因簇克隆在一个质粒上，然后分别地、或者同时地转入同一个酿酒酵母菌感受态细胞中，经筛选阳性克隆，得到β-丙氨酸基因工程菌。所述质粒载体可以是任何适合于在酿酒酵母BY4742中表达的质粒载体例如pUC57等。

在本文中，对于本发明，术语“β-丙氨酸基因工程生产菌”、“β-丙氨酸基因工程菌”、“丙氨酸生产菌”都表示构建的酿酒酵母BY4742工程菌，尤其是酿酒酵母BY4742(MATαhis3Δ1 leu2Δ0 lys2Δ0 ura3Δ0)工程菌。

为了在酿酒酵母BY4742中表达中最佳地表达甲基丙二酰-CoA变构酶SEQ ID NO:1、甲基丙二酰-CoA脱羧酶SEQ ID NO:3、丙酸CoA-转移酶SEQ ID NO:5、乳酰基-CoA脱水酶alfa亚基SEQ ID NO:7、乳酰基-CoA脱水酶beta亚基SEQ ID NO:9、硫酯裂解酶SEQ ID NO:11和氨基裂合酶SEQ ID NO:13，本发明对它们的表达基因进行了密码子优化。

密码子优化是可用于通过增加感兴趣基因的翻译效率使生物体中蛋白质表达最大化的一种技术。不同的生物体由于突变倾向和天然选择而通常示出对于编码相同氨基酸的一些密码子之一的特殊偏好性。例如，在生长快速的微生物如大肠杆菌中，优化密码子反映出其各自的基因组tRNA库的组成。因此，在生长快速的微生物中，氨基酸的低频率密码子可以用用于相同氨基酸的但高频率的密码子置换。因此，优化的DNA序列的表达在快速生长的微生物中得以改良。

经过针对酿酒酵母进行密码子优化，上述7种酶SEQ ID NO:1、3、5、7、9、11、13的编码基因分别可以是SEQ ID NO:2、4、6、8、10、12、14。

以下结合具体实施例对本发明做进一步详细说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。

本文中涉及到多种物质的添加量、含量及浓度，其中所述的百分含量，除特别说明外，皆指质量百分含量。

实施例

材料和方法

实施例中的全基因合成、引物合成及测序皆由苏州金唯智生物科技有限公司完成。

本文中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版)，J.萨姆布鲁克，D.W.拉塞尔(美)编著，黄培堂等译，科学出版社，北京，2002)进行。比如感受态细胞转化方法及感受态制备方法均参照第1章96页进行。必要时可以通过简单试验确定具体实验条件。

PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。

培养基：

LB培养基：5g/L酵母提取物，10g/L胰蛋白胨，10g/L氯化钠。(LB固体培养基另加20g/L琼脂粉。)

YPD培养基：10g/L酵母提取物，20g/L胰蛋白胨，20g/L葡萄糖。(固体培养基另加20g/L琼脂粉。)

YPD-NH4培养基：10g/L酵母提取物，20g/L胰蛋白胨，20g/L葡萄糖，根据需要在发酵培养至48h，添加15％的氨水。

以下实施例中，当使用含卡那霉素的培养基时，所述卡那霉素在培养基中的终浓度为50μg/ml。

20X电转母液：80g/L甘氨酸，2％吐温80。

丙烯酸HPLC检测方法：

检测仪器：安捷伦1260高效液相色谱仪，色谱柱：上海月旭OAA色谱柱，流动相A(100％)：10mM磷酸二氢钾(0.68g到500ml水)磷酸调至PH2.5，流速1mL/min，柱温箱20℃，检测时间20min，采集波长210nm，进样量5ul。

β-丙氨酸HPLC检测方法：

检测仪器：安捷伦1260高效液相色谱仪，色谱柱：安捷伦SB-C18色谱柱或依利特BDS-C18柱，流动相A：2.871g无水乙酸钠+700ml水，流动相B：甲醇，梯度程序见下表，流速1.0mL/min，柱温箱40℃，检测波长334nm，进样量5ul，检测时间20min。

衍生剂的配制：称取0.1372g邻苯二甲醛、0.0589g N-乙酰-L-半胱氨酸，加2ml无水乙醇，超声波震荡1min至溶解，加硼酸缓冲液至10ml(容量瓶定容)(P1F3位置)。

硼酸缓冲液配制：称取6.183g硼酸粉末，加250ml蒸馏水超声波震荡至溶解，然后用6mol NaOH溶液调pH至9.5，过滤，备用(P1F2位置)。P1F1位置是水针。衍生方法：1、使用默认补偿值以最大速度从位置P1F2上抽取5.00μl，在冲洗口清洗针3s。2、使用默认补偿值以最大速度从样品中抽取2.00μl。3、混合：将来自空气中的默认体积，以最大速度混合2次。4、等待0.1min，在冲洗口清洗针3s。5、使用默认补偿值以最大速度从位置P1F3上抽取1.00μl。6、混合：将来自空气中的默认体积，以最大速度混合6次。7、等待0.3min。8、在冲洗口清洗针3s。9、以最大速度，从针座上抽取32.00μl。10、将来自针座的默认体积，以最大速度混合2次。11、等待0.5min。12、进样。

