CN112280722A

CN112280722A - 用于生产光学纯1,3-丁二醇的重组菌及其应用

Info

Publication number: CN112280722A
Application number: CN201910664906.6A
Authority: CN
Inventors: 陈振; 刘德华; 刘煜
Original assignee: Guangdong Tsinghua Smart Biotech Co ltd; Tsinghua University
Current assignee: Guangdong Tsinghua Smart Biotech Co ltd; Tsinghua University
Priority date: 2019-07-23
Filing date: 2019-07-23
Publication date: 2021-01-29
Anticipated expiration: 2039-07-23
Also published as: CN112280722B

Abstract

本发明提供一种用于生产光学纯1,3‑丁二醇的重组菌及其应用。所述重组菌是将phaA、phaB、bld和yqhD基因通过质粒导入微生物中或通过基因工程手段整合到微生物染色体上而成。利用本发明的大肠杆菌工程菌在微氧的条件下发酵廉价有机碳源如葡萄糖、蔗糖或甘油等，1,3‑丁二醇相对于底物的得率可以达到0.4g/g以上，且R型1,3‑丁二醇纯度达到99％以上，具有重要的工业应用潜力。

Description

用于生产光学纯1,3-丁二醇的重组菌及其应用

技术领域

本发明属于生物化工技术领域，具体地说，涉及一种用于生产光学纯1,3-丁二醇的重组菌及其应用。

背景技术

1,3-丁二醇是一种具有重要工业应用价值的二元醇，其可以被用作化妆品的溶剂，同时可以用作单体来合成聚酯、聚氨酯及生物增塑剂。同时光学纯的1,3-丁二醇可以用作医药中间体来合成许多药物。但是化学法合成的1,3-丁二醇都是外消旋体，无法直接用作医药中间体。因此，利用生物法生产光学纯1,3-丁二醇具有重要应用价值。

CN201610481598.X和CN201080029715.X分别公开了两种1,3-丁二醇的生产方法，其主要是在厌氧条件下表达至少一种编码以足够的量表达的1,3-BDO通路酶的外源性核酸，以生产1,3-丁二醇。但该过程的生产效率低，1,3-丁二醇的最高产量低于2mM，不具有工业可行性。

发明内容

本发明的目的是提供一种用于生产光学纯1,3-丁二醇的重组菌及其应用。

本发明构思如下：提供一种重组大肠杆菌，其是通过上调大肠杆菌的pntAB基因并同时下调sthA基因从而极大提高细胞内的NADPH基因以满足1,3-丁二醇合成途径所需要的还原力NADPH，从而提高1,3-丁二醇的产量。本发明同时通过酶的设计构建了Clostridiumsaccharoperbutylacetonicum的丁醛脱氢酶突变体，其是将丁醛脱氢酶第273位亮氨酸突变为苏氨酸而成，在大肠杆菌中表达该突变体可以极大提高1,3-丁二醇的产量。本发明同时通过敲除adhE基因，ldhA基因，pta基因，ackA基因提高了乙酰-CoA流向1,3-丁二醇合成途径的代谢通量，进一步提高了1,3-丁二醇的产量。

为了实现本发明目的，第一方面，本发明提供一种用于生产光学纯1,3-丁二醇的重组菌，所述重组菌是将phaA、phaB、bld和yqhD基因通过质粒导入微生物中或通过基因工程手段整合到微生物染色体上而成。

其中，所述phaA基因为乙酰CoA酰基转移酶基因，来源于Cupriavidus necator，其为编码如下蛋白质(a)或(b)的基因：

(a)由SEQ ID NO:3所示的氨基酸序列组成的蛋白质；

(b)SEQ ID NO:3所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质。

所述phaB基因为3-氧酰基-(酰基载体蛋白)还原酶基因，来源于Cupriavidusnecator，其为编码如下蛋白质(c)或(d)的基因：

(c)由SEQ ID NO:4所示的氨基酸序列组成的蛋白质；

(d)SEQ ID NO:4所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(c)衍生的蛋白质。

所述bld基因来源于Clostridium saccharoperbutylacetonicum，其编码蛋白的氨基酸序列如SEQ ID NO:1或2所示。

所述yqhD基因为醇脱氢酶基因，来源于大肠杆菌(Escherichia coli)，其为编码如下蛋白质(e)或(f)的基因：

(e)由SEQ ID NO:6所示的氨基酸序列组成的蛋白质；

(f)SEQ ID NO:6所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(e)衍生的蛋白质。

所述微生物选自埃希氏菌属(Escherichia)、克雷伯氏菌属(Klebsiella)、棒杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)等中的菌种；优选大肠杆菌。

所述重组菌可按如下方法构建得到：phaA、phaB基因经密码子优化后，与bld、yqhD基因一起构建到表达载体上，用重组载体转化大肠杆菌，筛选阳性转化子。

优选地，密码子优化的phaA-phaB操纵子的序列如SEQ ID NO:5所示。

更优选地，所述重组菌的构建如下：将phaAB-bld2-yqhD串联基因表达盒构建到ptrc99a质粒上，用重组质粒转化大肠杆菌(如W3110)，筛选阳性转化子。

其中，bld-yqhD-phaAB串联基因表达盒的序列如SEQ ID NO:9和10所示。

第二方面，本发明提供一种用于生产光学纯1,3-丁二醇的重组大肠杆菌，所述重组大肠杆菌是以上述重组菌作为出发菌株，利用基因工程手段对出发菌株进行改造，得到的细胞内NADPH供给增强的工程菌。包括如下方案：

方案I：通过增强出发菌株中与NADPH生物合成途径相关的基因，来提高细胞内NADPH的供给。

优选地，所述与NADPH生物合成途径相关的基因为pntAB基因，核苷酸序列如下：

i)SEQ ID NO:7所示的核苷酸序列；

ii)SEQ ID NO:7所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；

iii)在严格条件下与SEQ ID NO:7所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或

iv)与i)、ii)或iii)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。

本发明中，所述增强的途径选自以下1)～6)，或任选的组合：

1)通过导入具有所述基因的质粒而增强；

2)通过增加染色体上所述基因的拷贝数而增强；

3)通过改变染色体上所述基因的启动子序列而增强；

4)通过将强启动子与所述基因可操作地连接而增强；

5)通过导入增强子而增强；

6)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强。

方案II：弱化出发菌株中sthA基因，来提高细胞内NADPH的供给；

sthA基因的核苷酸序列如下：

i)SEQ ID NO:8所示的核苷酸序列；

ii)SEQ ID NO:8所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；

iii)在严格条件下与SEQ ID NO:8所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或

本发明中，所述弱化包括敲除或降低基因的表达。

方案III：通过增强出发菌株(大肠杆菌)中pntAB基因，并弱化sthA基因，来提高细胞内NADPH的供给。

第三方面，本发明提供一种高产1,3-丁二醇的工程菌，所述工程菌是以上述重组菌，或者上述重组大肠杆菌作为原始菌株，利用基因工程手段对原始菌株做进一步改造，得到的细胞内乙酰CoA流向1,3-丁二醇合成途径的代谢通量增强的工程菌。

优选地，弱化原始菌株中adhE、ldhA、pta、ackA基因中的至少一种基因，来提高细胞内乙酰CoA流向1,3-丁二醇合成途径的代谢通量；所述弱化包括敲除或降低基因的表达。

adhE、ldhA、pta、ackA基因编码蛋白的氨基酸序列分别如SEQ ID NO:11-14所示。

优选地，弱化原始菌株(大肠杆菌)中adhE、ldhA、pta和ackA基因，来提高细胞内乙酰CoA流向1,3-丁二醇合成途径的代谢通量。

第四方面，本发明提供一种丁醛脱氢酶突变体，其是将丁醛脱氢酶(SEQ ID NO:1)第273位亮氨酸突变为苏氨酸而成，所述突变体的氨基酸序列如SEQ ID NO:2所示。

