CN109234295B

CN109234295B - 一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法

Info

Publication number: CN109234295B
Application number: CN201811033458.1A
Authority: CN
Inventors: 郭道义; 潘虹; 李勋; 李永东; 范小林
Original assignee: Gannan Normal University
Current assignee: Ningxia Wodidi Water And Fertilizer Technology Co ltd
Priority date: 2015-07-03
Filing date: 2015-07-03
Publication date: 2022-09-23
Anticipated expiration: 2035-07-03
Also published as: CN105331647A; CN105331647B; CN109234294A; CN109234294B; CN109234295A

Abstract

本发明公开了一种微生物内基于脂肪酸代谢途径三个中间产物为前体分子合成信息素脂肪醇乙酸酯的方法，其步骤为：首先通过微生物中高表达脂酰‑ACP还原酶AAR、羧酸还原酶CAR、脂酰‑CoA还原酶FAR及醛还原酶AHR，分别得到可还原脂酰‑ACP、脂肪酸或脂酰‑CoA产脂肪醇的工程菌。最后在产脂肪醇工程菌中高表达醇乙酰基转移酶ATF1，用于催化脂肪醇与乙酰辅酶A反应合成信息素脂肪醇乙酸酯。本发明可实现由葡萄糖到脂肪醇乙酸酯的微生物转化，为大规模的生物合成脂肪醇乙酸酯提供了一个可行道路。

Description

一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法

技术领域

本发明属于微生物基因工程领域，具体涉及改造微生物代谢途径，从单糖直接合成信息素脂肪醇乙酸酯的方法。

背景技术

1、昆虫性信息素，又称性外激素(人工合成用于防治害虫的又称为性引诱剂)，是由同种昆虫某一性别个体的特殊器官分泌于体外，能被同种异性个体的感受器所接受，并使其产生相应行为反应或生理效应(如觅偶、定向求偶、交配等)的微量化学物质。至今，已鉴定出约2000种昆虫性信息素，大多数性信息素为各种脂肪醇乙酸酯分子。目前已有少数昆虫性信息素实现了化学全合成。由于应用昆虫性信息素进行害虫监测和防治具有高效、无毒、无污染、不伤害天敌昆虫等优点，国内外学者十分重视对昆虫性信息素的研究和应用。

2、昆虫性信息素是昆虫本身的产物，因此其最大的优点就是使用非常安全，害虫不产生抗性、灵敏度高、用量少、专属性强、不污染环境、对天敌无害甚或有利，对生态环境的干扰小、不造成破坏。目前昆虫性信息素被用于虫情检测、大量诱捕、干扰交配、配合治虫、害虫检疫、区分近缘种等方面。但由于绝大多数的种类的昆虫性信息素还未能实现商品化生产，限制了其更广泛的应用。

3、目前已商品化的性信息素均为通过化学法合成。化学法合成性信息素需涉及到多个反应步骤导致合成成本较高。通过生物技术手段，实现微生物发酵单糖合成各种性信息素，可有效的降低成本。

发明内容

本发明的目的在于提供一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法，在微生物内构建一条由单糖转化到脂肪醇乙酸酯的代谢途径。

为实现上述目的，本发明提供了在大肠杆菌合成脂肪醇乙酸酯的方法，合成示意图如图1所示，主要步骤为：

(1)构建基于脂肪酸为前体分子产脂肪醇乙酸酯工程菌

将来自Mycobacterium marinum菌的羧酸还原酶CAR基因和酿酒酵母醇乙酰基转移酶ATF1基因，重组到载体pET28a(+)上，得到载体pDY10。将来自大肠杆菌的醛还原酶AHR及硫酯酶‘TESA基因、枯草芽孢杆菌磷酸泛酰巯基乙胺基转移酶Sfp基因，重组到载体pBBR1MCS1上，得到载体pDY09。将上述两个载体同时转入到大肠杆菌中，实现上述五个基因在大肠杆菌中的高量表达，得到大肠杆菌工程菌株GDY4。表达‘TESA用于水解脂酰-ACP产生脂肪酸，表达CAR用于催化脂肪酸还原为脂肪醛，表达Sfp用于活化CAR酶，表达AHR用于随后催化脂肪醛还原形成脂肪醇，表达ATF1用于最终催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。脂肪酸和乙酰辅酶A均可由大肠杆菌发酵单糖或甘油合成，为此上述构建的GDY4工程菌可有效的将单糖或甘油转化为脂肪醇乙酸酯。

相应的，还可以通过(2)构建基于脂酰-ACP为前体分子产脂肪醇乙酸酯工程菌

将来自Synechococcus elongates菌的脂酰-ACP还原酶AAR基因、大肠杆菌的醛还原酶AHR基因和酿酒酵母醇乙酰基转移酶ATF1基因，重组到载体pET28a(+)上，得到载体pDY05。将该载体转入到大肠杆菌中，实现上述三个基因在大肠杆菌中的高量表达，得到大肠杆菌工程菌株GDY2。表达AAR用于催化脂酰-ACP还原为脂肪醛，表达AHR用于随后催化脂肪醛还原形成脂肪醇，表达ATF1用于最终催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。脂酰-ACP和乙酰辅酶A均可由大肠杆菌发酵单糖或甘油合成，为此上述构建的GDY2工程菌可有效的将单糖或甘油转化为脂肪醇乙酸酯。

相应的，还可以通过(3)构建基于脂酰-ACP和脂酰-COA为前体分子产脂肪醇乙酸酯工程菌

将来自Marinobacter aquaeolei菌的脂酰-CoA还原酶FAR基因和酿酒酵母醇乙酰基转移酶ATF1基因，重组到载体pET28a(+)上，得到载体pDY12。将该载体转入到大肠杆菌中，实现上述两个基因在大肠杆菌中的高量表达，得到大肠杆菌工程菌株GDY6。表达FAR用于催化脂酰-ACP和脂酰-COA还原为脂肪醇，表达ATF1用于最终催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。脂酰-ACP、脂酰-COA和乙酰辅酶A均可由大肠杆菌发酵单糖或甘油合成，为此上述构建的GDY6工程菌可有效的将单糖或甘油转化为脂肪醇乙酸酯。

本发明的优点是在大肠杆菌引入外源基因构建一条合成脂肪醇乙酸酯的代谢途径，最终实现以单糖为原料合成脂肪醇乙酸酯的目的，不需要通过过多的化学合成反应，减少化学合成过程中有毒物质的释放，也减少了合成成本。同时，由于大肠杆菌生长速度快，遗传操作技术成熟，且发酵过程不会对环境造成污染破坏。

附图说明

图1.生物合成脂肪醇乙酸酯路径图。ATF1：醇乙酰基转移酶(alcoholacetyltransferase)来自S.cerevisiae菌；AAR：脂酰-ACP还原酶(fatty acyl-ACPreductase)来自S.elongates菌；CAR：羧酸还原酶(carboxylic acid reductase)来自M.marinum菌；FAR：脂酰-CoA还原酶(fatty acyl-CoAreductase)来自M.aquaeolei菌；AHR：醛还原酶(aldehyde reductase)来自E.coli菌；FadD：脂酰-CoA合酶(fatty acyl-CoAsynthetase)来自E.coli；'TesA：去除信号肽的硫酯酶(a truncated fatty acyl-ACPthioesterase)来自E.coli菌.