实施例中所使用的出发菌株为酿酒酵母BY4742(MATα his3Δ1 leu2Δ0 lys2Δ0ura3Δ0)，由上海工业生物技术研发中心惠赠。

实施例中所使用的质粒pUC57-pathway委托苏州金唯智生物科技有限公司合成，任何单位和个人都可以获得这些质粒用于验证本发明，但未经允许不得用作其他用途，包括开发利用、科学研究和教学。

实施例1：β-丙氨酸代谢工程菌的构建

1.1根据图2的设计，将7种基因搭配与酿酒酵母BY4742(MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0)适配的功能启动子和终止子，形成7个基因表达盒，其中：lcdA基因，使用启动子pENO2，终止子CPS1t，形成pENO2-lcdA-CPS1t；pct基因，使用启动子pTEF2，终止子IDP1t，形成pTEF2-pct-IDP1t；lcdB基因，使用启动子pGPM1，终止子IDP1t，形成pGPM1-lcdB-IDP1t；ygfG基因，使用启动子pTPI1，终止子PRM5t，形成pTPI1-ygfG-PRM5t；tesB基因，使用启动子pPGK3，终止子PRM9t，形成pPGK3-tesB-PRM9t；MMUT基因，使用启动子pTEF1，终止子ADH1t，形成pTEF1-MMUT-ADH1t；AspB-11-D4基因，使用启动子pTDH3，终止子SPG5t，形成pTDH3-AspB-11-D4-SPG5t。

将上述基因簇设计后，送苏州金唯智公司合成，装载到质粒载体pUC57上，形成pUC57-pathway质粒，全长序列为16192bp，如图2所示。

1.2将酿酒酵母BY4742(MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0)菌种在YPD固体培养基上划线，30℃培养2天，挑单菌落转接装有4ml YPD液体培养基的试管。30℃，220rpm过夜培养，转接装有25ml YPD液体培养基的250ml摇瓶，30℃，220rpm培养4～6h，培养至OD600为0.8～1.0，菌液用于制备酿酒酵母转化感受态。感受态的制作和转化，使用Frozen-EZ Yeast Transformation II Kit进行，严格参考其使用说明书。

利用电转化方法将质粒pUC57-pathway转入感受态细胞中。转化产物涂布SC-his平板(葡萄糖20g/l，YNB基本氮源1.7g/l，赖氨酸、亮氨酸、尿嘧啶各50mg/l)，30℃培养4天。使用HIS3作为后续转化酿酒酵母宿主的筛选标记，阳性转化子可在缺少组氨酸的筛选平板上生长。

1.3筛选平板上，挑取阳性转化子，进行基因组抽提，以基因组为模板，分别使用下述两对验证引物Yz-1-F/Yz-1-R和Yz-2-F/Yz-2-R对转化子进行PCR验证，PCR反应体系配置严格参考High efficient&High fidelity PCR enzyme KOD FX(购自东洋纺公司)。PCR产物进行DNA凝胶电泳检测，两个PCR产物分别对应7935bp和6928bp大小。

验证引物：

Yz-1-F：ATGAATACTGATGTTAGAAT，

Yz-1-R：ATGTCTTATCAATATGTCAA。

Yz-2-F：TTAATGACCAACAAAATTTG，

Yz-2-R：TTATTCAGCTGCAGCCATAT。

实施例2：β-丙氨酸工程菌发酵验证

对实施例1中鉴定获得的基因型阳性转化子进行摇瓶发酵验证。将转化子分别接种YPD-NH4培养基进行发酵，30℃、220rpm培养3天。阳性转化子的β-丙氨酸最高产量为3g/L。相反，未进行β-丙氨酸代谢途径整合的宿主菌不产生丙烯酸和β-丙氨酸。

上述实施例表明，发明人针对酿酒酵母BY4742(MATα his3Δ1 leu2Δ0 lys2Δ0ura3Δ0)设计的β-丙氨酸代谢途径是成功的。整合了该代谢途径的菌株能够实现发酵液中β-丙氨酸的积累，具有工业化开发潜力。

应理解，上述实施例仅用于举例说明目的，而不是对本发明的限制。本领域技术人员在阅读了本发明的构思之后，对其作出的各种改变或调整，均应落入本发明的保护范围内，这些等价形式同样属于本文所附权利要求书限定的范围。

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130 135 140

Ile His Asn Leu Thr Arg Asp Ala Gly Phe His Ile Val Lys Glu Leu

145 150 155 160

Ile Phe Thr Ala Ser Pro Ile Thr Ala Gln Arg Ala Leu Ala Val Gly

165 170 175

Ile Leu Asn His Val Val Glu Val Glu Glu Leu Glu Asp Phe Thr Leu

180 185 190

Gln Met Ala His His Ile Ser Glu Lys Ala Pro Leu Ala Ile Ala Val

195 200 205

Ile Lys Glu Glu Leu Arg Val Leu Gly Glu Ala His Thr Met Asn Ser

210 215 220

Asp Glu Phe Glu Arg Ile Gln Gly Met Arg Arg Ala Val Tyr Asp Ser

225 230 235 240

Glu Asp Tyr Gln Glu Gly Met Asn Ala Phe Leu Glu Lys Arg Lys Pro

245 250 255

Asn Phe Val Gly His

260

<210> 4

<211> 786

<212> DNA

<213> 人工序列()