第五方面，本发明提供含有上述丁醛脱氢酶突变体编码基因的生物材料，所述生物材料包括重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体或工程菌。

第六方面，本发明提供上述生物材料在微生物发酵生产1,3-丁二醇中的应用。

第七方面，本发明提供上述重组菌，或上述重组大肠杆菌，或者上述工程菌在发酵生产1,3-丁二醇中的应用。

第八方面，本发明提供一种生产光学纯1,3-丁二醇的方法，包括在发酵培养基中对上述重组菌，或上述重组大肠杆菌，或者上述工程菌进行培养以生产1,3-丁二醇。

可选地，所述发酵培养基为M9Y：葡萄糖20g/L,Na₂HPO₄ 6g/L，KH₂PO₄ 3g/L，NaCl0.5g/L，NH₄Cl 1g/L，MgSO₄ 0.5g/L,CaCl₂ 15mg/L,酵母粉2g/L,氨苄青霉素100mg/L，以水配制。

培养条件为：30℃，200rpm，控制溶氧低于10％的饱和溶氧值。

利用本发明的大肠杆菌工程菌在微氧条件下发酵廉价有机碳源如葡萄糖、蔗糖或甘油等，1,3-丁二醇相对于底物的得率可以达到0.4g/g以上，且R型1,3-丁二醇纯度达到99％以上，具有重要的工业应用潜力。

附图说明

图1为本发明质粒ptrc99a-bld2-yqhD-phaAB图谱。

图2为本发明质粒ptrc99a-bld-yqhD-phaAB图谱。

图3为本发明质粒ptrc99a-pduP-yqhD-phaAB图谱。

图4为本发明质粒ptrc99a-eutE-yqhD–phaAB图谱。

图5为本发明质粒ptrc99a-bld(L273T)-yqhD–phaAB图谱。

图6为本发明质粒ptrc99a-bld(L273T)-yqhD–phaAB-pntAB图谱。

具体实施方式

本发明具体的实施方式包括：(1)构建1,3-丁二醇合成质粒；(2)强化NADPH的供给；(3)敲除副产物的合成。

以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。

实施例1在大肠杆菌中构建1,3-丁二醇的生物合成途径

大肠杆菌本身不能直接合成1,3-丁二醇，为了引入1,3-丁二醇的合成途径，本发明对1,3-丁二醇合成过程的关键基因进行了大量的筛选和改造，经过优化后的1,3-丁二醇表达模块，其产量大大提高。

首先构建包含来源于Cupriavidus necator的phaA、phaB基因，来源于Clostridium beijerinckii的醛脱氢酶基因bld2基因(在uniprot的基因编号为Q9X681)，来源于大肠杆菌的yqhD基因(SEQ ID NO:6)的质粒ptrc99a–bld2-yqhD-phaAB。以phaA(SEQID NO:3)，phaB(SEQ ID NO:4)的氨基酸序列为基础，人工合成了密码子优化的phaA-phaB操纵子(SEQ ID NO:5)。将ptrc99a(购自Addgene公司)用EcoRI/XbaI进行双酶切，利用Gibson组装试剂盒将人工合成的phaA-phaB操纵子、bld2基因、yqhD基因插入到ptrc99a质粒骨架上EcoRI/XbaI双酶切位点之间，获得的质粒命名为ptrc99a-bld2-yqhD-phaAB(质粒图谱如图1所示)。

相应地，将ptrc99a(购自Addgene公司)用EcoRI/XbaI进行双酶切，利用Gibson组装试剂盒将人工合成的phaA-phaB操纵子、来源于Clostridiumsaccharoperbutylacetonicum的醛脱氢酶基因bld基因(编码蛋白的氨基酸序列见SEQ IDNO:1)、yqhD基因插入到ptrc99a质粒骨架上EcoRI/XbaI双酶切位点之间，获得的质粒命名为ptrc99a-bld-yqhD-phaAB(质粒图谱如图2所示)。

相应地，将ptrc99a(购自Addgene公司)用EcoRI/XbaI进行双酶切，利用Gibson组装试剂盒将人工合成的phaA-phaB操纵子、来源于Klebsiella pneumoniae的醛脱氢酶基因pduP基因(在uniprot的基因编号为B5XUS2)、yqhD基因插入到ptrc99a质粒骨架上EcoRI/XbaI双酶切位点之间，获得的质粒命名为ptrc99a-pduP-yqhD-phaAB(质粒图谱如图3所示)。

相应地，将ptrc99a(购自Addgene公司)用EcoRI/XbaI进行双酶切，利用Gibson组装试剂盒将人工合成的phaA-phaB操纵子、来源于Salmonella typhimurium的醛脱氢酶基因eutE基因(在uniprot的基因编号为P41793)、yqhD基因插入到ptrc99a质粒骨架上EcoRI/XbaI双酶切位点之间，获得的质粒命名为ptrc99a-eutE-yqhD-phaAB(质粒图谱如图4所示)。

将上述四个质粒分别转化到大肠杆菌W3110中，获得的重组菌命名为E.coli/ptrc99a-bld2-yqhD-phaAB,E.coli/ptrc99a-bld-yqhD-phaAB,E.coli/ptrc99a-pduP-yqhD-phaAB,E.coli/ptrc99a-eutE-yqhD-phaAB。将这四株菌分别在500mL的不带挡板摇瓶中进行培养，装液量为200ml，培养基为M9Y(葡萄糖20g/L,Na₂HPO₄ 6g/L，KH₂PO₄ 3g/L，NaCl0.5g/L，NH₄Cl 1g/L，MgSO₄ 0.5g/L,CaCl₂ 15mg/L,酵母粉2g/L,氨苄青霉素100mg/L)，培养温度为30℃，转速200rpm，待菌液OD₆₀₀＝0.6时，加入1mM IPTG进行诱导，发酵72h时取样，利用高效液相色谱检测四株菌生产1,3-丁二醇的情况。结果显示，仅有大肠杆菌E.coli/ptrc99a-bld-yqhD-phaAB生产了0.3g/L的1,3-丁二醇，而其他三株菌株并不能产生1,3-丁二醇。说明1,3-丁二醇途径中关键酶的筛选对于1,3-丁二醇的合成至关重要。

进一步地，本发明发现醛脱氢酶是影响1,3-丁二醇合成的关键制约因素。为此，本发明进行了大量的蛋白质工程改造，最终发现以ptrc99a-bld-yqhD-phaAB为模板，利用Biorad的基因突变试剂盒将bld基因的273位氨基酸由亮氨酸突变为苏氨酸(SEQ ID NO:2)，获得质粒ptrc99a-bld(L273T)-yqhD-phaAB(质粒图谱如图5所示)。将该质粒转化到大肠杆菌W3110中，获得的重组菌命名为E.coli/ptrc99a-bld(L273T)-yqhD-phaAB。将该菌株在M9Y培养基中进行培养，培养条件同上，72h时取样，利用高效液相色谱检测1,3-丁二醇的产量。结果显示，E.coli/ptrc99a-bld(L273T)-yqhD-phaAB的1,3-丁二醇的产量达到2.2g/L。