图2.基于脂酰-ACP为前体分子合成脂肪醇或脂肪醇乙酸酯的大肠杆菌工程菌GDY1和GDY2发酵产物GC-MS检测结果。1号峰代表内标碳十五脂肪酸甲酯(MethylPentadecanoate),2号峰代表碳十五脂肪醇内标(Pentadecanol),3号峰代表碳十六脂肪醇乙酸酯(Hexadecanol acetate ester)，4号峰代表碳十六脂肪醇(Hexadecanol)，5号峰代表Δ9-碳十八烯脂肪醇乙酸酯(9-Octadecen-1-ol acetate ester)，6号峰代表Δ9-碳十八烯脂肪醇(9-Octadecen-1-ol alcohol)。

图3.基于脂肪酸为前体分子合成脂肪醇或脂肪醇乙酸酯的大肠杆菌工程菌GDY3和GDY4发酵产物GC-MS检测结果。1号峰为碳十二脂肪醇乙酸酯(Dodecanol acetateester)，2号峰为碳十二脂肪醇(Dodecanol)，3号峰为Δ9-碳十二烯醇(9-Dodecanol-1-ol)，4号峰为碳十四脂肪醇乙酸酯(Tetradecanol acetate ester)，5号峰为碳十五脂肪酸甲酯内标(Methyl Pentadecanoate)，6号峰为碳十四脂肪醇(Tetradecanol)，7号峰为Δ9-碳十四烯醇(9-Tetradecen-1-ol)，8号峰为碳十五醇内标(Pentadecanol)，9号峰为Δ9-碳十六烯醇乙酸酯(9-Hexadecen-1-ol acetate ester)，10号峰为碳十六脂肪醇(Hexadecanol)，11号峰为Δ9-碳十六烯脂肪醇(9-Hexadecen-1-ol)，12号峰为Δ9-碳十八烯醇乙酸酯(9-Octadecen-1-ol acetate ester)，13号峰为Δ9-碳十八烯醇(9-Octadecen-1-ol)。

图4.基于脂酰-ACP和脂酰-CoA为前体分子合成脂肪醇或脂肪醇乙酸酯的大肠杆菌工程菌GDY5和GDY6发酵产物GC-MS检测结果。1号峰为碳十四脂肪醇乙酸酯(Tetradecanol acetate ester)，2号峰为碳十五脂肪酸甲酯内标(MethylPentadecanoate)，3号峰碳十四脂肪醇(Tetradecanol)，4号峰为Δ9-碳十四烯醇(9-Tetradecen-1-ol)，5号峰为碳十五醇内标(Pentadecanol)，6号峰为碳十六脂肪醇乙酸酯(Hexadecanol acetate ester)，7号峰为Δ9-碳十六烯醇乙酸酯(9-Hexadecen-1-olacetate ester)，8号峰为碳十六脂肪醇(Hexadecanol)，9号峰为Δ9-碳十六烯脂肪醇(9-Hexadecen-1-ol)，10号峰为Δ9-碳十八烯醇乙酸酯(9-Octadecen-1-ol acetate ester)，11号峰为Δ9-碳十八烯醇(9-Octadecen-1-ol)。

具体实施方式

本发明的目的通过以下措施来达到：

1、在微生物体内表达AAR和AHR基因，用于催化脂肪酸合成途径中间体脂酰-ACP还原为脂肪醇。表达ATF1基因用于催化脂肪醇和乙酰辅酶A两个底物合成脂肪醇乙酸酯。

2、在微生物体内表达‘TESA用于水解脂酰-ACP产生脂肪酸，表达CAR用于催化脂肪酸还原为脂肪醛，表达Sfp用于活化CAR酶，表达AHR用于随后催化脂肪醛还原形成脂肪醇，表达ATF1用于最终催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。

3、在微生物体内表达FAR用于催化脂酰-ACP和脂酰-COA还原为脂肪醇，表达ATF1用于最终催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。

4、本发明的三个工程菌，其细胞生长快速，能够利用单糖为唯一碳源合成脂肪醇乙酸酯。

以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。

实施例1构建基于脂酰ACP为前体分子产脂肪醇乙酸酯大肠杆菌工程菌(图1所示)

构建产脂肪醇工程菌

1、利用引物AAR-XbaI(GTATCTAGAAAGAGGAGATATAATGTTCGGTCTTATCGGTCATCTC)和AAR-SpeI-BamHI(TGTGGATCCACTAGTTCAAATTGCCAATGCCAAGG)PCR扩增来自Synechococcuselongates菌的AAR基因，随后将扩增的片段以XbaI和BamHI插入到pET28a(+)中得到载体pDY01。利用引物AHR-XbaI(ATATCTAGAAAGAGGAGATATAATGTCGATGATAAAAAGCTATGCCG)和AHR-SpeI-BamHI(GTAGGATCCACTAGTTCAAAAATCGGCTTTCAACACCAC)PCR扩增来自大肠杆菌的AHR基因，利用XbaI和Ba mHI插入到pET28a(+)中得到载体pDY02。利用XbaI和XhoI双酶切pDY01得到AAR表达框，将其插入以SpeI和XhoI双酶切的载体pDY02上，得到重组载体pDY03。

A)、将重组载体pDY03导入大肠杆菌GM1655中，得到GDY1工程菌。该工程菌实现了AAR和AHR基因的高表达。表达AAR酶用于催化脂肪酸合成中间体脂酰-ACP还原为脂肪醛醛，表达AHR酶用于催化脂肪醛还原为脂肪醇。

构建产脂肪醇乙酸酯工程菌

1、利用引物ATF1-XbaI(GGATCTAGAAACTTTAAGAAGGAGATATAATGAATGAAATCGATGAGAAAAATCAGG)and ATF1-SpeI-SacI(GATGAGCTCACTAGTCTAAGGGCCTAAAAGGAGAGCTTTGTAA)PCR扩增来自酿酒酵母菌的ATF1基因，随后将扩增的片段以XbaI和SacI双酶切插入到pET28a(+)载体中，得到载体pDY04。以XbaI和XhoI双酶切载体pDY03，得到AAR和AHR表达框，将其插入以SpeI和XhoI双酶切的载体pDY04上，得到重组载体pDY05。

2、将重组载体pDY05导入大肠杆菌GM1655中，得到GDY2工程菌。该工程菌实现了AAR、AHR和ATF1基因的高表达。表达AAR酶用于催化脂肪酸合成中间体脂酰-ACP还原为脂肪醛醛，表达AHR酶用于催化脂肪醛还原为脂肪醇醇，表达ATF1用于催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。

实施例2构建基于脂肪酸为前体分子产脂肪醇乙酸酯大肠杆菌工程菌(图1所示)

构建产脂肪醇工程菌

1、利用引物CAR-XbaI(ATATCTAGAAAGAGGAGATATAATGTCGCCAATCACGCGTGA)和CAR-SpeI-BamHI(AGAGGATCCACTAGTTCAGAGCAGGCCGAGTAGGC)PCR扩增来自Mycobacteriummarinum菌的CAR基因，随后将扩增的片段以XbaI和BamHI插入到pET28a(+)中得到载体pDY06。利用引物'TesA-SacI(AGATGAGCTCATGG CGGACACGTTATTGATTCTGG)和'TesA-SpeI(TGTACTAGTTTATGAGTCATGATTTACTAAAGGCTGC)PCR扩增来自大肠杆菌的'TesA基因，随后将扩增的片段以SacI和SpeI双酶切插入到pBBR1MCS1中得到载体pDY07。利用引物AHR-BamHI(ACTGGATCCAA GAAGGAGATATAATGTCGATGATAAAAAGCTATGCCG)和AHR-XhoI(TGTCTCGAGTCAAAAATCGGCTTTCAACACCAC)PCR扩增来自大肠杆菌的AHR基因,随后将扩增的片段以BamHI和XhoI双酶切插入到载体pDY07，得到载体pDY08。利用引物Sfp-XhoI(TT CCTCGAGAAGAAGGAGATATAATGAAGATTTACGGAATTTATATGGACCG)和Sfp-ApaI(ACAGGGCCCTTATAAAAGCTCTTCGTACGAGACCATTG)PCR扩增来自Bacillus subtilis subsp.subtilis str.168菌的Sfp基因，随后将扩增的片段以XhoI和ApaI双酶切插入到载体pDY08，得到载体pDY09。