<400> 4

atgtcttatc aatatgtcaa tgtcgtcact attaataaag ttgctgttat tgaattcaat 60

tacggaagga aattgaatgc tttgtctaag gttttcattg atgatttgat gcaagctttg 120

tctgatttga atagacctga aattagatgt attattttga gagctccatc tggttctaaa 180

gttttttctg ctggtcatga tattcatgaa ttgccatctg gtggtagaga tccattgtct 240

tatgatgacc cattgagaca aattacaaga atgattcaaa agtttccaaa accaattatt 300

tctatggttg aaggttctgt ttggggtggt gcttttgaaa tgattatgtc ttctgatttg 360

attattgctg cttctacttc tactttttct atgactcctg ttaatttggg tgttccatat 420

aatttggttg gtattcataa tttgactaga gatgctggtt ttcatattgt taaagaattg 480

atttttactg cttctccaat tactgctcaa agagctttgg ctgttggtat tttgaatcat 540

gttgttgaag ttgaagaatt ggaagatttt actttgcaaa tggctcatca tatttctgaa 600

aaagctccat tggctattgc tgttattaaa gaagaattga gagttttggg tgaagctcat 660

actatgaatt ctgatgaatt tgaaagaatt caaggtatga gaagagctgt ttatgattct 720

gaagattatc aagaaggtat gaatgctttt ttggaaaaaa gaaaaccaaa ttttgttggt 780

cattaa 786

<210> 5

<211> 524

<212> PRT

<213> Escherichia coli (strain K12)