因此，本发明发现1,3-丁二醇合成途径酶的选择对1,3-丁二醇的生产至关重要，通过系统的优化1,3-丁二醇的途径，极大提高了1,3-丁二醇的产量。

实施例2通过强化NADPH的合成提高1,3-丁二醇的产量

1,3-丁二醇合成途径需要消耗大量的NADPH，本发明创造性地发现，过表达pntAB基因及敲除大肠杆菌sthA基因可以显著提高细胞的1,3-丁二醇产量。

将ptrc99a-bld(L273T)-yqhD-phaAB用SbfI进行双酶切，利用Gibson组装试剂盒将大肠杆菌的pntAB基因(SEQ ID NO:7)插入到ptrc99a-bld(L273T)-yqhD-phaAB质粒骨架上，获得的质粒命名为ptrc99a-bld(L273T)-yqhD-phaAB-pntAB(质粒图谱如图6所示)。将该质粒转化到大肠杆菌W3110中，获得的重组菌命名为E.coli/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB。将该菌株在M9Y培养基中进行培养，发酵条件同上所述，72h时取样，利用高效液相色谱检测1,3-丁二醇的产量。结果显示，E.coli/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB的1,3-丁二醇的产量达到4.8g/L。检测该菌株胞内的NADPH含量显示，该菌株的NADPH含量较E.coli/ptrc99a-bld(L273T)-yqhD-phaAB提高了1.8倍。

进一步地，通过基因敲除大肠杆菌W3110的sthA基因(SEQ ID NO:8)以强化NADPH的供给并提高1,3-丁二醇的产量。具体方法是以大肠杆菌MG1655ΔsthA：：Kan(购自耶鲁大学CGSC大肠杆菌突变库，参见Baba et al.,Construction of Escherichia coli K-12in-frame,single-gene knockout mutants:the Keio collection,Mol Syst Biol.2006,2:2006.0008.)制备P1噬菌体溶菌产物，将溶菌产物与新鲜的大肠杆菌W3110培养物混合30分钟，加入1ml含有0.2M柠檬酸钠的LB培养基在37℃培养1h后涂布在包含50mg/L卡纳霉素抗性的LB平板上，筛选带抗性的重组菌株。进一步将带Flp酶的质粒pCP20(购自耶鲁大学CGSC大肠杆菌突变库，参见Datsenko&Wanner,One-step inactivation of chromosomal genesin Escherichia coli K-12 using PCR products,Proc Natl Acad Sci USA.2000,97(12):6640–6645.)转入该菌株，获得除去卡纳抗性标记的菌株命名为E.coli ΔsthA。将质粒ptrc99a-bld(L273T)-yqhD-phaAB-pntAB转化到大肠杆菌W3110中，获得的重组菌命名为E.coli ΔsthA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB。将该菌株在M9Y培养基中进行培养，发酵条件同上所述，72h时取样，利用高效液相色谱检测1,3-丁二醇的产量。结果显示，E.coli ΔsthA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB的1,3-丁二醇的产量达到5.8g/L。检测该菌株胞内的NADPH含量显示，该菌株的NADPH含量相比E.coli/ptrc99a-bld(L273T)-yqhD-phaAB提高了2.3倍。

因此，本发明发现大肠杆菌中NADPH的含量对1,3-丁二醇合成至关重要，通过系统的强化NADPH的含量，极大提高了1,3-丁二醇的产量。

实施例3强化乙酰辅酶A的供给提高1,3-丁二醇的产量

1,3-丁二醇合成途径的代谢前体是乙酰辅酶A，本发明发现通过减少乙酰辅酶A流向其他代谢支路可以进一步提高1,3-丁二醇的产量。利用P1噬菌体溶菌产物转染的方法，进一步敲除E.coli ΔsthA的乙醇合成基因adhE，乳酸合成基因ldhA，乙酸合成基因pta、ackA。其中，adhE、ldhA、pta、ackA基因编码蛋白的氨基酸序列分别如SEQ ID NO:11-14所示。获得的菌株分别命名为E.coli ΔsthAΔadhE，E.coli ΔsthAΔldhA，E.coli ΔsthAΔpta-ackA。同时利用P1噬菌体溶菌产物转染的方法，构建了同时包含这三个基因突变的菌株，命名为E.coli ΔsthAΔadhEΔldhAΔpta-ackA。将质粒ptrc99a-bld(L273T)-yqhD-phaAB-pntAB分别转化到这四株菌种，获得的菌株分别命名为E.coli ΔsthAΔadhE/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB，E.coli ΔsthAΔldhA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB，E.coli ΔsthAΔpta-ackA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB，E.coli ΔsthAΔadhEΔldhAΔpta-ackA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB。将该菌株在M9Y培养基中进行培养，发酵条件同上所述，72h时取样，利用高效液相色谱检测1,3-丁二醇的产量。结果显示，E.coli ΔsthAΔadhE/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB，E.coli ΔsthAΔldhA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB，E.coli ΔsthAΔpta-ackA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB，E.coli ΔsthAΔadhEΔldhAΔpta-ackA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB的1,3-丁二醇的产量分别达到6.1g/L,6.2g/L,6.8g/L,8.1g/L。

利用光学色谱检测，上述菌株生产的1,3-丁二醇均为R构型，纯度达到99％以上。

因此本发明通过系统地改造，获得1,3-丁二醇的高产菌株，1,3-丁二醇对底物的得率可以达到0.4g/g以上，且R型1,3-丁二醇纯度达到99％以上，具有重要的工业应用潜力。

实施例4重组大肠杆菌利用其它碳源生产1,3-丁二醇

进一步测试了重组菌株E.coli ΔsthAΔadhEΔldhAΔpta-ackA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB利用其它碳源生产1,3-丁二醇的情况。以甘油为碳源，利用发酵培养基M9Y-G(甘油20g/L,Na₂HPO₄ 6g/L，KH₂PO₄ 3g/L，NaCl 0.5g/L，NH₄Cl 1g/L，MgSO₄0.5g/L,CaCl₂ 15mg/L,酵母粉2g/L,氨苄青霉素100mg/L)进行培养，培养温度为30℃，转速200rpm，待菌液OD₆₀₀＝0.6时，加入1mM IPTG进行诱导，发酵72h时取样，利用高效液相色谱检测该菌生产1,3-丁二醇的情况，其1,3-丁二醇产量达到8.3g/L，说明本发明构建的重组菌株同样能高效利用甘油高产1,3-丁二醇。

虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

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tgcgcagaat ccggaattaa tgcaaaaact tgcggaacgt aacgtgaccg tgatggcgat 360

ggactctgtg ccgcgtatct cacgcgcaca atcgctggac gcactaagct cgatggcgaa 420

catcgccggt tatcgcgcca ttgttgaagc ggcacatgaa tttgggcgct tctttaccgg 480

gcaaattact gcggccggga aagtgccacc ggcaaaagtg atggtgattg gtgcgggtgt 540

tgcaggtctg gccgccattg gcgcagcaaa cagtctcggc gcgattgtgc gtgcattcga 600

cacccgcccg gaagtgaaag aacaagttca aagtatgggc gcggaattcc tcgagctgga 660

ttttaaagag gaagctggca gcggcgatgg ctatgccaaa gtgatgtcgg acgcgttcat 720

caaagcggaa atggaactct ttgccgccca ggcaaaagag gtcgatatca ttgtcaccac 780

cgcgcttatt ccaggcaaac cagcgccgaa gctaattacc cgtgaaatgg ttgactccat 840

gaaggcgggc agtgtgattg tcgacctggc agcccaaaac ggcggcaact gtgaatacac 900

cgtgccgggt gaaatcttca ctacggaaaa tggtgtcaaa gtgattggtt ataccgatct 960

tccgggccgt ctgccgacgc aatcctcaca gctttacggc acaaacctcg ttaatctgct 1020

gaaactgttg tgcaaagaga aagacggcaa tatcactgtt gattttgatg atgtggtgat 1080

tcgcggcgtg accgtgatcc gtgcgggcga aattacctgg ccggcaccgc cgattcaggt 1140

atcagctcag ccgcaggcgg cacaaaaagc ggcaccggaa gtgaaaactg aggaaaaatg 1200

tacctgctca ccgtggcgta aatacgcgtt gatggcgctg gcaatcattc tttttggctg 1260

gatggcaagc gttgcgccga aagaattcct tgggcacttc accgttttcg cgctggcctg 1320

cgttgtcggt tattacgtgg tgtggaatgt atcgcacgcg ctgcatacac cgttgatgtc 1380

ggtcaccaac gcgatttcag ggattattgt tgtcggagca ctgttgcaga ttggccaggg 1440

cggctgggtt agcttcctta gttttatcgc ggtgcttata gccagcatta atattttcgg 1500

tggcttcacc gtgactcagc gcatgctgaa aatgttccgc aaaaattaag gggtaacata 1560

tgtctggagg attagttaca gctgcataca ttgttgccgc gatcctgttt atcttcagtc 1620

tggccggtct ttcgaaacat gaaacgtctc gccagggtaa caacttcggt atcgccggga 1680

tggcgattgc gttaatcgca accatttttg gaccggatac gggtaatgtt ggctggatct 1740

tgctggcgat ggtcattggt ggggcaattg gtatccgtct ggcgaagaaa gttgaaatga 1800

ccgaaatgcc agaactggtg gcgatcctgc atagcttcgt gggtctggcg gcagtgctgg 1860

ttggctttaa cagctatctg catcatgacg cgggaatggc accgattctg gtcaatattc 1920

acctgacgga agtgttcctc ggtatcttca tcggggcggt aacgttcacg ggttcggtgg 1980

tggcgttcgg caaactgtgt ggcaagattt cgtctaaacc attgatgctg ccaaaccgtc 2040

acaaaatgaa cctggcggct ctggtcgttt ccttcctgct gctgattgta tttgttcgca 2100

cggacagcgt cggcctgcaa gtgctggcat tgctgataat gaccgcaatt gcgctggtat 2160

tcggctggca tttagtcgcc tccatcggtg gtgcagatat gccagtggtg gtgtcgatgc 2220

tgaactcgta ctccggctgg gcggctgcgg ctgcgggctt tatgctcagc aacgacctgc 2280

tgattgtgac cggtgcgctg gtcggttctt cgggggctat cctttcttac attatgtgta 2340

aggcgatgaa ccgttccttt atcagcgtta ttgcgggtgg tttcggcacc gacggctctt 2400

ctactggcga tgatcaggaa gtgggtgagc accgcgaaat caccgcagaa gagacagcgg 2460

aactgctgaa aaactcccat tcagtgatca ttactccggg gtacggcatg gcagtcgcgc 2520

aggcgcaata tcctgtcgct gaaattactg agaaattgcg cgctcgtggt attaatgtgc 2580

gtttcggtat ccacccggtc gcggggcgtt tgcctggaca tatgaacgta ttgctggctg 2640

aagcaaaagt accgtatgac atcgtgctgg aaatggacga gatcaatgat gactttgctg 2700

ataccgatac cgtactggtg attggtgcta acgatacggt taacccggcg gcgcaggatg 2760

atccgaagag tccgattgct ggtatgcctg tgctggaagt gtggaaagcg cagaacgtga 2820

ttgtctttaa acgttcgatg aacactggct atgctggtgt gcaaaacccg ctgttcttca 2880

aggaaaacac ccacatgctg tttggtgacg ccaaagccag cgtggatgca atcctgaaag 2940

ctctgtaa 2948

<210> 8

<211> 1401

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<400> 8

atgccacatt cctacgatta cgatgccata gtaataggtt ccggccccgg cggcgaaggc 60

gctgcaatgg gcctggttaa gcaaggtgcg cgcgtcgcag ttatcgagcg ttatcaaaat 120

gttggcggcg gttgcaccca ctggggcacc atcccgtcga aagctctccg tcacgccgtc 180

agccgcatta tagaattcaa tcaaaaccca ctttacagcg accattcccg actgctccgc 240

tcttcttttg ccgatatcct taaccatgcc gataacgtga ttaatcaaca aacgcgcatg 300

cgtcagggat tttacgaacg taatcactgt gaaatattgc agggaaacgc tcgctttgtt 360

gacgagcata cgttggcgct ggattgcccg gacggcagcg ttgaaacact aaccgctgaa 420

aaatttgtta ttgcctgcgg ctctcgtcca tatcatccaa cagatgttga tttcacccat 480

ccacgcattt acgacagcga ctcaattctc agcatgcacc acgaaccgcg ccatgtactt 540

atctatggtg ctggagtgat cggctgtgaa tatgcgtcga tcttccgcgg tatggatgta 600

aaagtggatc tgatcaacac ccgcgatcgc ctgctggcat ttctcgatca agagatgtca 660

gattctctct cctatcactt ctggaacagt ggcgtagtga ttcgtcacaa cgaagagtac 720

gagaagatcg aaggctgtga cgatggtgtg atcatgcatc tgaagtcggg taaaaaactg 780

aaagctgact gcctgctcta tgccaacggt cgcaccggta ataccgattc gctggcgtta 840

cagaacattg ggctagaaac tgacagccgc ggacagctga aggtcaacag catgtatcag 900

accgcacagc cacacgttta cgcggtgggc gacgtgattg gttatccgag cctggcgtcg 960

gcggcctatg accaggggcg cattgccgcg caggcgctgg taaaaggcga agccaccgca 1020

catctgattg aagatatccc taccggtatt tacaccatcc cggaaatcag ctctgtgggc 1080

aaaaccgaac agcagctgac cgcaatgaaa gtgccatatg aagtgggccg cgcccagttt 1140

aaacatctgg cacgcgcaca aatcgtcggc atgaacgtgg gcacgctgaa aattttgttc 1200

catcgggaaa caaaagagat tctgggtatt cactgctttg gcgagcgcgc tgccgaaatt 1260

attcatatcg gtcaggcgat tatggaacag aaaggtggcg gcaacactat tgagtacttc 1320

gtcaacacca cctttaacta cccgacgatg gcggaagcct atcgggtagc tgcgttaaac 1380

ggtttaaacc gcctgtttta a 1401

<210> 9

<211> 4627

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 9

atgaacaaag atactttaat cccgacgacc aaagatttaa aacttaaaac gaatgtcgaa 60

aatatcaatt tgaaaaatta taaagataat tcctcttgct ttggggtctt cgaaaatgtg 120

