CN112159769A

CN112159769A - 一种产l-薄荷醇的基因工程菌及其构建方法与应用

Info

Publication number: CN112159769A
Application number: CN202011060993.3A
Authority: CN
Inventors: 刘龙; 陈坚; 吕雪芹; 堵国成; 李江华; 于文文; 马骏; 房峻
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-01-01
Anticipated expiration: 2040-09-30
Also published as: CN112159769B

Abstract

本发明公开了一种产L‑薄荷醇的基因工程菌及其构建方法与应用，属于代谢工程技术领域。本发明以酵母菌为出发菌株，在酵母菌内构建了L‑薄荷醇的代谢途径，首次实现了发酵法生产L‑薄荷醇。本发明构建的产L‑薄荷醇的基因工程菌，利用廉价的发酵培养基培养4d左右，L‑薄荷醇的发酵产量达到5.03mg/L。与当前L‑薄荷醇的生产技术相比，发酵法生产L‑薄荷醇周期短，反馈市场需求快，生产过程仅需常见的发酵原料，无需添加长叶薄荷酮、百里酚等植物来源前体物质，克服了植物原料的限制性。

Description

一种产L-薄荷醇的基因工程菌及其构建方法与应用

技术领域

本发明涉及一种产L-薄荷醇的基因工程菌及其构建方法与应用，属于代谢工程技术领域。

背景技术

L-薄荷醇(L-menthol)是具有高挥发性单环单萜物质，白色针状结晶，易溶于石油醚、氯仿等有机溶剂，微溶于水。L-薄荷醇是世界范围内销量最大的香料之一，全球年需求量超过2万吨。L-薄荷醇因为具有清凉、杀菌、止痒、镇痛等作用，被广泛的应用于食品、日化、医药、烟草等领域；且还可以作为前体物质合成其他香味物质。目前L-薄荷醇的工业化生产方式较为单一，主要为天然提取与化学合成，天然提取方法需要大量的薄荷属植物原料，化学合成L-薄荷醇同样需要植物来源的百里酚等化合物为底物进行反应拆分。但植物原料生长周期长，市场反馈慢，且容易受到气候、地域等自然环境因素的影响出现产量不稳定。另有以长叶薄荷酮等为底物进行全细胞或酶催化制备L-薄荷醇的方法，仍无法解决植物原料的限制因素。

发明内容

为解决上述问题，本发明提供一种产L-薄荷醇的基因工程菌及其构建方法与应用，以酵母为出发菌株，在酵母内构建了L-薄荷醇的代谢途径，首次实现了发酵法生产L-薄荷醇。

本发明的第一个目的是提供一种产L-薄荷醇的基因工程菌，所述的基因工程菌是以酵母菌为宿主，过表达内源MVA合成途径中的甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1，并异源表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H、细胞色素P450还原酶基因CPR、反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR、类固醇异构酶基因KSI、长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR。

进一步地，所述的甲羟戊酸焦磷酸脱羧酶的氨基酸序列如SEQ ID NO.1所示，所述的3-羟基-3-甲基戊二酰辅酶A还原酶的氨基酸序列如SEQ ID NO.2所示。

进一步地，所述的截短的3-羟基-3-甲基戊二酰辅酶A还原酶是将3-羟基-3-甲基戊二酰辅酶A还原酶前530个氨基酸序列删除得到。

进一步地，所述的甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1采用组成型启动子进行过表达。

进一步地，所述的柠檬烯合成酶的氨基酸序列如SEQ ID NO.3所示，柠檬烯羟基化酶的氨基酸序列如SEQ ID NO.4所示，细胞色素P450还原酶的氨基酸序列如SEQ ID NO.5所示，反式异胡椒醇脱氢酶的氨基酸序列如SEQ ID NO.6所示，异薄荷二烯酮还原酶的氨基酸序列如SEQ ID NO.7所示，类固醇异构酶的氨基酸序列如SEQ ID NO.8所示，长叶薄荷酮还原酶的氨基酸序列如SEQ ID NO.9所示，薄荷醇还原酶的氨基酸序列如SEQ ID NO.10所示。