<400> 5

Met Arg Lys Val Pro Ile Ile Thr Ala Asp Glu Ala Ala Lys Leu Ile

1 5 10 15

Lys Asp Gly Asp Thr Val Thr Thr Ser Gly Phe Val Gly Asn Ala Ile

20 25 30

Pro Glu Ala Leu Asp Arg Ala Val Glu Lys Arg Phe Leu Glu Thr Gly

35 40 45

Glu Pro Lys Asn Ile Thr Tyr Val Tyr Cys Gly Ser Gln Gly Asn Arg

50 55 60

Asp Gly Arg Gly Ala Glu His Phe Ala His Glu Gly Leu Leu Lys Arg

65 70 75 80

Tyr Ile Ala Gly His Trp Ala Thr Val Pro Ala Leu Gly Lys Met Ala

85 90 95

Met Glu Asn Lys Met Glu Ala Tyr Asn Val Ser Gln Gly Ala Leu Cys

100 105 110

His Leu Phe Arg Asp Ile Ala Ser His Lys Pro Gly Val Phe Thr Lys

115 120 125

Val Gly Ile Gly Thr Phe Ile Asp Pro Arg Asn Gly Gly Gly Lys Val

130 135 140

Asn Asp Ile Thr Lys Glu Asp Ile Val Glu Leu Val Glu Ile Lys Gly

145 150 155 160

Gln Glu Tyr Leu Phe Tyr Pro Ala Phe Pro Ile His Val Ala Leu Ile

165 170 175

Arg Gly Thr Tyr Ala Asp Glu Ser Gly Asn Ile Thr Phe Glu Lys Glu

180 185 190

Val Ala Pro Leu Glu Gly Thr Ser Val Cys Gln Ala Val Lys Asn Ser

195 200 205

Gly Gly Ile Val Val Val Gln Val Glu Arg Val Val Lys Ala Gly Thr

210 215 220

Leu Asp Pro Arg His Val Lys Val Pro Gly Ile Tyr Val Asp Tyr Val

225 230 235 240

Val Val Ala Asp Pro Glu Asp His Gln Gln Ser Leu Asp Cys Glu Tyr

245 250 255

Asp Pro Ala Leu Ser Gly Glu His Arg Arg Pro Glu Val Val Gly Glu

260 265 270

Pro Leu Pro Leu Ser Ala Lys Lys Val Ile Gly Arg Arg Gly Ala Ile

275 280 285

Glu Leu Glu Lys Asp Val Ala Val Asn Leu Gly Val Gly Ala Pro Glu

290 295 300

Tyr Val Ala Ser Val Ala Asp Glu Glu Gly Ile Val Asp Phe Met Thr

305 310 315 320

Leu Thr Ala Glu Ser Gly Ala Ile Gly Gly Val Pro Ala Gly Gly Val

325 330 335

Arg Phe Gly Ala Ser Tyr Asn Ala Asp Ala Leu Ile Asp Gln Gly Tyr

340 345 350

Gln Phe Asp Tyr Tyr Asp Gly Gly Gly Leu Asp Leu Cys Tyr Leu Gly

355 360 365

Leu Ala Glu Cys Asp Glu Lys Gly Asn Ile Asn Val Ser Arg Phe Gly

370 375 380

Pro Arg Ile Ala Gly Cys Gly Gly Phe Ile Asn Ile Thr Gln Asn Thr

385 390 395 400

Pro Lys Val Phe Phe Cys Gly Thr Phe Thr Ala Gly Gly Leu Lys Val

405 410 415

Lys Ile Glu Asp Gly Lys Val Ile Ile Val Gln Glu Gly Lys Gln Lys

420 425 430

Lys Phe Leu Lys Ala Val Glu Gln Ile Thr Phe Asn Gly Asp Val Ala

435 440 445

Leu Ala Asn Lys Gln Gln Val Thr Tyr Ile Thr Glu Arg Cys Val Phe

450 455 460

Leu Leu Lys Glu Asp Gly Leu His Leu Ser Glu Ile Ala Pro Gly Ile

465 470 475 480

Asp Leu Gln Thr Gln Ile Leu Asp Val Met Asp Phe Ala Pro Ile Ile

485 490 495

Asp Arg Asp Ala Asn Gly Gln Ile Lys Leu Met Asp Ala Ala Leu Phe

500 505 510

Ala Glu Gly Leu Met Gly Leu Lys Glu Met Lys Ser

515 520

<210> 6

<211> 1575

<212> DNA

<213> 人工序列()