gaaaatgcga ttaatagcgc agttcacgct caaaaaatcc tttcgttgca ttacaccaag 180

gaacagcgtg agaaaattat caccgaaatt cgtaaagctg cacttgagaa taaggaggtg 240

cttgctacta tgattttgga ggaaacacat atgggccgct acgaagacaa aatccttaaa 300

catgaactgg ttgcgaaata taccccggga accgaagacc ttacaaccac tgcgtggagt 360

ggtgacaacg gcttgactgt tgtagagatg tcgccctatg gggtaattgg ggcgattacc 420

ccgtctacca acccgacgga gaccgtgatc tgcaactcaa ttggaatgat tgctgcagga 480

aatgcagtcg tattcaacgg ccacccgggt gccaaaaagt gcgttgcatt cgcgattgaa 540

atgattaata aggcgattat ttcttgcggt ggtccggaaa atctggtcac cacgatcaag 600

aacccgacaa tggaaagtct ggatgcgatc atcaaacacc ctttgattaa gcttctttgc 660

gggacgggtg gaccgggtat ggttaagact ttgctgaact caggtaagaa agcgattggt 720

gctggcgcag gtaacccgcc tgtgatcgtc gacgatactg ccgacatcga gaaagcaggg 780

aagtccatta ttgaaggatg tagctttgac aacaatttgc cttgcatcgc agagaaagag 840

gtattcgttt ttgagaacgt cgcggacgat ctgattagta acatgctgaa gaacaacgca 900

gtcattatta acgaggatca ggtgtccaaa ctgatcgact tggtgttgca aaaaaataat 960

gaaactcagg agtacttcat caacaagaaa tgggttggaa aggatgccaa gttattcagt 1020

gatgaaattg atgttgaatc gccctcgaac attaagtgta ttgtctgtga ggtcaacgct 1080

aaccatccat ttgttatgac cgaattaatg atgcctattc tgcccatcgt tcgcgtgaag 1140

gacattgatg aagctgtaaa gtataccaaa atcgcggaac aaaaccgtaa gcacagcgca 1200

tacatctaca gtaaaaacat tgacaacctt aaccgttttg agcgcgaaat tgatacgacg 1260

attttcgtta agaacgcaaa aagtttcgct ggggtagggt atgaggcaga aggatttaca 1320

acttttacaa ttgcgggatc tacgggagaa gggattacct ccgcacgcaa tttcacgcgc 1380

caacgtcgtt gtgtgttggc ggggtgaaac tttaagaagg agatatacat gaacaacttt 1440

aatctgcaca ccccaacccg cattctgttt ggtaaaggcg caatcgctgg tttacgcgaa 1500

caaattcctc acgatgctcg cgtattgatt acctacggcg gcggcagcgt gaaaaaaacc 1560

ggcgttctcg atcaagttct ggatgccctg aaaggcatgg acgtgctgga atttggcggt 1620

attgagccaa acccggctta tgaaacgctg atgaacgccg tgaaactggt tcgcgaacag 1680

aaagtgactt tcctgctggc ggttggcggc ggttctgtac tggacggcac caaatttatc 1740

gccgcagcgg ctaactatcc ggaaaatatc gatccgtggc acattctgca aacgggcggt 1800

aaagagatta aaagcgccat cccgatgggc tgtgtgctga cgctgccagc aaccggttca 1860

gaatccaacg caggcgcggt gatctcccgt aaaaccacag gcgacaagca ggcgttccat 1920

tctgcccatg ttcagccggt atttgccgtg ctcgatccgg tttataccta caccctgccg 1980

ccgcgtcagg tggctaacgg cgtagtggac gcctttgtac acaccgtgga acagtatgtt 2040

accaaaccgg ttgatgccaa aattcaggac cgtttcgcag aaggcatttt gctgacgcta 2100

atcgaagatg gtccgaaagc cctgaaagag ccagaaaact acgatgtgcg cgccaacgtc 2160

atgtgggcgg cgactcaggc gctgaacggt ttgattggcg ctggcgtacc gcaggactgg 2220

gcaacgcata tgctgggcca cgaactgact gcgatgcacg gtctggatca cgcgcaaaca 2280

ctggctatcg tcctgcctgc actgtggaat gaaaaacgcg ataccaagcg cgctaagctg 2340

ctgcaatatg ctgaacgcgt ctggaacatc actgaaggtt ccgatgatga gcgtattgac 2400

gccgcgattg ccgcaacccg caatttcttt gagcaattag gcgtgccgac ccacctctcc 2460

gactacggtc tggacggcag ctccatcccg gctttgctga aaaaactgga agagcacggc 2520

atgacccaac tgggcgaaaa tcatgacatt acgttggatg tcagccgccg tatatacgaa 2580

gccgcccgct aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 2640

aacaatttca cccattgaaa ggactacaca atgactgacg ttgtcatcgt atccgccgcc 2700

cgcaccgcgg tcggcaagtt tggcggctcg ctggccaaga tcccggcacc ggaactgggt 2760

gccgtggtca tcaaggccgc gctggagcgc gccggcgtca agccggagca ggtgagcgaa 2820

gtcatcatgg gccaggtgct gaccgccggt tcgggccaga accccgcacg ccaggccgcg 2880

atcaaggccg gcctgccggc gatggtgccg gccatgacca tcaacaaggt gtgcggctcg 2940

ggcctgaagg ccgtgatgct ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg 3000

gtggccggcg gccaggaaaa catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat 3060

ggtttccgca tgggcgatgc caagctggtc gacaccatga tcgtcgacgg cctgtgggac 3120

gtgtacaacc agtaccacat gggcatcacc gccgagaacg tggccaagga atacggcatc 3180

acacgcgagg cgcaggatga gttcgccgtc ggctcgcaga acaaggccga agccgcgcag 3240

aaggccggca agtttgacga agagatcgtc ccggtgctga tcccgcagcg caagggcgac 3300

ccggtggcct tcaagaccga cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc 3360

ggcctcaagc ccgccttcga caaggccggc acggtgaccg cggccaacgc ctcgggcctg 3420

aacgacggcg ccgccgcggt ggtggtgatg tcggcggcca aggccaagga actgggcctg 3480

accccgctgg ccacgatcaa gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc 3540

atgggcccgg tgccggcctc caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac 3600

ctggacctga tggagatcaa cgaggccttt gccgcgcagg cgctggcggt gcaccagcag 3660

atgggctggg acacctccaa ggtcaatgtg aacggcggcg ccatcgccat cggccacccg 3720

atcggcgcgt cgggctgccg tatcctggtg acgctgctgc acgagatgaa gcgccgtgac 3780

gcgaagaagg gcctggcctc gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc 3840

gagcgcaaat aacggcgacg ataacgaagc caatcaagga gtggacatga ctcagcgcat 3900

tgcgtatgtg accggcggca tgggtggtat cggaaccgcc atttgccagc ggctggccaa 3960

ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg ccgcgccgcg aaaagtggct 4020

ggagcagcag aaggccctgg gcttcgattt cattgcctcg gaaggcaatg tggctgactg 4080

ggactcgacc aagaccgcat tcgacaaggt caagtccgag gtcggcgagg ttgatgtgct 4140

gatcaacaac gccggtatca cccgcgacgt ggtgttccgc aagatgaccc gcgccgactg 4200

ggatgcggtg atcgacacca acctgacctc gctgttcaac gtcaccaagc aggtgatcga 4260

cggcatggcc gaccgtggct ggggccgcat cgtcaacatc tcgtcggtga acgggcagaa 4320

gggccagttc ggccagacca actactccac cgccaaggcc ggcctgcatg gcttcaccat 4380

ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc aacacggtct ctccgggcta 4440