2、将重组载体pDY06和pDY09导入大肠杆菌GM1655中，得到GDY3工程菌。该工程菌实现了CAR、AHR、Sfp和'TesA基因的高表达。表达‘TesA用于水解脂酰-ACP产生脂肪酸，表达CAR用于催化脂肪酸还原为脂肪醛，表达Sfp用于活化CAR酶，表达AHR用于随后催化脂肪醛还原形成脂肪醇。

构建产脂肪醇乙酸酯工程菌

1、以XbaI和XhoI双酶切载体pDY06，得到CAR表达框，将其插入以SpeI和XhoI双酶切的载体pDY04上，得到重组载体pDY10。

2、将重组载体pDY10和pDY09导入大肠杆菌GM1655中，得到GDY4工程菌。该工程菌实现了CAR、AHR、Sfp、'TesA和ATF1基因的高表达。表达‘TesA用于水解脂酰-ACP产生脂肪酸，表达CAR用于催化脂肪酸还原为脂肪醛，表达Sfp用于活化CAR酶，表达AHR用于随后催化脂肪醛还原形成脂肪醇，表达ATF1用于催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。

实施例3构建基于脂酰ACP和脂酰-CoA为前体分子产脂肪醇乙酸酯大肠杆菌工程菌(图1所示)

构建产脂肪醇工程菌

1、利用引物FAR-XbaI(CAATCTAGAAAGAGGAGATATAATGGCAATACAGCAGGTACATCAC)和FAR-SpeI-BamHI(CGAGGATCCACTAGTTCAGGCAGCTTTTTTGCGCT)PCR扩增来自M.aquaeolei菌的FAR基因，随后将扩增的片段以XbaI和BamHI插入到pET28a(+)中得到载体pDY11。

2、将重组载体pDY11导入大肠杆菌GM1655中，得到GDY5工程菌。该工程菌实现了FAR基因的高表达。表达FAR酶用于催化脂肪酸合成中间体脂酰-ACP和脂酰-CoA直接还原为脂肪醇醇。

构建产脂肪醇乙酸酯工程菌

1、以XbaI和XhoI双酶切载体pDY11，得到FAR表达框，将其插入以SpeI和XhoI双酶切的载体pDY04上，得到重组载体pDY12。

2、将重组载体pDY12导入大肠杆菌GM1655中，得到GDY6工程菌。该工程菌实现了FAR、和ATF1基因的高表达。表达FAR酶用于催化脂肪酸合成中间体脂酰-ACP和脂酰-CoA直接还原为脂肪醇醇，表达ATF1用于催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯。

实施例4大肠杆菌工程菌生产脂肪醇或脂肪醇乙酸酯的发酵实验

1、将30度过夜培养的大肠杆菌工程菌，接种到50ml M9发酵培养基中(包含2％葡萄糖)。

2、培养24小时后收集菌体，超声波破细胞，利用乙酸乙酯萃取产物。

3、利用旋转蒸发仪将溶剂乙酸乙酯旋干，再用正己烷溶解产物。

4、GC-MS(气相色谱-质谱联用仪)检测经正己烷溶解的样品.

实验结果：如图2所示大肠杆菌工程菌GDY1和GDY2可分别直接利用葡萄糖基于脂酰-ACP为前体分子合成各种脂肪醇或脂肪醇乙酸酯；如图3所示大肠杆菌工程菌GDY3和GDY4可分别直接利用葡萄糖基于脂肪酸为前体分子合成各种脂肪醇或脂肪醇乙酸酯；如图4所示大肠杆菌工程菌GDY5和GDY6可分别直接利用葡萄糖基于脂酰-ACP和脂酰-CoA为前体分子合成各种脂肪醇或脂肪醇乙酸酯。