<400> 6

atgagaaaag ttccaattat tactgctgat gaagctgcta aattgattaa agatggtgat 60

actgttacta cttctggttt tgttggtaat gctattcctg aagctttgga tagagctgtt 120

gaaaaaagat ttttggaaac tggtgaacca aagaatatta catatgttta ttgtggttct 180

caaggtaata gagatggtag aggtgctgaa cattttgctc atgaaggttt gttgaaaaga 240

tatattgctg gtcattgggc tactgttcct gctttgggta aaatggctat ggaaaataaa 300

atggaagctt ataatgtttc tcaaggtgct ttgtgtcatt tgtttagaga tattgcttct 360

cataaacctg gtgtttttac taaagttggt attggtactt ttattgatcc aaggaatggt 420

ggaggtaaag ttaatgatat tactaaggag gatattgttg aattagttga aattaagggt 480

caagaatatt tgttttatcc tgcttttcca attcatgttg ctttgattag aggtacttat 540

gctgatgaat ctggtaatat tacttttgaa aaagaagttg ctccattgga aggtacttct 600

gtttgtcaag ctgttaaaaa ttctggtggt attgttgtcg ttcaagttga aagagttgtt 660

aaagctggta ctttggatcc aagacatgtt aaagttcctg gtatttatgt tgattatgtt 720

gtcgttgctg atcctgaaga tcatcaacaa tctttggatt gtgaatatga tcctgctttg 780

tctggtgaac atagaagacc tgaagttgtt ggtgaaccat tgccattgtc tgctaaaaaa 840

gttattggta gaagaggtgc tattgaattg gaaaaagatg ttgctgttaa tttgggtgtt 900

ggtgctcctg aatatgttgc ttctgttgct gatgaagaag gtattgttga ttttatgact 960

ttgactgctg aatctggtgc tattggtggt gttcctgctg gtggtgttag atttggtgct 1020

tcttataatg ctgatgcttt gattgatcaa ggttatcaat ttgattatta tgatggtgga 1080

ggtttggatt tgtgttattt gggtttggct gaatgtgatg aaaaaggtaa tattaatgtt 1140

tctagatttg gtccaagaat tgctggttgt ggtggtttta ttaatattac tcaaaatact 1200

ccaaaagttt ttttttgtgg tacttttact gctggtggat tgaaggttaa aatcgaagat 1260

ggaaaggtca ttatcgttca agaaggtaag cagaaaaaat tcttgaaagc tgttgaacaa 1320

attactttta atggtgatgt tgctttggct aataaacaac aagtcactta tattactgaa 1380

agatgtgttt ttttgttgaa agaagatggt ttgcatttgt ctgaaattgc tcctggtatt 1440

gatttgcaaa ctcaaatttt ggatgttatg gattttgctc caattattga tagagatgct 1500

aatggtcaaa ttaaattgat ggatgctgct ttgtttgctg aaggtttgat gggtttgaaa 1560

gaaatgaaat cttaa 1575

<210> 7

<211> 422

<212> PRT

<213> Anaerotignum propionicum DSM 1682

<400> 7

Met Ser Leu Thr Gln Gly Met Lys Ala Lys Gln Leu Leu Ala Tyr Phe

1 5 10 15

Gln Gly Lys Ala Asp Gln Asp Ala Arg Glu Ala Lys Ala Arg Gly Glu

20 25 30

Leu Val Cys Trp Ser Ala Ser Val Ala Pro Pro Glu Phe Cys Val Thr

35 40 45

Met Gly Ile Ala Met Ile Tyr Pro Glu Thr His Ala Ala Gly Ile Gly

50 55 60

Ala Arg Lys Gly Ala Met Asp Met Leu Glu Val Ala Asp Arg Lys Gly

65 70 75 80

Tyr Asn Val Asp Cys Cys Ser Tyr Gly Arg Val Asn Met Gly Tyr Met

85 90 95

Glu Cys Leu Lys Glu Ala Ala Ile Thr Gly Val Lys Pro Glu Val Leu

100 105 110

Val Asn Ser Pro Ala Ala Asp Val Pro Leu Pro Asp Leu Val Ile Thr

115 120 125

Cys Asn Asn Ile Cys Asn Thr Leu Leu Lys Trp Tyr Glu Asn Leu Ala

130 135 140

Ala Glu Leu Asp Ile Pro Cys Ile Val Ile Asp Val Pro Phe Asn His

145 150 155 160

Thr Met Pro Ile Pro Glu Tyr Ala Lys Ala Tyr Ile Ala Asp Gln Phe

165 170 175

Arg Asn Ala Ile Ser Gln Leu Glu Val Ile Cys Gly Arg Pro Phe Asp

180 185 190

Trp Lys Lys Phe Lys Glu Val Lys Asp Gln Thr Gln Arg Ser Val Tyr

195 200 205

His Trp Asn Arg Ile Ala Glu Met Ala Lys Tyr Lys Pro Ser Pro Leu

210 215 220

Asn Gly Phe Asp Leu Phe Asn Tyr Met Ala Leu Ile Val Ala Cys Arg

225 230 235 240

Ser Leu Asp Tyr Ala Glu Ile Thr Phe Lys Ala Phe Ala Asp Glu Leu

245 250 255

Glu Glu Asn Leu Lys Ala Gly Ile Tyr Ala Phe Lys Gly Ala Glu Lys

260 265 270

Thr Arg Phe Gln Trp Glu Gly Ile Ala Val Trp Pro His Leu Gly His

275 280 285

Thr Phe Lys Ser Met Lys Asn Leu Asn Ser Ile Met Thr Gly Thr Ala

290 295 300

Tyr Pro Ala Leu Trp Asp Leu His Tyr Asp Ala Asn Asp Glu Ser Met

305 310 315 320

His Ser Met Ala Glu Ala Tyr Thr Arg Ile Tyr Ile Asn Thr Cys Leu

325 330 335

Gln Asn Lys Val Glu Val Leu Leu Gly Ile Met Glu Lys Gly Gln Val

340 345 350

Asp Gly Thr Val Tyr His Leu Asn Arg Ser Cys Lys Leu Met Ser Phe

355 360 365

Leu Asn Val Glu Thr Ala Glu Ile Ile Lys Glu Lys Asn Gly Leu Pro

370 375 380

Tyr Val Ser Ile Asp Gly Asp Gln Thr Asp Pro Arg Val Phe Ser Pro

385 390 395 400

Ala Gln Phe Asp Thr Arg Val Gln Ala Leu Val Glu Met Met Glu Ala

405 410 415

Asn Met Ala Ala Ala Glu

420

<210> 8

<211> 1269

<212> DNA

<213> 人工序列()