tatcgccacc gacatggtca aggcgatccg ccaggacgtg ctcgacaaga tcgtcgcgac 4500

gatcccggtc aagcgcctgg gcctgccgga agagatcgcc tcgatctgcg cctggttgtc 4560

gtcggaggag tccggtttct cgaccggcgc cgacttctcg ctcaacggcg gcctgcatat 4620

gggctga 4627

<210> 10

<211> 4627

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

atgatcaagg acaccctggt ttcgattaca aaagacttaa agttgaaaac aaacgtagaa 60

aacgccaacc ttaaaaacta taaagatgac tcgtcttgct tcggggtgtt tgagaacgtg 120

gaaaatgcga tttcgaacgc agtccacgcc cagaagattt tatcgcttca ctacactaag 180

gagcagcgcg aaaagattat cacggagatt cgtaaagctg cacttgagaa caaagagatt 240

ttggcaacca tgatcttgga ggagactcat atggggcgtt atgaagataa aattttaaag 300

catgaacttg tcgctaaata cactccaggg actgaagact taacgaccac ggcatggagc 360

ggagataatg ggcttactgt tgttgaaatg agcccctacg gggtgattgg ggcaatcact 420

cccagcacaa atcctaccga gactgtgatt tgtaattcta ttggcatgat tgcggctggc 480

aatacggtgg tcttcaatgg acatccgggg gccaagaagt gtgttgcatt tgctgttgag 540

atgatcaaca aagccattat ttcatgtggt gggcccgaga atttggttac aaccattaag 600

aatccaacta tggactctct ggacgctatt attaaacacc cgtcgattaa acttttatgc 660

ggaaccggag gaccgggaat ggtgaaaacc cttctgaatt ccgggaaaaa ggcgatcggt 720

gcgggtgccg gcaacccacc tgtcattgtt gatgacacag cagacattga aaaggcgggc 780

aagagcatca tcgagggctg tagttttgat aacaacacgc cctgcattgc tgaaaaggaa 840

gttttcgtct tcgagaatgt tgcggacgac cttatttcca atatgcttaa gaacaacgcg 900

gtaatcatta acgaagacca agtatcaaag ttaatcgatt tggtcctgca gaagaacaat 960

gagactcaag agtatagcat taataaaaaa tgggttggga aggacgcaaa attgtttctt 1020

gatgagatcg atgtggaatc cccttcctct gtcaaatgca tcatttgcga agtatctgcg 1080

cgtcacccat tcgtcatgac agagctgatg atgcccattc tgcctattgt acgcgttaaa 1140

gatatcgacg aagctattga atatgcgaaa atcgccgagc agaaccgtaa gcactcagcg 1200

tatatttatt ccaaaaatat cgataactta aatcgttttg agcgcgaaat cgatacaact 1260

atcttcgtca agaacgccaa aagctttgct ggcgtgggct atgaagctga agggtttacc 1320

acgttcacca tcgcagggag caccggcgaa gggattacaa gtgcgcgcaa tttcactcgt 1380

caacgtcgct gcgtgttagc cggttaaaac tttaagaagg agatatacat gaacaacttt 1440

aatctgcaca ccccaacccg cattctgttt ggtaaaggcg caatcgctgg tttacgcgaa 1500

caaattcctc acgatgctcg cgtattgatt acctacggcg gcggcagcgt gaaaaaaacc 1560

ggcgttctcg atcaagttct ggatgccctg aaaggcatgg acgtgctgga atttggcggt 1620

attgagccaa acccggctta tgaaacgctg atgaacgccg tgaaactggt tcgcgaacag 1680

aaagtgactt tcctgctggc ggttggcggc ggttctgtac tggacggcac caaatttatc 1740

gccgcagcgg ctaactatcc ggaaaatatc gatccgtggc acattctgca aacgggcggt 1800

aaagagatta aaagcgccat cccgatgggc tgtgtgctga cgctgccagc aaccggttca 1860

gaatccaacg caggcgcggt gatctcccgt aaaaccacag gcgacaagca ggcgttccat 1920

tctgcccatg ttcagccggt atttgccgtg ctcgatccgg tttataccta caccctgccg 1980

ccgcgtcagg tggctaacgg cgtagtggac gcctttgtac acaccgtgga acagtatgtt 2040

accaaaccgg ttgatgccaa aattcaggac cgtttcgcag aaggcatttt gctgacgcta 2100

atcgaagatg gtccgaaagc cctgaaagag ccagaaaact acgatgtgcg cgccaacgtc 2160

atgtgggcgg cgactcaggc gctgaacggt ttgattggcg ctggcgtacc gcaggactgg 2220

gcaacgcata tgctgggcca cgaactgact gcgatgcacg gtctggatca cgcgcaaaca 2280

ctggctatcg tcctgcctgc actgtggaat gaaaaacgcg ataccaagcg cgctaagctg 2340

ctgcaatatg ctgaacgcgt ctggaacatc actgaaggtt ccgatgatga gcgtattgac 2400

gccgcgattg ccgcaacccg caatttcttt gagcaattag gcgtgccgac ccacctctcc 2460

gactacggtc tggacggcag ctccatcccg gctttgctga aaaaactgga agagcacggc 2520

atgacccaac tgggcgaaaa tcatgacatt acgttggatg tcagccgccg tatatacgaa 2580

gccgcccgct aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 2640

aacaatttca cccattgaaa ggactacaca atgactgacg ttgtcatcgt atccgccgcc 2700

cgcaccgcgg tcggcaagtt tggcggctcg ctggccaaga tcccggcacc ggaactgggt 2760

gccgtggtca tcaaggccgc gctggagcgc gccggcgtca agccggagca ggtgagcgaa 2820

gtcatcatgg gccaggtgct gaccgccggt tcgggccaga accccgcacg ccaggccgcg 2880

atcaaggccg gcctgccggc gatggtgccg gccatgacca tcaacaaggt gtgcggctcg 2940

ggcctgaagg ccgtgatgct ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg 3000

gtggccggcg gccaggaaaa catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat 3060

ggtttccgca tgggcgatgc caagctggtc gacaccatga tcgtcgacgg cctgtgggac 3120

gtgtacaacc agtaccacat gggcatcacc gccgagaacg tggccaagga atacggcatc 3180

acacgcgagg cgcaggatga gttcgccgtc ggctcgcaga acaaggccga agccgcgcag 3240

aaggccggca agtttgacga agagatcgtc ccggtgctga tcccgcagcg caagggcgac 3300

ccggtggcct tcaagaccga cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc 3360

ggcctcaagc ccgccttcga caaggccggc acggtgaccg cggccaacgc ctcgggcctg 3420

aacgacggcg ccgccgcggt ggtggtgatg tcggcggcca aggccaagga actgggcctg 3480

accccgctgg ccacgatcaa gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc 3540

atgggcccgg tgccggcctc caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac 3600

ctggacctga tggagatcaa cgaggccttt gccgcgcagg cgctggcggt gcaccagcag 3660

atgggctggg acacctccaa ggtcaatgtg aacggcggcg ccatcgccat cggccacccg 3720

atcggcgcgt cgggctgccg tatcctggtg acgctgctgc acgagatgaa gcgccgtgac 3780

gcgaagaagg gcctggcctc gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc 3840

gagcgcaaat aacggcgacg ataacgaagc caatcaagga gtggacatga ctcagcgcat 3900

tgcgtatgtg accggcggca tgggtggtat cggaaccgcc atttgccagc ggctggccaa 3960

ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg ccgcgccgcg aaaagtggct 4020

ggagcagcag aaggccctgg gcttcgattt cattgcctcg gaaggcaatg tggctgactg 4080

ggactcgacc aagaccgcat tcgacaaggt caagtccgag gtcggcgagg ttgatgtgct 4140

gatcaacaac gccggtatca cccgcgacgt ggtgttccgc aagatgaccc gcgccgactg 4200

ggatgcggtg atcgacacca acctgacctc gctgttcaac gtcaccaagc aggtgatcga 4260

cggcatggcc gaccgtggct ggggccgcat cgtcaacatc tcgtcggtga acgggcagaa 4320

gggccagttc ggccagacca actactccac cgccaaggcc ggcctgcatg gcttcaccat 4380

ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc aacacggtct ctccgggcta 4440

tatcgccacc gacatggtca aggcgatccg ccaggacgtg ctcgacaaga tcgtcgcgac 4500

gatcccggtc aagcgcctgg gcctgccgga agagatcgcc tcgatctgcg cctggttgtc 4560

gtcggaggag tccggtttct cgaccggcgc cgacttctcg ctcaacggcg gcctgcatat 4620

gggctga 4627

<210> 11

<211> 891

<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 11

Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val

1 5 10 15

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

20 25 30

Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro

35 40 45

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

50 55 60

Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr

65 70 75 80

Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly

85 90 95

Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly 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 Ile Phe Ser Pro His Pro Arg Ala Lys Asp