<110> 赣南师范学院

<120> 脂肪醇乙酸酯的生物合成

<160> 14

<170> PatentIn Version 2.1

<210> 1

<211> 1026

<212> DNA

<213> 蓝藻（Synechococcus elongates ）

<400> 1

atgttcggtc ttatcggtca tctcaccagt ttggagcagg cccgcgacgt ttctcgcagg 60

atgggctacg acgaatacgc cgatcaagga ttggagtttt ggagtagcgc tcctcctcaa 120

atcgttgatg aaatcacagt caccagtgcc acaggcaagg tgattcacgg tcgctacatc 180

gaatcgtgtt tcttgccgga aatgctggcg gcgcgccgct tcaaaacagc cacgcgcaaa 240

gttctcaatg ccatgtccca tgcccaaaaa cacggcatcg acatctcggc cttggggggc 300

tttacctcga ttattttcga gaatttcgat ttggccagtt tgcggcaagt gcgcgacact 360

accttggagt ttgaacggtt caccaccggc aatactcaca cggcctacgt aatctgtaga 420

caggtggaag ccgctgctaa aacgctgggc atcgacatta cccaagcgac agtagcggtt 480

gtcggcgcga ctggcgatat cggtagcgct gtctgccgct ggctcgacct caaactgggt 540

gtcggtgatt tgatcctgac ggcgcgcaat caggagcgtt tggataacct gcaggctgaa 600

ctcggccggg gcaagattct gcccttggaa gccgctctgc cggaagctga ctttatcgtg 660

tgggtcgcca gtatgcctca gggcgtagtg atcgacccag caaccctgaa gcaaccctgc 720

gtcctaatcg acgggggcta ccccaaaaac ttgggcagca aagtccaagg tgagggcatc 780

tatgtcctca atggcggggt agttgaacat tgcttcgaca tcgactggca gatcatgtcc 840

gctgcagaga tggcgcggcc cgagcgccag atgtttgcct gctttgccga ggcgatgctc 900

ttggaatttg aaggctggca tactaacttc tcctggggcc gcaaccaaat cacgatcgag 960

aagatggaag cgatcggtga ggcatcggtg cgccacggct tccaaccctt ggcattggca 1200

atttga 1026

<210> 2

<211> 341

<212> PRT

<213>蓝藻（Synechococcus elongates ）

<400> 2

Met Phe Gly Leu Ile Gly His Leu Thr Ser Leu Glu Gln Ala Arg Asp

1 5 10 15

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

20 25 30

Phe Trp Ser Ser Ala Pro Pro Gln Ile Val Asp Glu Ile Thr Val Thr

35 40 45

Ser Ala Thr Gly Lys Val Ile His Gly Arg Tyr Ile Glu Ser Cys Phe

50 55 60

Leu Pro Glu Met Leu Ala Ala Arg Arg Phe Lys Thr Ala Thr Arg Lys

65 70 75 80

Val Leu Asn Ala Met Ser His Ala Gln Lys His Gly Ile Asp Ile Ser

85 90 95

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

100 105 110

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

115 120 125

Thr Gly Asn Thr His Thr Ala Tyr Val Ile Cys Arg Gln Val Glu Ala

130 135 140

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

145 150 155 160

Val Gly Ala Thr Gly Asp Ile Gly Ser Ala Val Cys Arg Trp Leu Asp

165 170 175

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

180 185 190

Arg Leu Asp Asn Leu Gln Ala Glu Leu Gly Arg Gly Lys Ile Leu Pro

195 200 205

Leu Glu Ala Ala Leu Pro Glu Ala Asp Phe Ile Val Trp Val Ala Ser

210 215 220

Met Pro Gln Gly Val Val Ile Asp Pro Ala Thr Leu Lys Gln Pro Cys

225 230 235 240

Val Leu Ile Asp Gly Gly Tyr Pro Lys Asn Leu Gly Ser Lys Val Gln

245 250 255

Gly Glu Gly Ile Tyr Val Leu Asn Gly Gly Val Val Glu His Cys Phe

260 265 270

Asp Ile Asp Trp Gln Ile Met Ser Ala Ala Glu Met Ala Arg Pro Glu

275 280 285

Arg Gln Met Phe Ala Cys Phe Ala Glu Ala Met Leu Leu Glu Phe Glu

290 295 300

Gly Trp His Thr Asn Phe Ser Trp Gly Arg Asn Gln Ile Thr Ile Glu

305 310 315 320

Lys Met Glu Ala Ile Gly Glu Ala Ser Val Arg His Gly Phe Gln Pro

325 330 335

Leu Ala Leu Ala Ile

340

<210> 3

<211> 1026

<212> DNA

<213> 大肠杆菌（Escherichia coli）

<400> 3

atgtcgatga taaaaagcta tgccgcaaaa gaagcgggcg gcgaactgga agtttatgag 60

tacgatcccg gtgagctgag gccacaagat gttgaagtgc aggtggatta ctgcgggatc 120

tgccattccg atctgtcgat gatcgataac gaatggggat tttcacaata tccgctggtt 180

gccgggcatg aggtgattgg gcgcgtggtg gcactcggga gcgccgcgca ggataaaggt 240

ttgcaggtcg gtcagcgtgt cgggattggc tggacggcgc gtagctgtgg tcactgcgac 300

gcctgtatta gcggtaatca gatcaactgc gagcaaggtg cggtgccgac gattatgaat 360

cgcggtggct ttgccgagaa gttgcgtgcg gactggcaat gggtgattcc actgccagaa 420

aatattgata tcgagtccgc cgggccgctg ttgtgcggcg gtatcacggt ctttaaacca 480

ctgttgatgc accatatcac tgctaccagc cgcgttgggg taattggtat tggcgggctg 540

gggcatatcg ctataaaact tctgcacgca atgggatgcg aggtgacagc ctttagttct 600

aatccggcga aagagcagga agtgctggcg atgggtgccg ataaagtggt gaatagccgc 660

gatccgcagg cactgaaagc actggcgggg cagtttgatc tcattatcaa caccgtcaac 720

gtcagcctcg actggcagcc ctattttgag gcgctgacct atggcggtaa tttccatacg 780

gtcggtgcgg ttctcacgcc gctgtctgtt ccggccttta cgttaattgc gggcgatcgc 840

agcgtctctg gttctgctac cggcacgcct tatgagctgc gtaagctgat gcgttttgcc 900

gcccgcagca aggttgcgcc gaccaccgaa ctgttcccga tgtcgaaaat taacgacgcc 960

atccagcatg tgcgcgacgg taaggcgcgt taccgcgtgg tgttgaaagc cgatttttga 1020

<210> 4

<211> 339

<212> PRT

<213>大肠杆菌（Escherichia coli）

<400> 4

Met Ser Met Ile Lys Ser Tyr Ala Ala Lys Glu Ala Gly Gly Glu Leu

1 5 10 15

Glu Val Tyr Glu Tyr Asp Pro Gly Glu Leu Arg Pro Gln Asp Val Glu

20 25 30

Val Gln Val Asp Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile

35 40 45

Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu

50 55 60

Val Ile Gly Arg Val Val Ala Leu Gly Ser Ala Ala Gln Asp Lys Gly

65 70 75 80

Leu Gln Val Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Arg Ser Cys

85 90 95

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

100 105 110

Gly Ala Val Pro Thr Ile Met Asn Arg Gly Gly Phe Ala Glu Lys Leu

115 120 125

Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Asn Ile Asp Ile

130 135 140

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

145 150 155 160

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

165 170 175

Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly

180 185 190

Cys Glu Val Thr Ala Phe Ser Ser Asn Pro Ala Lys Glu Gln Glu Val