进一步地，所述的截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H、细胞色素P450还原酶基因CPR、反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR、类固醇异构酶基因KSI、长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR通过诱导型启动子进行表达。

进一步地，所述的截短的柠檬烯合成酶是将柠檬烯合成酶前56个氨基酸序列删除得到。

进一步地，所述的酵母菌为酿酒酵母、异常汉逊氏酵母、粟酒裂殖酵母、黏红酵母、热带假丝酵母、产朊假丝酵母、解脂假丝酵母、巴斯德毕赤酵母等。

本发明的第二个目的是提供所述的产L-薄荷醇的基因工程菌的构建方法，包括如下步骤：强化酵母菌内源性MVA代谢途径的关键基因IDI与tHMG1，然后异源表达L-薄荷醇代谢途径的必需基因LIS、L3H、CPR、IPDH、IPR、KSI、PGR和MMR。

本发明的第三个目的是提供所述的产L-薄荷醇的基因工程菌在发酵生产L-薄荷醇中的应用。

本发明的有益效果：

本发明构建了一株产L-薄荷醇的基因工程菌，利用廉价的发酵培养基培养4d左右，L-薄荷醇的发酵产量达到5.03mg/L。与当前L-薄荷醇的生产技术相比，发酵法生产L-薄荷醇周期短，反馈市场需求快，生产过程仅需常见的发酵原料，无需添加长叶薄荷酮、百里酚等植物来源前体物质，克服了植物原料的限制性。

具体实施方式

下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。

气相-质谱联用检测L-薄荷醇：使用带有FID检测器和Chirasil-DEX-CB色谱柱(Agilent；25m，0.32mm，0.25μm)的Agilent Technologies 7890A仪器进行检测。进样器温度为180℃，载气为氦气，流速为1mL/min，压力为5.8psi。程序从70℃开始，以20℃/min的速率将温度升至150℃，保持3分钟，然后以2℃/min的速度将温度升至190℃，保持3分钟。

实施例1：优化内源性MVA途径

(a)以酿酒酵母S288C基因组为模版，采用引物tHMG1-F、tHMG1-R扩增得到基因tHMG1，tHMG1是基因HMG1去除前530个氨基酸序列所形成，采用引物GPD-F，GPD-R扩增得到启动子基因GPD(SEQ ID NO.11)，采用引物ADH1-F1，ADH1-R1扩增得到终止子基因ADH1-1(SEQ ID NO.12)；采用引物IDI-F、IDI-R扩增得到基因IDI，采用引物TEF1-F，TEF1-R扩增得到启动子基因TEF1(SEQ ID NO.13)，采用引物CYC1-F1，CYC1-R1扩增得到终止子基因CYC1-1(SEQ ID NO.14)，采用引物208a-D-F，208a-D-R扩增得到基因片段208a-D，采用引物208a-U-F，208a-U-R扩增得到基因片段208a-F。

(b)将步骤(a)中获得的六个片段tHMG1、GPD、ADH1-1和IDI、TEF1、CYC1-1基因片段分为两组进行重叠延伸PCR，PCR条件：98℃，5min预变性，然后98℃，变性10s，55℃，退火5s，72℃，延伸2min，总共30个循环，切胶回收大小正确的片段，得到融合基因片段P_GPD-tHMG1-T_ADH1与P_TEF1-IDI-T_CYC1基因片段。

(c)以pML104质粒为模板，采用引物208a-F，208a-R扩增后得到片段208a，热击转入大肠杆菌JM109感受态后，采用引物208a-YZ-F与L4440菌落PCR验证，并进行测序，测序正确的单菌落接入2mL LB培养基内培养16h后利用质粒提取试剂盒提取得到质粒pML104-208a。

(d)制备酿酒酵母CEN.PK2–1C感受态，并将构建好的融合片段P_GPD-tHMG1-T_ADH1、P_TEF1-IDI-T_CYC1与基因片段208a-D、208a-F与质粒pML104-208a共同转入CEN.PK2–1C感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物208a-U-F，208a-D-R进行菌落PCR验证。