<400> 8

atgtctttga ctcaaggtat gaaagctaaa caattgttgg cttattttca aggtaaagct 60

gatcaagatg ctagagaagc taaagctaga ggtgaattgg tttgttggtc tgcttctgtt 120

gctccacctg aattttgtgt tactatgggt attgctatga tttatcctga aactcatgct 180

gctggtattg gtgctagaaa aggtgctatg gatatgttgg aagttgctga tagaaaaggt 240

tataatgttg attgttgttc ttatggtaga gttaatatgg gttatatgga atgtttgaaa 300

gaagctgcta ttactggtgt taaacctgaa gttttggtta attctcctgc tgctgatgtt 360

cctttgcctg atttagtcat tacttgcaat aatatctgta atactttgtt gaaatggtat 420

gaaaatttgg ctgctgaatt ggatattcca tgtattgtta ttgatgttcc attcaatcat 480

actatgccaa tccctgaata tgctaaggct tatattgctg atcagtttag aaatgctatt 540

tctcagttgg aagttatttg tggtagacca ttcgattgga aaaaatttaa agaggtcaaa 600

gaccaaactc aaagatctgt ttatcattgg aatagaattg ctgaaatggc aaaatataaa 660

ccttctccat tgaatggttt tgatttgttt aattacatgg ctttgattgt tgcttgtaga 720

tctttggatt atgctgaaat tacttttaaa gcttttgctg atgaattgga agaaaatttg 780

aaagctggta tttatgcttt taaaggtgct gaaaaaacta gatttcaatg ggaaggtatt 840

gctgtttggc cacatttggg tcatactttt aaatctatga aaaatttgaa ttctattatg 900

actggtactg cttatcctgc tttgtgggat ttgcattatg atgctaatga tgaatcaatg 960

cactctatgg ctgaagctta cactaggatt tatattaata catgtttgca aaataaagtt 1020

gaagttttgt tgggtattat ggaaaaaggt caagttgacg gaactgttta tcacttgaac 1080

agatcttgca aattaatgtc tttcttaaat gttgaaactg cagaaattat taaggagaaa 1140

aatggtttgc catatgtttc tattgatggt gatcaaactg atccaagagt tttttctcct 1200

gctcaatttg atactagagt tcaagctttg gttgaaatga tggaagctaa tatggctgca 1260

gctgaataa 1269

<210> 9

<211> 374

<212> PRT

<213> Anaerotignum propionicum DSM 1682

<400> 9

Met Ser Arg Val Glu Ala Ile Leu Ser Gln Leu Lys Asp Val Ala Ala

1 5 10 15

Asn Pro Lys Lys Ala Met Asp Asp Tyr Lys Ala Glu Thr Gly Lys Gly

20 25 30

Ala Val Gly Ile Met Pro Ile Tyr Ser Pro Glu Glu Met Val His Ala

35 40 45

Ala Gly Tyr Leu Pro Met Gly Ile Trp Gly Ala Gln Gly Lys Thr Ile

50 55 60

Ser Lys Ala Arg Thr Tyr Leu Pro Ala Phe Ala Cys Ser Val Met Gln

65 70 75 80

Gln Val Met Glu Leu Gln Cys Glu Gly Ala Tyr Asp Asp Leu Ser Ala

85 90 95

Val Ile Phe Ser Val Pro Cys Asp Thr Leu Lys Cys Leu Ser Gln Lys

100 105 110

Trp Lys Gly Thr Ser Pro Val Ile Val Phe Thr His Pro Gln Asn Arg

115 120 125

Gly Leu Glu Ala Ala Asn Gln Phe Leu Val Thr Glu Tyr Glu Leu Val

130 135 140

Lys Ala Gln Leu Glu Ser Val Leu Gly Val Lys Ile Ser Asn Ala Ala

145 150 155 160

Leu Glu Asn Ser Ile Ala Ile Tyr Asn Glu Asn Arg Ala Val Met Arg

165 170 175

Glu Phe Val Lys Val Ala Ala Asp Tyr Pro Gln Val Ile Asp Ala Val

180 185 190

Ser Arg His Ala Val Phe Lys Ala Arg Gln Phe Met Leu Lys Glu Lys

195 200 205

His Thr Ala Leu Val Lys Glu Leu Ile Ala Glu Ile Lys Ala Thr Pro

210 215 220

Val Gln Pro Trp Asp Gly Lys Lys Val Val Val Thr Gly Ile Leu Leu

225 230 235 240

Glu Pro Asn Glu Leu Leu Asp Ile Phe Asn Glu Phe Lys Ile Ala Ile

245 250 255

Val Asp Asp Asp Leu Ala Gln Glu Ser Arg Gln Ile Arg Val Asp Val

260 265 270

Leu Asp Gly Glu Gly Gly Pro Leu Tyr Arg Met Ala Lys Ala Trp Gln

275 280 285

Gln Met Tyr Gly Cys Ser Leu Ala Thr Asp Thr Lys Lys Gly Arg Gly

290 295 300

Arg Met Leu Ile Asn Lys Thr Ile Gln Thr Gly Ala Asp Ala Ile Val

305 310 315 320

Val Ala Met Met Lys Phe Cys Asp Pro Glu Glu Trp Asp Tyr Pro Val

325 330 335

Met Tyr Arg Glu Phe Glu Glu Lys Gly Val Lys Ser Leu Met Ile Glu

340 345 350

Val Asp Gln Glu Val Ser Ser Phe Glu Gln Ile Lys Thr Arg Leu Gln

355 360 365

Ser Phe Val Glu Met Leu

370

<210> 10

<211> 1125

<212> DNA

<213> 人工序列()

<400> 10

atgtctagag ttgaagctat tttgtctcaa ttgaaagatg ttgctgctaa tccaaaaaaa 60

gctatggatg attataaagc tgaaactggt aaaggtgctg ttggtattat gccaatttat 120

tctcctgaag aaatggttca tgctgctggt tatttgccaa tgggtatttg gggtgctcaa 180

ggtaaaacta tttctaaagc tagaacttat ttgcctgctt ttgcttgttc tgttatgcaa 240

caagttatgg aattgcaatg tgaaggtgct tatgatgatt tgtctgctgt tattttttct 300

gttccatgtg atactttgaa atgtttgtct caaaaatgga aaggtacttc tcctgttatt 360

gtttttactc atccacaaaa tagaggtttg gaagctgcta atcaattttt ggttactgaa 420

tatgaattgg ttaaagctca attggaatct gtcttgggag ttaaaatctc taatgctgct 480

ttggagaatt caattgctat ctataatgaa aatagagctg ttatgagaga atttgttaaa 540

gttgctgctg attatccaca agttattgat gctgtttcta gacatgctgt ttttaaagct 600

agacaattta tgttgaaaga aaaacatact gctttggtta aagaattgat tgctgaaatt 660

aaagctactc ctgttcaacc atgggatggt aaaaaagttg ttgtcactgg tattttgtta 720

gaaccaaacg agttgttaga tatctttaat gaattcaaaa ttgctattgt tgatgacgat 780

ttggctcaag aatctagaca aattagagtt gatgttttgg atggtgaagg tggtccattg 840

tatagaatgg ctaaagcttg gcaacaaatg tatggttgtt ctttggctac tgatactaaa 900

aaaggtagag gtagaatgtt gattaataaa actattcaaa ctggtgctga tgctattgtt 960

gttgctatga tgaaattttg tgatcctgaa gaatgggatt atcctgttat gtatagagaa 1020

ttcgaagaga aaggagttaa atctttaatg attgaagttg accaagaagt ttcatctttt 1080

gagcagatta aaactagatt gcaatctttt gttgaaatgt tgtaa 1125

<210> 11

<211> 286

<212> PRT

<213> Escherichia coli (strain K12)