195 200 205

Leu Ala Met Gly Ala Asp Lys Val Val Asn Ser Arg Asp Pro Gln Ala

210 215 220

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

225 230 235 240

Val Ser Leu Asp Trp Gln Pro Tyr Phe Glu Ala Leu Thr Tyr Gly Gly

245 250 255

Asn Phe His Thr Val Gly Ala Val Leu Thr Pro Leu Ser Val Pro Ala

260 265 270

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

275 280 285

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

290 295 300

Val Ala Pro Thr Thr Glu Leu Phe Pro Met Ser Lys Ile Asn Asp Ala

305 310 315 320

Ile Gln His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Leu Lys

325 330 335

Ala Asp Phe

<210> 5

<211> 1578

<212> DNA

<213> 酿酒酵母（Saccharomyces cerevisiae）

<400> 5

atgaatgaaa tcgatgagaa aaatcaggcc cccgtgcaac aagaatgcct gaaagagatg 60

attcagaatg ggcatgctcg gcgtatggga tctgttgaag atctgtatgt tgctctcaac 120

agacaaaact tatatcgaaa cttctgcaca tatggagaat tgagtgatta ctgtactagg 180

gatcagctca cattagcttt gagggaaatc tgcctgaaaa atccaactct tttacatatt 240

gttctaccaa caagatggcc aaatcatgaa aattattatc gcagttccga atactattca 300

cggccacatc cagtgcatga ttatatttca gtattacaag aattgaaact gagtggtgtg 360

gttctcaatg aacaacctga gtacagtgca gtaatgaagc aaatattaga agaattcaaa 400

aatagtaagg gttcctatac tgcaaaaatt tttaaactta ctaccacttt gactattcct 480

tactttggac caacaggacc gagttggcgg ctaatttgtc ttccagaaga gcacacagaa 540

aagtggaaaa aatttatctt tgtatctaat cattgcatgt ctgatggtcg gtcttcgatc 600

cacttttttc atgatttaag agacgaatta aataatatta aaactccacc aaaaaaatta 660

gattacattt tcaagtacga ggaggattac caattattga ggaaacttcc agaaccgatc 720

gaaaaggtga tagactttag accaccgtac ttgtttattc cgaagtcact tctttcgggt 780

ttcatctaca atcatttgag attttcttca aaaggtgtct gtatgagaat ggatgatgtg 840

gaaaaaaccg atgatgttgt caccgagatc atcaatattt caccaacaga atttcaagcg 900

attaaagcaa atattaaatc aaatatccaa ggtaagtgta ctatcactcc gtttttacat 960

gtttgttggt ttgtatctct tcataaatgg ggtaaatttt tcaaaccatt gaacttcgaa 1020

tggcttacgg atatttttat ccccgcagat tgccgctcac aactaccaga tgatgatgaa 1080

atgagacaga tgtacagata tggcgctaac gttggattta ttgacttcac cccctggata 1140

agcgaatttg acatgaatga taacaaagaa aatttttggc cacttattga gcactaccat 1200

gaagtaattt cggaagcttt aagaaataaa aagcatctcc atggcttagg gttcaatata 1260

caaggcttcg ttcaaaaata tgtgaacatt gacaaggtaa tgtgcgatcg tgccatcggg 1320

aaaagacgcg gaggtacatt gttaagcaat gtaggtctgt ttaatcagtt agaggagccc 1380

gatgccaaat attctatatg cgatttggca tttggccaat ttcaaggatc ctggcaccaa 1440

gcattttcct tgggtgtttg ttcgactaat gtaaagggga tgaatattgt tgttgcttca 1500

acaaagaatg ttgttggtag tcaagaatct ctcgaagagc tttgctccat ttacaaagct 1560

ctccttttag gcccttag

<210> 6

<211> 525

<212> PRT

<213>酿酒酵母（Saccharomyces cerevisiae）

<400> 6

Met Asn Glu Ile Asp Glu Lys Asn Gln Ala Pro Val Gln Gln Glu Cys

1 5 10 15

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

20 25 30

Glu Asp Leu Tyr Val Ala Leu Asn Arg Gln Asn Leu Tyr Arg Asn Phe

35 40 45

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

50 55 60

Leu Ala Leu Arg Glu Ile Cys Leu Lys Asn Pro Thr Leu Leu His Ile

65 70 75 80

Val Leu Pro Thr Arg Trp Pro Asn His Glu Asn Tyr Tyr Arg Ser Ser

85 90 95

Glu Tyr Tyr Ser Arg Pro His Pro Val His Asp Tyr Ile Ser Val Leu

100 105 110

Gln Glu Leu Lys Leu Ser Gly Val Val Leu Asn Glu Gln Pro Glu Tyr

115 120 125

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

130 135 140

Ser Tyr Thr Ala Lys Ile Phe Lys Leu Thr Thr Thr Leu Thr Ile Pro

145 150 155 160

Tyr Phe Gly Pro Thr Gly Pro Ser Trp Arg Leu Ile Cys Leu Pro Glu

165 170 175

Glu His Thr Glu Lys Trp Lys Lys Phe Ile Phe Val Ser Asn His Cys

180 185 190

Met Ser Asp Gly Arg Ser Ser Ile His Phe Phe His Asp Leu Arg Asp

195 200 205

Glu Leu Asn Asn Ile Lys Thr Pro Pro Lys Lys Leu Asp Tyr Ile Phe

210 215 220

Lys Tyr Glu Glu Asp Tyr Gln Leu Leu Arg Lys Leu Pro Glu Pro Ile

225 230 235 240

Glu Lys Val Ile Asp Phe Arg Pro Pro Tyr Leu Phe Ile Pro Lys Ser

245 250 255

Leu Leu Ser Gly Phe Ile Tyr Asn His Leu Arg Phe Ser Ser Lys Gly

260 265 270

Val Cys Met Arg Met Asp Asp Val Glu Lys Thr Asp Asp Val Val Thr

275 280 285

Glu Ile Ile Asn Ile Ser Pro Thr Glu Phe Gln Ala Ile Lys Ala Asn

290 295 300

Ile Lys Ser Asn Ile Gln Gly Lys Cys Thr Ile Thr Pro Phe Leu His

305 310 315 320

Val Cys Trp Phe Val Ser Leu His Lys Trp Gly Lys Phe Phe Lys Pro

325 330 335

Leu Asn Phe Glu Trp Leu Thr Asp Ile Phe Ile Pro Ala Asp Cys Arg

340 345 350

Ser Gln Leu Pro Asp Asp Asp Glu Met Arg Gln Met Tyr Arg Tyr Gly

355 360 365

Ala Asn Val Gly Phe Ile Asp Phe Thr Pro Trp Ile Ser Glu Phe Asp

370 375 380

Met Asn Asp Asn Lys Glu Asn Phe Trp Pro Leu Ile Glu His Tyr His

385 390 395 400

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

405 410 415

Gly Phe Asn Ile Gln Gly Phe Val Gln Lys Tyr Val Asn Ile Asp Lys

420 425 430

Val Met Cys Asp Arg Ala Ile Gly Lys Arg Arg Gly Gly Thr Leu Leu

435 440 445

Ser Asn Val Gly Leu Phe Asn Gln Leu Glu Glu Pro Asp Ala Lys Tyr

450 455 460

Ser Ile Cys Asp Leu Ala Phe Gly Gln Phe Gln Gly Ser Trp His Gln

465 470 475 480

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

485 490 495

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

500 505 510

Glu Leu Cys Ser Ile Tyr Lys Ala Leu Leu Leu Gly Pro

515 520 525

<210> 7

<211> 3525

<212> DNA

<213> 分枝杆菌（Mycobacterium marinum）

<400> 7

atgtcgccaa tcacgcgtga agagcggctc gagcgccgca tccaggacct ctacgccaac 60