(e)将步骤(d)中菌落PCR验证正确的单菌落接入YPD液体培养基内培养16h，之后划线于含有5-FOA的YPD固体平板上，30℃培养3d，之后将长出的单菌落分别转板于YPD固体平板与SD Ura筛选固体平板上对比验证，YPD平板上正常生长但SD Ura筛选平板无法生长的单菌落即为正确的基因工程菌，并命名为WMT1。

表1引物序列

实施例2：重建L-薄荷醇代谢合成途径

(a)人工合成基因片段P_GAL1-tLIS-T_ADH1-P_GAL10-L3H-T_CYC1-P_GAL7-CPR-T_TDH3、P_GAL1-IPDH-T_ADH1-P_GAL10-IPR-T_CYC1-P_GAL7-KSI-T_TDH3、P_GAL1-PGR-T_ADH1-P_GAL10-MMR-T_CYC1，其中tLIS基因是基因LIS去除前56个氨基酸序列所形成。以酿酒酵母S288C基因组为模版，采用引物911b-U-F，911b-U-R扩增得到基因片段911b-U，采用引物911b-D-F，911b-D-R扩增得到基因片段911b-D，采用引物308a-U-F，308a-U-R扩增得到基因片段308a-U，采用引物308a-D-F，308a-D-R扩增得到基因片段308a-D，采用引物1622b-U-F，1622b-U-R扩增得到基因片段1622b-U，采用引物1622b-D-F，1622b-D-R扩增得到基因片段1622b-D。

(b)以质粒pML104为模版，采用引物308a-F，308a-R、911b-F，911b-R和1622b-F，1622b-R分别扩增得到片段308a，911b和1622b，分别利用引物308a-YZ-F，L4440、911b-YZ-F，L4440和1622b-YZ-F，L4440，采用与实施例1(c)步骤类似的方法，构建获得质粒pML104-308a、pML104-911b和pML104-1622b。

(c)制备菌株WMT1的感受态，并将合成的基因片段P_GAL1-tLIS-T_ADH1、P_GAL10-L3H-T_CYC1、P_GAL7-CPR-T_TDH3和基因片段911b-U，911b-D和质粒pML104-911b转入WMT1感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物911b-U-F，911b-D-R进行菌落PCR验证，采用实施例1(e)类似的方法筛选得到正确的工程菌，并命名为WMT2。

(d)制备菌株WMT2的感受态，并将合成的基因片段P_GAL1-IPDH-T_ADH1、P_GAL10-IPR-T_CYC1、P_GAL7-KSI-T_TDH3和基因片段308a-U，308a-D和质粒pML104-308a转入WMT2感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物308a-U-F，308a-D-R进行菌落PCR验证，采用实施例1(e)类似的方法筛选得到正确的工程菌，并命名为WMT3(其中，启动子基因GAL1如SEQ ID NO.15所示，启动子基因GAL10如SEQ ID NO.16所示，启动子基因GAL7如SEQ IDNO.17所示，终止子基因TDH3如SEQ ID NO.18所示)。

(e)制备菌株WMT3的感受态，并将合成的基因片段P_GAL1-PGR-T_ADH1、P_GAL10-MMR-T_CYC1和基因片段1622b-U，1622b-D和质粒pML104-1622b转入WMT3感受态中，待SD Ura筛选固体平板上长出单菌落后采用引物1622b-U-F，1622b-D-R进行菌落PCR验证，采用实施例1(e)类似的方法筛选得到正确的工程菌，并命名为WMT4。

表2引物序列

实施例3：利用基因工程菌株WMT4发酵产L-薄荷醇

在2ml YPD培养基中接种固体YPD平板上的WMT4单菌落，30℃，220rpm培养16-20h后，按接种量1％接入含有25mL YPD液体培养基的250mL圆底摇瓶内，30℃，220rpm培养96h。发酵至16h时，加入10％半乳糖水溶液。发酵结束后，取600μL菌液加入0.5mm玻璃珠与等体积乙酸乙酯破碎，之后高速离心取上层乙酸乙酯层进行气相质谱联用检测，L-薄荷醇的发酵产量为5.03mg/L。