130 135 140

Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala

145 150 155 160

Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu

165 170 175

Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala

180 185 190

Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro

195 200 205

Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr

210 215 220

Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe

225 230 235 240

Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp

245 250 255

Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly Gly Tyr

260 265 270

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

275 280 285

Asn Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile

290 295 300

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

305 310 315 320

Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys

325 330 335

Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala

340 345 350

Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr

355 360 365

Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ser Tyr

370 375 380

Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala

385 390 395 400

Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser

405 410 415

Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn

420 425 430

Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala

435 440 445

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

450 455 460

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

465 470 475 480

Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala

485 490 495

Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr Glu Val

500 505 510

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

515 520 525

Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly

530 535 540

Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu

545 550 555 560

His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile

565 570 575

Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met

580 585 590

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

595 600 605

Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp

610 615 620

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

625 630 635 640

Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr

645 650 655

His Ala Met Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp

660 665 670

Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala

675 680 685

Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His

690 695 700

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

705 710 715 720

Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile Pro

725 730 735

His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg Tyr Asn

740 745 750

Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg

755 760 765

Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu

770 775 780

Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala

785 790 795 800

Trp Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg

805 810 815

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

820 825 830

Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr

835 840 845

Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly

850 855 860

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

865 870 875 880

Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala

885 890

<210> 12

<211> 329

<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 12

Met Lys Leu Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu

1 5 10 15

Gln Gln Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe

20 25 30

Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val

35 40 45

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

50 55 60

Lys Lys His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn

65 70 75 80

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

85 90 95

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

100 105 110

Met Met Thr Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg

115 120 125

Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly

130 135 140

Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu

145 150 155 160

Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr

165 170 175

Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro

180 185 190

Thr Leu Phe Ser Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr

195 200 205

Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Glu Gln Met Lys

210 215 220

Asn Gly Val Met Ile Val Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser

225 230 235 240

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

245 250 255

Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser

260 265 270

Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His

275 280 285

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

290 295 300

Thr Ser Ile Ser Gln Thr Thr Leu Gln Asn Leu Ser Asn Leu Glu Lys

305 310 315 320

Gly Glu Thr Cys Pro Asn Glu Leu Val

325

<210> 13

<211> 714

<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 13

Met Ser Arg Ile Ile Met Leu Ile Pro Thr Gly Thr Ser Val Gly Leu

1 5 10 15

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

20 25 30

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

35 40 45

Ala Pro Asp Gln Thr Thr Thr Ile Val Arg Ala Asn Ser Ser Thr Thr

50 55 60

Thr Ala Ala Glu Pro Leu Lys Met Ser Tyr Val Glu Gly Leu Leu Ser

65 70 75 80

Ser Asn Gln Lys Asp Val Leu Met Glu Glu Ile Val Ala Asn Tyr His

85 90 95

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

100 105 110

Thr Arg Lys His Gln Phe Ala Gln Ser Leu Asn Tyr Glu Ile Ala Lys

115 120 125

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

130 135 140

Pro Glu Gln Leu Lys Glu Arg Ile Glu Leu Thr Arg Asn Ser Phe Gly

145 150 155 160

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

165 170 175

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

180 185 190

Phe Asp Asp Ser Ser Lys Ala Lys Val Asn Asn Val Asp Pro Ala Lys

195 200 205

Leu Gln Glu Ser Ser Pro Leu Pro Val Leu Gly Ala Val Pro Trp Ser

210 215 220