gacccgcagt tcgccgccgc caaacccgcc acggcgatca ccgcagcaat cgagcggccg 120

ggtctaccgc taccccagat catcgagacc gtcatgaccg gatacgccga tcggccggct 180

ctcgctcagc gctcggtcga attcgtgacc gacgccggca ccggccacac cacgctgcga 240

ctgctccccc acttcgaaac catcagctac ggcgagcttt gggaccgcat cagcgcactg 300

gccgacgtgc tcagcaccga acagacggtg aaaccgggcg accgggtctg cttgttgggc 360

ttcaacagcg tcgactacgc cacgatcgac atgactttgg cgcggctggg cgcggtggcc 420

gtaccactgc agaccagcgc ggcgataacc cagctgcagc cgatcgtcgc cgagacccag 480

cccaccatga tcgcggccag cgtcgacgca ctcgctgacg ccaccgaatt ggctctgtcc 540

ggtcagaccg ctacccgagt cctggtgttc gaccaccacc ggcaggttga cgcacaccgc 600

gcagcggtcg aatccgcccg ggagcgcctg gccggctcgg cggtcgtcga aaccctggcc 660

gaggccatcg cgcgcggcga cgtgccccgc ggtgcgtccg ccggctcggc gcccggcacc 721

gatgtgtccg acgactcgct cgcgctactg atctacacct cgggcagcac gggtgcgccc 780

aagggcgcga tgtacccccg acgcaacgtt gcgaccttct ggcgcaagcg cacctggttc 840

gaaggcggct acgagccgtc gatcacgctg aacttcatgc caatgagcca cgtcatgggc 900

cgccaaatcc tgtacggcac gctgtgcaat ggcggcaccg cctacttcgt ggcgaaaagc 960

gatctctcca ccttgttcga agacctggcg ctggtgcggc ccaccgagct gaccttcgtg 1020

ccgcgcgtgt gggacatggt gttcgacgag tttcagagtg aggtcgaccg ccgcctggtc 1080

gacggcgccg accgggtcgc gctcgaagcc caggtcaagg ccgagatacg caacgacgtg 1140

ctcggtggac ggtataccag cgcactgacc ggctccgccc ctatctccga cgagatgaag 1200

gcgtgggtcg aggagctgct cgacatgcat ctggtcgagg gctacggctc caccgaggcc 1260

gggatgatcc tgatcgacgg agccattcgg cgcccggcgg tactcgacta caagctggtc 1320

gatgttcccg acctgggtta cttcctgacc gaccggccac atccgcgggg cgagttgctg 1380

gtcaagaccg atagtttgtt cccgggctac taccagcgag ccgaagtcac cgccgacgtg 1440

ttcgatgctg acggcttcta ccggaccggc gacatcatgg ccgaggtcgg ccccgaacag 1500

ttcgtgtacc tcgaccgccg caacaacgtg ttgaagctgt cgcagggcga gttcgtcacc 1560

gtctccaaac tcgaagcggt gtttggcgac agcccactgg tacggcagat ctacatctac 1620

ggcaacagcg cccgtgccta cctgttggcg gtgatcgtcc ccacccagga ggcgctggac 1680

gccgtgcctg tcgaggagct caaggcgcgg ctgggcgact cgctgcaaga ggtcgcaaag 1740

gccgccggcc tgcagtccta cgagatcccg cgcgacttca tcatcgaaac aacaccatgg 1800

acgctggaga acggcctgct caccggcatc cgcaagttgg ccaggccgca gctgaaaaag 1860

cattacggcg agcttctcga gcagatctac acggacctgg cacacggcca ggccgacgaa 1920

ctgcgctcgc tgcgccaaag cggtgccgat gcgccggtgc tggtgacggt gtgccgtgcg 1980

gcggccgcgc tgttgggcgg cagcgcctct gacgtccagc ccgatgcgca cttcaccgat 2040

ttgggcggcg actcgctgtc ggcgctgtcg ttcaccaacc tgctgcacga gatcttcgac 2100

atcgaagtgc cggtgggcgt catcgtcagc cccgccaacg acttgcaggc cctggccgac 2160

tacgtcgagg cggctcgcaa acccggctcg tcacggccga ccttcgcctc ggtccacggc 2220

gcctcgaatg ggcaggtcac cgaggtgcat gccggtgacc tgtccctgga caaattcatc 2280

gatgccgcaa ccctggccga agctccccgg ctgcccgccg caaacaccca agtgcgcacc 2340

gtgctgctga ccggcgccac cggcttcctc gggcgctacc tggccctgga atggctggag 2400

cggatggacc tggtcgacgg caaactgatc tgcctggtcc gggccaagtc cgacaccgaa 2460

gcacgggcgc ggctggacaa gacgttcgac agcggcgacc ccgaactgct ggcccactac 2520

cgcgcactgg ccggcgacca cctcgaggtg ctcgccggtg acaagggcga agccgacctc 2580

ggactggacc ggcagacctg gcaacgcctg gccgacacgg tcgacctgat cgtcgacccc 2640

gcggccctgg tcaaccacgt actgccatac agccagctgt tcgggcccaa cgcgctgggc 2700

accgccgagc tgctgcggct ggcgctcacc tccaagatca agccctacag ctacacctcg 2760

acaatcggtg tcgccgacca gatcccgccg tcggcgttca ccgaggacgc cgacatccgg 2820

gtcatcagcg ccacccgcgc ggtcgacgac agctacgcca atggctactc gaacagcaag 2880

tgggccggcg aggtgctgtt gcgcgaggcg catgacctgt gtggcctgcc ggttgcggtg 2940

ttccgctgcg acatgatcct ggccgacacc acatgggcgg gacagctcaa tgtgccggac 3000

atgttcaccc ggatgatcct gagcctggcg gccaccggta tcgcgccggg ttcgttctat 3060

gagcttgcgg ccgacggcgc ccggcaacgc gcccactatg acggtctgcc cgtcgagttc 3120

atcgccgagg cgatttcgac tttgggtgcg cagagccagg atggtttcca cacgtatcac 3180

gtgatgaacc cctacgacga cggcatcgga ctcgacgagt tcgtcgactg gctcaacgag 3240

tccggttgcc ccatccagcg catcgctgac tatggcgact ggctgcagcg cttcgaaacc 3300

gcactgcgcg cactgcccga tcggcagcgg cacagctcac tgctgccgct gttgcacaac 3360

tatcggcagc cggagcggcc cgtccgcggg tcgatcgccc ctaccgatcg cttccgggca 3420

gcggtgcaag aggccaagat cggccccgac aaagacattc cgcacgtcgg cgcgccgatc 3480

atcgtgaagt acgtcagcga cctgcgccta ctcggcctgc tctga

<210> 8

<211> 1174

<212> PRT

<213>分枝杆菌（Mycobacterium marinum）

<400> 8

Met Ser Pro Ile Thr Arg Glu Glu Arg Leu Glu Arg Arg Ile Gln Asp

1 5 10 15

Leu Tyr Ala Asn Asp Pro Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala

20 25 30

Ile Thr Ala Ala Ile Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile Ile

35 40 45

Glu Thr Val Met Thr Gly Tyr Ala Asp Arg Pro Ala Leu Ala Gln Arg

50 55 60

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

65 70 75 80

Leu Leu Pro His Phe Glu Thr Ile Ser Tyr Gly Glu Leu Trp Asp Arg

85 90 95

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

100 105 110

Gly Asp Arg Val Cys Leu Leu Gly Phe Asn Ser Val Asp Tyr Ala Thr

115 120 125

Ile Asp Met Thr Leu Ala Arg Leu Gly Ala Val Ala Val Pro Leu Gln

130 135 140

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

145 150 155 160

Pro Thr Met Ile Ala Ala Ser Val Asp Ala Leu Ala Asp Ala Thr Glu

165 170 175

Leu Ala Leu Ser Gly Gln Thr Ala Thr Arg Val Leu Val Phe Asp His

180 185 190

His Arg Gln Val Asp Ala His Arg Ala Ala Val Glu Ser Ala Arg Glu

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Thr Gly Ala Pro Lys Gly Ala Met Tyr Pro Arg Arg Asn Val Ala Thr