以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

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Gly Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr Ser Arg Phe Ala

770 775 780

Arg Leu Gln His Ile Gln Thr Cys Leu Ala Gly Asp Leu Leu Phe Met

785 790 795 800

Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser

805 810 815

Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp

820 825 830

Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys

835 840 845

Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val

850 855 860

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

865 870 875 880

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

885 890 895

Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala Ala Asn

900 905 910

Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala Gln Asn

915 920 925

Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr His

1010 1015 1020

Asn Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala

1025 1030 1035

Thr Asp Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys

1040 1045 1050

Ser

<210> 3

<211> 599

<212> PRT

<213> （人工序列）

<400> 3

Met Ala Leu Lys Val Leu Ser Val Ala Thr Gln Met Ala Ile Pro Ser

1 5 10 15

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

20 25 30

Leu Leu Ser Ser Thr Asn Ser Ser Ser Arg Ser Arg Leu Arg Val Tyr

35 40 45

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

50 55 60

Pro Ser Arg Trp Asp Val Asn Phe Ile Gln Ser Leu Leu Ser Asp Tyr

65 70 75 80

Lys Glu Asp Lys His Val Ile Arg Ala Ser Glu Leu Val Thr Leu Val

85 90 95

Lys Met Glu Leu Glu Lys Glu Thr Asp Gln Ile Arg Gln Leu Glu Leu

100 105 110

Ile Asp Asp Leu Gln Arg Met Gly Leu Ser Asp His Phe Gln Asn Glu

115 120 125

Phe Lys Glu Ile Leu Ser Ser Ile Tyr Leu Asp His His Tyr Tyr Lys

130 135 140

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

145 150 155 160

Phe Arg Leu Leu Arg Glu His Gly Phe Gln Val Ala Gln Glu Val Phe

165 170 175

Asp Ser Phe Lys Asn Glu Glu Gly Glu Phe Lys Glu Ser Leu Ser Asp

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Arg Ile Ala Tyr Ser Leu Asp Ile Pro Leu His Trp Arg Ile Lys Arg