<400> 11

Met Ser Gln Ala Leu Lys Asn Leu Leu Thr Leu Leu Asn Leu Glu Lys

1 5 10 15

Ile Glu Glu Gly Leu Phe Arg Gly Gln Ser Glu Asp Leu Gly Leu Arg

20 25 30

Gln Val Phe Gly Gly Gln Val Val Gly Gln Ala Leu Tyr Ala Ala Lys

35 40 45

Glu Thr Val Pro Glu Glu Arg Leu Val His Ser Phe His Ser Tyr Phe

50 55 60

Leu Arg Pro Gly Asp Ser Lys Lys Pro Ile Ile Tyr Asp Val Glu Thr

65 70 75 80

Leu Arg Asp Gly Asn Ser Phe Ser Ala Arg Arg Val Ala Ala Ile Gln

85 90 95

Asn Gly Lys Pro Ile Phe Tyr Met Thr Ala Ser Phe Gln Ala Pro Glu

100 105 110

Ala Gly Phe Glu His Gln Lys Thr Met Pro Ser Ala Pro Ala Pro Asp

115 120 125

Gly Leu Pro Ser Glu Thr Gln Ile Ala Gln Ser Leu Ala His Leu Leu

130 135 140

Pro Pro Val Leu Lys Asp Lys Phe Ile Cys Asp Arg Pro Leu Glu Val

145 150 155 160

Arg Pro Val Glu Phe His Asn Pro Leu Lys Gly His Val Ala Glu Pro

165 170 175

His Arg Gln Val Trp Ile Arg Ala Asn Gly Ser Val Pro Asp Asp Leu

180 185 190

Arg Val His Gln Tyr Leu Leu Gly Tyr Ala Ser Asp Leu Asn Phe Leu

195 200 205

Pro Val Ala Leu Gln Pro His Gly Ile Gly Phe Leu Glu Pro Gly Ile

210 215 220

Gln Ile Ala Thr Ile Asp His Ser Met Trp Phe His Arg Pro Phe Asn

225 230 235 240

Leu Asn Glu Trp Leu Leu Tyr Ser Val Glu Ser Thr Ser Ala Ser Ser

245 250 255

Ala Arg Gly Phe Val Arg Gly Glu Phe Tyr Thr Gln Asp Gly Val Leu

260 265 270

Val Ala Ser Thr Val Gln Glu Gly Val Met Arg Asn His Asn

275 280 285

<210> 12

<211> 861

<212> DNA

<213> 人工序列()

<400> 12

atgtctcaag ctttgaaaaa tttgttgact ttgttgaatt tggaaaaaat tgaagaaggt 60

ttgtttagag gtcaatctga agatttgggt ttgagacaag tttttggtgg tcaagttgtt 120

ggtcaagctt tgtatgctgc taaagaaact gttcctgaag aaagattggt tcattctttt 180

cactcttatt ttttgagacc tggtgattct aaaaaaccaa ttatttatga cgttgaaact 240

ttgagagatg gtaattcttt ttctgctaga agagttgctg ctattcaaaa tggtaaacca 300

attttttata tgactgcttc ttttcaagct cctgaagctg gttttgaaca tcaaaaaact 360

atgccatctg ctcctgctcc tgatggtttg ccatctgaaa ctcaaattgc tcaatctttg 420

gctcatttgt tgccacctgt tttgaaagat aaatttattt gtgatagacc attggaagtt 480

agacctgttg aatttcataa tccattgaaa ggtcatgttg ctgaaccaca tagacaagtt 540

tggattagag ctaatggttc tgttcctgat gatttgagag ttcatcaata tttgttgggt 600

tatgcttctg atttgaattt tttgcctgtt gctttgcaac cacatggtat tggttttttg 660

gaacctggta ttcaaattgc tactattgat cattctatgt ggtttcatag accatttaat 720

ttgaatgaat ggttgttgta ttctgttgaa tctacttctg cttcttctgc tagaggtttt 780

gttagaggtg aattttatac tcaagatggt gttttggttg cttctactgt tcaagaaggt 840

gttatgagaa atcataatta a 861

<210> 13

<211> 468

<212> PRT

<213> 人工序列()