260 265 270

Phe Trp Arg Lys Arg Thr Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile

275 280 285

Thr Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gln Ile Leu

290 295 300

Tyr Gly Thr Leu Cys Asn Gly Gly Thr Ala Tyr Phe Val Ala Lys Ser

305 310 315 320

Asp Leu Ser Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu

325 330 335

Leu Thr Phe Val Pro Arg Val Trp Asp Met Val Phe Asp Glu Phe Gln

340 345 350

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

355 360 365

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

370 375 380

Tyr Thr Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Asp Glu Met Lys

385 390 395 400

Ala Trp Val Glu Glu Leu Leu Asp Met His Leu Val Glu Gly Tyr Gly

405 410 415

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

420 425 430

Ala Val Leu Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe

435 440 445

Leu Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Val Lys Thr Asp

450 455 460

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

465 470 475 480

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

485 490 495

Gly Pro Glu Gln Phe Val Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys

500 505 510

Leu Ser Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe

515 520 525

Gly Asp Ser Pro Leu Val Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala

530 535 540

Arg Ala Tyr Leu Leu Ala Val Ile Val Pro Thr Gln Glu Ala Leu Asp

545 550 555 560

Ala Val Pro Val Glu Glu Leu Lys Ala Arg Leu Gly Asp Ser Leu Gln

565 570 575

Glu Val Ala Lys Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp

580 585 590

Phe Ile Ile Glu Thr Thr Pro Trp Thr Leu Glu Asn Gly Leu Leu Thr

595 600 605

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

610 615 620

Leu Leu Glu Gln Ile Tyr Thr Asp Leu Ala His Gly Gln Ala Asp Glu

625 630 635 640

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

645 650 655

Val Cys Arg Ala Ala Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp Val

660 665 670

Gln Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala

675 680 685

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

690 695 700

Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Gln Ala Leu Ala Asp

705 710 715 720

Tyr Val Glu Ala Ala Arg Lys Pro Gly Ser Ser Arg Pro Thr Phe Ala

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu

785 790 795 800

Arg Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu Val Arg Ala Lys

805 810 815

Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly

820 825 830

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

835 840 845

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

850 855 860

Gln Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Leu Ile Val Asp Pro

865 870 875 880

Ala Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu Phe Gly Pro

885 890 895

Asn Ala Leu Gly Thr Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys

900 905 910

Ile Lys Pro Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp Gln Ile

915 920 925

Pro Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg Val Ile Ser Ala

930 935 940

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

945 950 955 960

Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu

965 970 975

Pro Val Ala Val Phe Arg Cys Asp Met Ile Leu Ala Asp Thr Thr Trp

980 985 990

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

995 1000 1005

Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala Ala

1010 1015 1020

Asp Gly Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val Glu Phe

1025 1030 1035 1040

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

1045 1050 1055

His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly Ile Gly Leu Asp

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

Leu Pro Asp Arg Gln Arg His Ser Ser Leu Leu Pro Leu Leu His Asn

1105 1110 1115 1120

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

1125 1130 1135

Arg Phe Arg Ala Ala Val Gln Glu Ala Lys Ile Gly Pro Asp Lys Asp

1140 1145 1150

Ile Pro His Val Gly Ala Pro Ile Ile Val Lys Tyr Val Ser Asp Leu

1155 1160 1165

Arg Leu Leu Gly Leu Leu

1170

<210> 9

<211> 675

<212> DNA

<213> 枯草芽孢杆菌（Bacillus subtilis）

<400> 9

atgaagattt acggaattta tatggaccgc ccgctttcac aggaagaaaa tgaacggttc 60

atgactttca tatcacctga aaaacgggag aaatgccgga gattttatca taaagaagat 120

gctcaccgca ccctgctggg agatgtgctc gttcgctcag tcataagcag gcagtatcag 180

ttggacaaat ccgatatccg ctttagcacg caggaatacg ggaagccgtg catccctgat 240

cttcccgacg ctcatttcaa catttctcac tccggccgct gggtcattgg tgcgtttgat 300

tcacagccga tcggcataga tatcgaaaaa acgaaaccga tcagccttga gatcgccaag 360

cgcttctttt caaaaacaga gtacagcgac cttttagcaa aagacaagga cgagcagaca 420

gactattttt atcatctatg gtcaatgaaa gaaagcttta tcaaacagga aggcaaaggc 480

ttatcgcttc cgcttgattc cttttcagtg cgcctgcatc aggacggaca agtatccatt 540

gagcttccgg acagccattc cccatgctat atcaaaacgt atgaggtcga tcccggctac 600

aaaatggctg tatgcgccgc acaccctgat ttccccgagg atatcacaat ggtctcgtac 660

gaagagcttt tataa

<210> 10

<211> 224

<212> PRT

<213>枯草芽孢杆菌（Bacillus subtilis）

<400> 10

Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu

1 5 10 15

Asn Glu Arg Phe Met Thr Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys

20 25 30

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

35 40 45

Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser

50 55 60

Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp

65 70 75 80

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

85 90 95

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

100 105 110

Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr

115 120 125

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

130 135 140

His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly

145 150 155 160

Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly

165 170 175

Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys

180 185 190

Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His

195 200 205

Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu

210 215 220

<210> 11

<211> 549

<212> DNA

<213> 大肠杆菌（Escherichia coli）

<400> 11

gcggacacgt tattgattct gggtgatagc ctgagcgccg ggtatcgaat gtctgccagc 60

gcggcctggc ctgccttgtt gaatgataag tggcagagta aaacgtcggt agttaatgcc 120

agcatcagcg gcgacacctc gcaacaagga ctggcgcgcc ttccggctct gctgaaacag 180

catcagccgc gttgggtgct ggttgaactg ggcggcaatg acggtttgcg tggttttcag 240

ccacagcaaa ccgagcaaac gctgcgccag attttgcagg atgtcaaagc cgccaacgct 300

gaaccattgt taatgcaaat acgtctgcct gcaaactatg gtcgccgtta taatgaagcc 360

tttagcgcca tttaccccaa actcgccaaa gagtttgatg ttccgctgct gccctttttt 420