245 250 255

Pro Asn Ala Pro Val Trp Ile Glu Trp Tyr Arg Lys Arg Pro Asp Met

260 265 270

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

275 280 285

Ala Gln Phe Gln Glu Glu Leu Lys Glu Ser Phe Arg Trp Trp Arg Asn

290 295 300

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

305 310 315 320

Cys Tyr Phe Trp Asn Thr Gly Ile Ile Glu Pro Arg Gln His Ala Ser

325 330 335

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

340 345 350

Asp Ile Tyr Asp Val Tyr Gly Thr Leu Glu Glu Leu Glu Gln Phe Thr

355 360 365

Asp Leu Ile Arg Arg Trp Asp Ile Asn Ser Ile Asp Gln Leu Pro Asp

370 375 380

Tyr Met Gln Leu Cys Phe Leu Ala Leu Asn Asn Phe Val Asp Asp Thr

385 390 395 400

Ser Tyr Asp Val Met Lys Glu Lys Gly Val Asn Val Ile Pro Tyr Leu

405 410 415

Arg Gln Ser Trp Val Asp Leu Ala Asp Lys Tyr Met Val Glu Ala Arg

420 425 430

Trp Phe Tyr Gly Gly His Lys Pro Ser Leu Glu Glu Tyr Leu Glu Asn

435 440 445

Ser Trp Gln Ser Ile Ser Gly Pro Cys Met Leu Thr His Ile Phe Phe

450 455 460

Arg Val Thr Asp Ser Phe Thr Lys Glu Thr Val Asp Ser Leu Tyr Lys

465 470 475 480

Tyr His Asp Leu Val Arg Trp Ser Ser Phe Val Leu Arg Leu Ala Asp

485 490 495

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

500 505 510

Ser Leu Gln Cys Tyr Met Ser Asp Tyr Asn Ala Ser Glu Ala Glu Ala

515 520 525

Arg Lys His Val Lys Trp Leu Ile Ala Glu Val Trp Lys Lys Met Asn

530 535 540

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

545 550 555 560

Cys Ala Val Asp Leu Gly Arg Met Ala Gln Leu Met Tyr His Asn Gly

565 570 575

Asp Gly His Gly Thr Gln His Pro Ile Ile His Gln Gln Met Thr Arg

580 585 590

Thr Leu Phe Glu Pro Phe Ala

595

<210> 4

<211> 500

<212> PRT

<213> （人工序列）

<400> 4

Met Glu Leu Gln Ile Ser Ser Ala Ile Ile Ile Leu Val Val Thr Tyr

1 5 10 15

Thr Ile Ser Leu Leu Ile Ile Lys Gln Trp Arg Lys Pro Lys Pro Gln

20 25 30

Glu Asn Leu Pro Pro Gly Pro Pro Lys Leu Pro Leu Ile Gly His Leu

35 40 45

His Leu Leu Trp Gly Lys Leu Pro Gln His Ala Leu Ala Ser Val Ala

50 55 60

Lys Gln Tyr Gly Pro Val Ala His Val Gln Leu Gly Glu Val Phe Ser

65 70 75 80

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

85 90 95

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

100 105 110

Trp Tyr Asp Asn Asp Asp Ile Ile Phe Ser Pro Tyr Ser Val His Trp

115 120 125

Arg Gln Met Arg Lys Ile Cys Val Ser Glu Leu Leu Ser Ala Arg Asn

130 135 140

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

145 150 155 160

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

165 170 175

Glu Arg Ile Ala Thr Leu Thr Cys Ser Ile Ile Cys Arg Ala Ala Phe

180 185 190

Gly Ser Val Ile Arg Asp His Glu Glu Leu Val Glu Leu Val Lys Asp

195 200 205

Ala Leu Ser Met Ala Ser Gly Phe Glu Leu Ala Asp Met Phe Pro Ser

210 215 220

Ser Lys Leu Leu Asn Leu Leu Cys Trp Asn Lys Ser Lys Leu Trp Arg

225 230 235 240

Met Arg Arg Arg Val Asp Ala Ile Leu Glu Ala Ile Val Glu Glu His

245 250 255

Lys Leu Lys Lys Ser Gly Glu Phe Gly Gly Glu Asp Ile Ile Asp Val

260 265 270

Leu Phe Arg Met Gln Lys Asp Ser Gln Ile Lys Val Pro Ile Thr Thr

275 280 285

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

290 295 300

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

305 310 315 320

Pro Glu Val Met Ala Lys Ala Gln Ala Glu Val Arg Ala Ala Leu Lys

325 330 335

Gly Lys Thr Asp Trp Asp Val Asp Asp Val Gln Glu Leu Lys Tyr Met

340 345 350

Lys Ser Val Val Lys Glu Thr Met Arg Met His Pro Pro Ile Pro Leu

355 360 365

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

370 375 380

Pro Asn Lys Ala Arg Ile Met Ile Asn Val Trp Ser Met Gly Arg Asn

385 390 395 400

Pro Leu Tyr Trp Glu Lys Pro Glu Thr Phe Trp Pro Glu Arg Phe Asp

405 410 415

Gln Val Ser Arg Asp Phe Met Gly Asn Asp Phe Glu Phe Ile Pro Phe

420 425 430