Phe Asp Leu Ile Ala Thr Arg Ala Ile Asp Met Ala Arg His Leu Asn

225 230 235 240

Ala Thr Ile Ile Asn Glu Gly Asp Ile Asn Thr Arg Arg Val Lys Ser

245 250 255

Val Thr Phe Cys Ala Arg Ser Ile Pro His Met Leu Glu His Phe Arg

260 265 270

Ala Gly Ser Leu Leu Val Thr Ser Ala Asp Arg Pro Asp Val Leu Val

275 280 285

Ala Ala Cys Leu Ala Ala Met Asn Gly Val Glu Ile Gly Ala Leu Leu

290 295 300

Leu Thr Gly Gly Tyr Glu Met Asp Ala Arg Ile Ser Lys Leu Cys Glu

305 310 315 320

Arg Ala Phe Ala Thr Gly Leu Pro Val Phe Met Val Asn Thr Asn Thr

325 330 335

Trp Gln Thr Ser Leu Ser Leu Gln Ser Phe Asn Leu Glu Val Pro Val

340 345 350

Asp Asp His Glu Arg Ile Glu Lys Val Gln Glu Tyr Val Ala Asn Tyr

355 360 365

Ile Asn Ala Asp Trp Ile Glu Ser Leu Thr Ala Thr Ser Glu Arg Ser

370 375 380

Arg Arg Leu Ser Pro Pro Ala Phe Arg Tyr Gln Leu Thr Glu Leu Ala

385 390 395 400

Arg Lys Ala Gly Lys Arg Ile Val Leu Pro Glu Gly Asp Glu Pro Arg

405 410 415

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

420 425 430

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

435 440 445

Gly Val Glu Leu Gly Ala Gly Ile Glu Ile Val Asp Pro Glu Val Val

450 455 460

Arg Glu Ser Tyr Val Gly Arg Leu Val Glu Leu Arg Lys Asn Lys Gly

465 470 475 480

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

485 490 495

Gly Thr Leu Met Leu Glu Gln Asp Glu Val Asp Gly Leu Val Ser Gly

500 505 510

Ala Val His Thr Thr Ala Asn Thr Ile Arg Pro Pro Leu Gln Leu Ile

515 520 525

Lys Thr Ala Pro Gly Ser Ser Leu Val Ser Ser Val Phe Phe Met Leu

530 535 540

Leu Pro Glu Gln Val Tyr Val Tyr Gly Asp Cys Ala Ile Asn Pro Asp

545 550 555 560

Pro Thr Ala Glu Gln Leu Ala Glu Ile Ala Ile Gln Ser Ala Asp Ser

565 570 575

Ala Ala Ala Phe Gly Ile Glu Pro Arg Val Ala Met Leu Ser Tyr Ser

580 585 590

Thr Gly Thr Ser Gly Ala Gly Ser Asp Val Glu Lys Val Arg Glu Ala

595 600 605

Thr Arg Leu Ala Gln Glu Lys Arg Pro Asp Leu Met Ile Asp Gly Pro

610 615 620

Leu Gln Tyr Asp Ala Ala Val Met Ala Asp Val Ala Lys Ser Lys Ala

625 630 635 640

Pro Asn Ser Pro Val Ala Gly Arg Ala Thr Val Phe Ile Phe Pro Asp

645 650 655

Leu Asn Thr Gly Asn Thr Thr Tyr Lys Ala Val Gln Arg Ser Ala Asp

660 665 670

Leu Ile Ser Ile Gly Pro Met Leu Gln Gly Met Arg Lys Pro Val Asn

675 680 685

Asp Leu Ser Arg Gly Ala Leu Val Asp Asp Ile Val Tyr Thr Ile Ala

690 695 700

Leu Thr Ala Ile Gln Ser Ala Gln Gln Gln

705 710

<210> 14

<211> 400

<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 14

Met Ser Ser Lys Leu Val Leu Val Leu Asn Cys Gly Ser Ser Ser Leu

1 5 10 15

Lys Phe Ala Ile Ile Asp Ala Val Asn Gly Glu Glu Tyr Leu Ser Gly

20 25 30

Leu Ala Glu Cys Phe His Leu Pro Glu Ala Arg Ile Lys Trp Lys Met

35 40 45

Asp Gly Asn Lys Gln Glu Ala Ala Leu Gly Ala Gly Ala Ala His Ser

50 55 60

Glu Ala Leu Asn Phe Ile Val Asn Thr Ile Leu Ala Gln Lys Pro Glu

65 70 75 80

Leu Ser Ala Gln Leu Thr Ala Ile Gly His Arg Ile Val His Gly Gly

85 90 95

Glu Lys Tyr Thr Ser Ser Val Val Ile Asp Glu Ser Val Ile Gln Gly

100 105 110

Ile Lys Asp Ala Ala Ser Phe Ala Pro Leu His Asn Pro Ala His Leu

115 120 125

Ile Gly Ile Glu Glu Ala Leu Lys Ser Phe Pro Gln Leu Lys Asp Lys

130 135 140

Asn Val Ala Val Phe Asp Thr Ala Phe His Gln Thr Met Pro Glu Glu

145 150 155 160

Ser Tyr Leu Tyr Ala Leu Pro Tyr Asn Leu Tyr Lys Glu His Gly Ile

165 170 175

Arg Arg Tyr Gly Ala His Gly Thr Ser His Phe Tyr Val Thr Gln Glu

180 185 190

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

195 200 205

Cys His Leu Gly Asn Gly Gly Ser Val Ser Ala Ile Arg Asn Gly Lys

210 215 220

Cys Val Asp Thr Ser Met Gly Leu Thr Pro Leu Glu Gly Leu Val Met

225 230 235 240

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

245 250 255

Asp Thr Leu Gly Met Ser Val Asp Ala Ile Asn Lys Leu Leu Thr Lys

260 265 270

Glu Ser Gly Leu Leu Gly Leu Thr Glu Val Thr Ser Asp Cys Arg Tyr

275 280 285

Val Glu Asp Asn Tyr Ala Thr Lys Glu Asp Ala Lys Arg Ala Met Asp

290 295 300

Val Tyr Cys His Arg Leu Ala Lys Tyr Ile Gly Ala Tyr Thr Ala Leu

305 310 315 320

Met Asp Gly Arg Leu Asp Ala Val Val Phe Thr Gly Gly Ile Gly Glu

325 330 335

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

340 345 350

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

355 360 365

Ser Gly Phe Ile Asn Lys Glu Gly Thr Arg Pro Ala Val Val Ile Pro

370 375 380

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

385 390 395 400

Claims

1.用于生产光学纯1,3-丁二醇的重组菌，其特征在于，所述重组菌是将phaA、phaB、bld和yqhD基因通过质粒导入微生物中或通过基因工程手段整合到微生物染色体上而成；

其中，所述phaA基因来源于Cupriavidus necator，其为编码如下蛋白质(a)或(b)的基因：

(a)由SEQ ID NO:3所示的氨基酸序列组成的蛋白质；

(b)SEQ ID NO:3所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质；

所述phaB基因来源于Cupriavidus necator，其为编码如下蛋白质(c)或(d)的基因：

(c)由SEQ ID NO:4所示的氨基酸序列组成的蛋白质；

(d)SEQ ID NO:4所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(c)衍生的蛋白质；

所述bld基因来源于Clostridium saccharoperbutylacetonicum，其编码蛋白的氨基酸序列如SEQ ID NO:1或2所示；

所述yqhD基因来源于大肠杆菌(Escherichia coli)，其为编码如下蛋白质(e)或(f)的基因：

(e)由SEQ ID NO:6所示的氨基酸序列组成的蛋白质；

(f)SEQ ID NO:6所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(e)衍生的蛋白质；

其中，所述微生物选自埃希氏菌属(Escherichia)、克雷伯氏菌属(Klebsiella)、棒杆菌属(Corynebacterium)、短杆菌属(Brevibacterium)中的菌种；优选大肠杆菌。

2.根据权利要求1所述的重组菌，其特征在于，所述重组菌的构建如下：phaA、phaB基因经密码子优化后，与bld、yqhD基因一起构建到表达载体上，用重组载体转化大肠杆菌，筛选阳性转化子；

3.根据权利要求2所述的重组菌，其特征在于，所述重组菌的构建如下：将bld-yqhD-phaAB串联基因表达盒构建到ptrc99a质粒上，用重组质粒转化大肠杆菌，筛选阳性转化子；

4.用于生产光学纯1,3-丁二醇的重组大肠杆菌，其特征在于，所述重组大肠杆菌是以权利要求2或3所述的重组菌作为出发菌株，利用基因工程手段对出发菌株进行改造，得到的细胞内NADPH供给增强的工程菌。

5.根据权利要求4所述的重组大肠杆菌，其特征在于，通过增强出发菌株中与NADPH生物合成途径相关的基因，来提高细胞内NADPH的供给；

i)SEQ ID NO:7所示的核苷酸序列；

6.根据权利要求4或5所述的重组大肠杆菌，其特征在于，弱化出发菌株中sthA基因，来提高细胞内NADPH的供给；所述弱化包括敲除或降低基因的表达；

sthA基因的核苷酸序列如下：

i)SEQ ID NO:8所示的核苷酸序列；

7.高产1,3-丁二醇的工程菌，其特征在于，所述工程菌是以权利要求2或3所述的重组菌，或者权利要求4-6任一项所述的重组大肠杆菌作为原始菌株，利用基因工程手段对原始菌株做进一步改造，得到的细胞内乙酰CoA流向1,3-丁二醇合成途径的代谢通量增强的工程菌。

8.根据权利要求7所述的工程菌，其特征在于，弱化原始菌株中adhE、ldhA、pta、ackA基因中的至少一种基因，来提高细胞内乙酰CoA流向1,3-丁二醇合成途径的代谢通量；所述弱化包括敲除或降低基因的表达；

adhE、ldhA、pta、ackA基因编码蛋白的氨基酸序列分别如SEQ ID NO:11-14所示；

优选地，弱化原始菌株中adhE、ldhA、pta和ackA基因，来提高细胞内乙酰CoA流向1,3-丁二醇合成途径的代谢通量。

9.权利要求1-3任一项所述的重组菌，或权利要求4-6任一项所述的重组大肠杆菌，或者权利要求7或8所述的工程菌在发酵生产1,3-丁二醇中的应用。

10.丁醛脱氢酶突变体，其特征在于，所述丁醛脱氢酶突变体的氨基酸序列如SEQ IDNO:2所示。