<400> 13

Met Asn Thr Asp Val Arg Ile Glu Lys Asp Phe Leu Gly Glu Lys Glu

1 5 10 15

Ile Pro Lys Asp Ala Tyr Tyr Gly Val Gln Thr Ile Arg Ala Thr Glu

20 25 30

Asn Phe Pro Ile Thr Gly Tyr Arg Ile His Pro Glu Leu Ile Lys Ser

35 40 45

Leu Gly Ile Val Lys Lys Ser Ala Ala Leu Ala Asn Met Glu Val Gly

50 55 60

Leu Leu Asp Lys Glu Val Gly Gln Tyr Ile Val Lys Ala Ala Asp Glu

65 70 75 80

Val Ile Glu Gly Lys Trp Asn Asp Gln Phe Ile Val Asp Pro Ile Gln

85 90 95

Gly Gly Ala Gly Thr Ser Ile Asn Met Asn Ala Asn Glu Val Ile Ala

100 105 110

Asn Arg Ala Leu Glu Leu Met Gly Glu Glu Lys Gly Asn Tyr Ser Lys

115 120 125

Ile Ser Pro Asn Ser His Val Asn Met Ser Gln Ser Thr Val Asp Ala

130 135 140

Phe Pro Thr Ala Thr His Ile Ala Val Leu Ser Leu Leu Asn Gln Leu

145 150 155 160

Ile Glu Thr Thr Lys Tyr Met Gln Gln Glu Phe Met Lys Lys Ala Asp

165 170 175

Glu Phe Ala Gly Val Ile Lys Met Gly Arg Cys Ala Leu Gln Asp Ala

180 185 190

Val Pro Ile Leu Leu Gly Gln Glu Phe Glu Ala Tyr Ala Arg Val Ile

195 200 205

Ala Arg Asp Ile Glu Arg Ile Ala Asn Thr Arg Asn Asn Leu Tyr Asp

210 215 220

Ile Asn Met Gly Ala Thr Ala Val Gly Thr Gly Leu Asn Ala Asp Pro

225 230 235 240

Glu Tyr Ile Ser Ile Val Thr Glu His Leu Ala Lys Phe Ser Gly His

245 250 255

Pro Leu Arg Ser Ala Gln His Leu Val Asp Ala Thr Gln Asn Thr Asp

260 265 270

Cys Tyr Thr Glu Val Ser Ser Ala Leu Lys Val Cys Met Ile Asn Met

275 280 285

Ser Lys Ile Ala Asn Asp Leu Arg Leu Met Ala Ser Gly Pro Arg Ala

290 295 300

Gly Leu Ser Glu Ile Val Leu Pro Ala Arg Gln Pro Gly Ser Ser Ile

305 310 315 320

Ile Pro Gly Leu Val Ala Pro Val Met Pro Glu Val Met Asn Gln Val

325 330 335

Ala Phe Gln Val Phe Gly Asn Asp Leu Thr Ile Thr Ser Ala Ser Glu

340 345 350

Ala Gly Gln Phe Glu Leu Asn Val Met Glu Pro Val Leu Phe Phe Asn

355 360 365

Leu Ile Gln Ser Ile Ser Ile Met Thr Asn Val Phe Lys Ser Phe Thr

370 375 380

Glu Asn Cys Leu Lys Gly Ile Lys Ala Asn Glu Glu Arg Met Lys Glu

385 390 395 400

Tyr Val Glu Lys Ser Ile Gly Ile Ile Thr Ala Ile Asn Pro His Val

405 410 415

Gly Tyr Glu Thr Ala Ala Lys Leu Ala Arg Glu Ala Tyr Leu Thr Gly

420 425 430

Glu Ser Ile Arg Glu Leu Cys Ile Lys Tyr Gly Val Leu Thr Glu Glu

435 440 445

Gln Leu Asn Glu Ile Leu Asn Pro Tyr Glu Met Thr His Pro Gly Ile

450 455 460

Ala Gly Arg Lys

465

<210> 14

<211> 1407

<212> DNA

<213> 人工序列()

<400> 14

atgaatactg atgttagaat tgaaaaagat tttttgggtg aaaaagaaat tccaaaagat 60

gcttattacg gtgttcaaac tattagagct actgagaatt ttccaattac tggttataga 120

attcatcctg aattgattaa atctttgggt attgttaaaa aatctgctgc tttggctaat 180

atggaagttg gtttgttgga taaagaagtt ggtcaatata ttgttaaagc tgctgatgag 240

gtcattgaag gtaaatggaa tgatcaattt attgttgatc caattcaagg tggtgctggt 300

acttctatta atatgaatgc taatgaagtt attgctaata gagctttgga attgatggga 360

gaagaaaaag gtaactattc taaaatctct ccaaattctc atgttaatat gtctcaatct 420

actgttgatg cttttccaac tgctactcat attgcagttt tatctttgtt aaatcaattg 480

attgaaacaa ctaaatatat gcagcaagaa tttatgaaaa aagctgatga atttgctggt 540

gttattaaaa tgggtagatg tgctttgcaa gatgctgttc caattttgtt gggtcaagaa 600

tttgaagctt atgctagggt tatcgctagg gatattgaaa gaattgcaaa tacaaggaat 660

aatttgtatg atattaatat gggtgctact gctgttggta ctggtttgaa tgctgatcct 720

gaatatattt ctattgttac tgaacatttg gctaaatttt ctggtcatcc attgagatct 780

gctcaacatt tggttgatgc tactcaaaat actgattgtt atactgaggt ctcttctgct 840

ttgaaagttt gtatgattaa catgtctaaa attgctaatg atttgagatt gatggcttct 900

ggtccaagag ctggtttgtc tgaaattgtt ttgcctgcta gacaacctgg ttcttctatt 960

attcctggtt tggttgctcc tgttatgcct gaagttatga atcaagttgc ttttcaagtt 1020

tttggtaatg atttgactat tacttctgct tctgaagctg gtcagttcga attgaacgtc 1080

atggagcctg ttttattttt caatttgatt cagtctatct caattatgac taacgttttc 1140

aagtctttta ctgaaaattg tttgaaggga attaaagcta atgaggaaag aatgaaggaa 1200

tatgtcgaaa agtctattgg tattatcact gctattaatc cacatgttgg ttatgaaact 1260

gctgctaaat tggctagaga agcttatttg actggtgaat ctattagaga attgtgtatt 1320

aagtatggtg tcttgactga agaacaattg aatgaaattt tgaatccata tgaaatgact 1380

catcctggta ttgctggtag aaaataa 1407