atggaagagg tctacctcaa gccacaatgg atgcaggatg acggtattca tcccaaccgc 480

gacgcccagc cgtttattgc cgactggatg gcgaagcagt tgcagccttt agtaaatcat 540

gactcataa

<210> 12

<211> 182

<212> PRT

<213>大肠杆菌（Escherichia coli）

<400> 12

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

1 5 10 15

Met Ser Ala Ser Ala Ala Trp Pro Ala Leu Leu Asn Asp Lys Trp Gln

20 25 30

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

35 40 45

Gln Gly Leu Ala Arg Leu Pro Ala Leu Leu Lys Gln His Gln Pro Arg

50 55 60

Trp Val Leu Val Glu Leu Gly Gly Asn Asp Gly Leu Arg Gly Phe Gln

65 70 75 80

Pro Gln Gln Thr Glu Gln Thr Leu Arg Gln Ile Leu Gln Asp Val Lys

85 90 95

Ala Ala Asn Ala Glu Pro Leu Leu Met Gln Ile Arg Leu Pro Ala Asn

100 105 110

Tyr Gly Arg Arg Tyr Asn Glu Ala Phe Ser Ala Ile Tyr Pro Lys Leu

115 120 125

Ala Lys Glu Phe Asp Val Pro Leu Leu Pro Phe Phe Met Glu Glu Val

130 135 140

Tyr Leu Lys Pro Gln Trp Met Gln Asp Asp Gly Ile His Pro Asn Arg

145 150 155 160

Asp Ala Gln Pro Phe Ile Ala Asp Trp Met Ala Lys Gln Leu Gln Pro

165 170 175

Leu Val Asn His Asp Ser

180

<210> 13

<211> 1542

<212> DNA

<213> 海杆菌（Marinobacter aquaeolei）

<400>13

ATGGCAATAC AGCAGGTACA TCACGCTGAC ACTTCATCAT CAAAGGTGCT CGGACAGCTC 60

CGTGGCAAGC GGGTTCTGAT CACCGGTACC ACTGGCTTTC TGGGCAAGGT GGTCCTCGAA 120

AGGCTGATTC GGGCGGTGCC TGATATCGGC GCAATTTACC TGCTGATCCG GGGCAATAAA 180

CGGCATCCGG ATGCTCGTTC CCGTTTCCTG GAAGAAATTG CCACCTCCTC GGTGTTTGAC 240

CGTCTTCGCG AGGCCGATTC AGAGGGATTT GACGCCTTTC TGGAAGAGCG CATTCACTGC 300

GTGACCGGTG AGGTGACCGA AGCGGGTTTC GGGATAGGGC AGGAAGACTA TCGCAAACTC 360

GCCACCGAAC TGGATGCGGT GATCAACTCC GCTGCAAGCG TGAATTTCCG TGAAGAGCTC 420

GACAAGGCGC TGGCCATCAA CACCCTGTGC CTTCGGAATA TTGCCGGCAT GGTGGATTTG 480

AATCCGAAGC TTGCGGTCCT GCAGGTCTCC ACCTGCTATG TCAATGGCAT GAACTCGGGG 540

CAGGTAACCG AATCGGTGAT CAAGCCGGCA GGCGAGGCCG TGCCGCGTTC CCCGGACGGC 600

TTCTATGAGA TAGAAGAGCT TGTTCGCCTG CTTCAGGATA AAATTGAAGA CGTTCAGGCC 660

CGTTATTCCG GCAAAGTGCT GGAGAGGAAG CTGGTGGACC TGGGGATTCG GGAAGCCAAC 720

CGCTATGGCT GGAGCGATAC CTACACCTTT ACCAAGTGGC TGGGCGAACA GTTGCTGATG 780

AAGGCGTTAA ACGGGCGCAC GCTGACCATT CTGCGTCCTT CGATTATCGA AAGTGCCCTG 840

GAGGAACCAG CGCCCGGCTG GATTGAGGGG GTGAAGGTGG CAGATGCCAT CATCCTGGCT 900

TACGCACGGG AAAAAGTCAC CCTCTTCCCG GGCAAACGCT CCGGTATCAT CGATGTGATT 960

CCAGTGGACC TGGTGGCCAA CTCCATCATC CTTTCCCTGG CGGAAGCTCT TGGAGAACCC 1020

GGTCGACGTC GCATCTATCA ATGTTGCAGC GGGGGCGGCA ATCCAATCTC CCTGGGTGAG 1080

TTCATCGATC ATCTCATGGC GGAATCAAAA GCCAATTACG CTGCCTACGA TCACCTGTTC 1140

TACCGGCAGC CCAGCAAGCC GTTTCTGGCG GTTAACCGGG CGCTGTTTGA TTTGGTGATC 1200

AGTGGTGTTC GCTTACCGCT CTCCCTGACG GACCGTGTGC TCAAATTACT GGGAAATTCC 1260

CGGGACCTGA AAATGCTCAG GAATCTGGAT ACCACCCAGT CGCTGGCAAC CATTTTTGGT 1320

TTCTACACCG CGCCGGATTA TATCTTCCGG AACGATGAGC TGATGGCGCT GGCGAACCGG 1380

ATGGGTGAGG TCGATAAAGG GCTGTTCCCG GTGGATGCCC GCCTGATTGA CTGGGAGCTC 1440

TACCTGCGCA AGATTCACCT GGCCGGGCTC AATCGCTATG CCCTGAAAGA ACGAAAGGTG 1500

TACAGTCTGA AAACCGCGCG CCAGCGCAAA AAAGCTGCCT GA

<210> 14

<211> 513

<212> PRT

<213>海杆菌（Marinobacter aquaeolei）

<400>14

MET ALA ILE GLN GLN VAL HIS HIS ALA ASP THR SER SER SER LYS VAL

1 5 10 15

LEU GLY GLN LEU ARG GLY LYS ARG VAL LEU ILE THR GLY THR THR GLY

20 25 30

PHE LEU GLY LYS VAL VAL LEU GLU ARG LEU ILE ARG ALA VAL PRO ASP

35 40 45

ILE GLY ALA ILE TYR LEU LEU ILE ARG GLY ASN LYS ARG HIS PRO ASP

50 55 60

ALA ARG SER ARG PHE LEU GLU GLU ILE ALA THR SER SER VAL PHE ASP

65 70 75 80

ARG LEU ARG GLU ALA ASP SER GLU GLY PHE ASP ALA PHE LEU GLU GLU

85 90 95

ARG ILE HIS CYS VAL THR GLY GLU VAL THR GLU ALA GLY PHE GLY ILE

100 105 110

GLY GLN GLU ASP TYR ARG LYS LEU ALA THR GLU LEU ASP ALA VAL ILE

115 120 125

ASN SER ALA ALA SER VAL ASN PHE ARG GLU GLU LEU ASP LYS ALA LEU

130 135 140

ALA ILE ASN THR LEU CYS LEU ARG ASN ILE ALA GLY MET VAL ASP LEU

145 150 155 160

ASN PRO LYS LEU ALA VAL LEU GLN VAL SER THR CYS TYR VAL ASN GLY

165 170 175

MET ASN SER GLY GLN VAL THR GLU SER VAL ILE LYS PRO ALA GLY GLU

180 185 190

ALA VAL PRO ARG SER PRO ASP GLY PHE TYR GLU ILE GLU GLU LEU VAL

195 200 205

ARG LEU LEU GLN ASP LYS ILE GLU ASP VAL GLN ALA ARG TYR SER GLY

210 215 220

LYS VAL LEU GLU ARG LYS LEU VAL ASP LEU GLY ILE ARG GLU ALA ASN

225 230 235 240

ARG TYR GLY TRP SER ASP THR TYR THR PHE THR LYS TRP LEU GLY GLU

245 250 255

GLN LEU LEU MET LYS ALA LEU ASN GLY ARG THR LEU THR ILE LEU ARG

260 265 270

PRO SER ILE ILE GLU SER ALA LEU GLU GLU PRO ALA PRO GLY TRP ILE

275 280 285

GLU GLY VAL LYS VAL ALA ASP ALA ILE ILE LEU ALA TYR ALA ARG GLU

290 295 300

LYS VAL THR LEU PHE PRO GLY LYS ARG SER GLY ILE ILE ASP VAL ILE

305 310 315 320

PRO VAL ASP LEU VAL ALA ASN SER ILE ILE LEU SER LEU ALA GLU ALA

325 330 335

LEU GLY GLU PRO GLY ARG ARG ARG ILE TYR GLN CYS CYS SER GLY GLY

340 345 350

GLY ASN PRO ILE SER LEU GLY GLU PHE ILE ASP HIS LEU MET ALA GLU

355 360 365

SER LYS ALA ASN TYR ALA ALA TYR ASP HIS LEU PHE TYR ARG GLN PRO

370 375 380

SER LYS PRO PHE LEU ALA VAL ASN ARG ALA LEU PHE ASP LEU VAL ILE

385 390 395 400

SER GLY VAL ARG LEU PRO LEU SER LEU THR ASP ARG VAL LEU LYS LEU

405 410 415

LEU GLY ASN SER ARG ASP LEU LYS MET LEU ARG ASN LEU ASP THR THR

420 425 430

GLN SER LEU ALA THR ILE PHE GLY PHE TYR THR ALA PRO ASP TYR ILE

435 440 445

PHE ARG ASN ASP GLU LEU MET ALA LEU ALA ASN ARG MET GLY GLU VAL

450 455 460

ASP LYS GLY LEU PHE PRO VAL ASP ALA ARG LEU ILE ASP TRP GLU LEU

465 470 475 480

TYR LEU ARG LYS ILE HIS LEU ALA GLY LEU ASN ARG TYR ALA LEU LYS

485 490 495

GLU ARG LYS VAL TYR SER LEU LYS THR ALA ARG GLN ARG LYS LYS ALA

500 505 510

ALA

Claims

1.一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法，其特征在于基于脂肪酸为前体分子产脂肪醇乙酸酯：

将羧酸还原酶CAR基因和醇乙酰基转移酶ATF1基因、醛还原酶AHR及硫酯酶‘TESA基因、磷酸泛酰巯基乙胺基转移酶Sfp基因，重组到载体上；将上述载体转入到微生物中，实现上述五个基因在微生物中的高量表达，得到工程菌株；表达‘TESA用于水解脂酰-ACP产生脂肪酸，表达CAR用于催化脂肪酸还原为脂肪醛，表达Sfp用于活化CAR酶，表达AHR用于随后催化脂肪醛还原形成脂肪醇，表达ATF1用于最终催化脂肪醇与乙酰辅酶A反应合成脂肪醇乙酸酯，脂肪酸和乙酰辅酶A均可由微生物发酵单糖或甘油合成，为此上述构建的工程菌可有效的将单糖或甘油转化为脂肪醇乙酸酯，所述微生物为细菌或酵母中的任意一种。

2.根据权利要求1所述的一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法，其特征在于，

所述醛还原酶AHR基因核苷酸序列如SEQ ID NO：3所示；所述醛还原酶AHR基因编码的氨基酸序列如SEQ ID NO：4所示；

所述醇乙酰基转移酶ATF1基因核苷酸序列如SEQ ID NO：5所示；所述醇乙酰基转移酶ATF1基因编码的氨基酸序列如SEQ ID NO：6所示；

所述羧酸还原酶CAR基因核苷酸序列如SEQ ID NO：7所示；所述羧酸还原酶CAR基因编码的氨基酸序列如SEQ ID NO：8所示；

所述磷酸泛酰巯基乙胺基转移酶Sfp基因核苷酸序列如SEQ ID NO：9所示；所述磷酸泛酰巯基乙胺基转移酶Sfp基因编码的氨基酸序列如SEQ ID NO：10所示；

所述硫酯酶‘TESA核苷酸序列如SEQ ID NO：11所示；所述‘TESA编码的氨基酸序列如SEQ ID NO：12所示。

3.根据权利要求1所述的一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法，其特征在于：所述微生物包含醇乙酰基转移酶ATF1基因的核酸序列；所述编码醇乙酰基转移酶ATF1基因的核酸序列利用乙酰辅酶A和醇类化合物生成脂肪醇乙酸酯；所述醇类化合物为碳源子数大于5但小于30的饱和一元醇和不饱和一元醇。

4.根据权利要求1所述的一种微生物体内基于脂肪酸合成脂肪醇乙酸酯的方法，其特征在于：所述单糖为葡萄糖、半乳糖或果糖中的任意一种。