Gly Ala Gly Arg Arg Ile Cys Pro Gly Leu Asn Phe Gly Leu Ala Asn

435 440 445

Val Glu Val Pro Leu Ala Gln Leu Leu Tyr His Phe Asp Trp Lys Leu

450 455 460

Ala Glu Gly Met Asn Pro Ser Asp Met Asp Met Ser Glu Ala Glu Gly

465 470 475 480

Leu Thr Gly Ile Arg Lys Asn Asn Leu Leu Leu Val Pro Thr Pro Tyr

485 490 495

Asp Pro Ser Ser

500

<210> 5

<211> 710

<212> PRT

<213> （人工序列）

<400> 5

Met Gln Ser Asp Ser Val Lys Val Ser Pro Phe Asp Leu Val Ser Ala

1 5 10 15

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

20 25 30

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

35 40 45

Leu Leu Thr Leu Phe Thr Thr Ser Phe Ala Val Leu Ile Gly Cys Leu

50 55 60

Val Phe Leu Met Trp Arg Arg Ser Ser Ser Lys Lys Leu Val Gln Asp

65 70 75 80

Pro Val Pro Gln Val Ile Val Val Lys Lys Lys Glu Lys Glu Ser Glu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Ala Asp Asp Asp Glu Tyr Glu Glu Lys Leu Lys Lys Glu Ser Leu Ala

145 150 155 160

Phe Phe Phe Leu Ala Thr Tyr Gly Asp Gly Glu Pro Thr Asp Asn Ala

165 170 175

Ala Asn Phe Tyr Lys Trp Phe Thr Glu Gly Asp Asp Lys Gly Glu Trp

180 185 190

Leu Lys Lys Leu Gln Tyr Gly Val Phe Gly Leu Gly Asn Arg Gln Tyr

195 200 205

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

210 215 220

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

225 230 235 240

Cys Ile Glu Asp Asp Phe Thr Ala Trp Lys Glu Leu Val Trp Pro Glu

245 250 255

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

260 265 270

Tyr Thr Ala Ala Val Leu Glu Tyr Arg Val Val Tyr His Asp Lys Pro

275 280 285

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

290 295 300

His Asp Ala Gln His Pro Ser Arg Ser Asn Val Ala Phe Lys Lys Glu

305 310 315 320

Leu His Thr Ser Gln Ser Asp Arg Ser Cys Thr His Leu Glu Phe Asp

325 330 335

Ile Ser His Thr Gly Leu Ser Tyr Glu Thr Gly Asp His Val Gly Val

340 345 350

Tyr Ser Glu Asn Leu Ser Glu Val Val Asp Glu Ala Leu Lys Leu Leu

355 360 365

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

370 375 380

Gly Thr Pro Ile Gly Gly Ala Ser Leu Pro Pro Pro Phe Pro Pro Cys

385 390 395 400

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

405 410 415

Lys Lys Val Ala Leu Leu Ala Leu Ala Ala His Ala Ser Asp Pro Ser

420 425 430

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

435 440 445

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

450 455 460

Gln Ser Phe Pro Ser Ala Lys Pro Pro Leu Gly Val Phe Phe Ala Ala

465 470 475 480

Val Ala Pro Arg Leu Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro

485 490 495

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

500 505 510

Thr Thr Pro Ala Gly Arg Ile His Arg Gly Leu Cys Ser Thr Trp Met

515 520 525

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

530 535 540

Ile Phe Val Arg Thr Ser Asn Phe Arg Leu Pro Val Asp Pro Lys Val

545 550 555 560

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

565 570 575

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

580 585 590

Ser Ser Ile Phe Phe Phe Gly Cys Arg Asn Arg Lys Val Asp Phe Ile

595 600 605

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

610 615 620

Leu Ile Val Ala Phe Ser Arg Glu Gly Thr Ala Lys Glu Tyr Val Gln

625 630 635 640

His Lys Met Ser Gln Lys Ala Ser Asp Ile Trp Lys Leu Leu Ser Glu

645 650 655

Gly Ala Tyr Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala Lys Asp

660 665 670

Val His Arg Thr Leu His Thr Ile Val Gln Glu Gln Gly Ser Leu Asp

675 680 685

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

690 695 700

Tyr Leu Arg Asp Val Trp

705 710

<210> 6

<211> 265

<212> PRT

<213> （人工序列）

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Asp Ile Thr Asp Glu Gln Gln Val Arg Ser Val Val Asp Trp Thr Ala

65 70 75 80

Ala Thr Tyr Gly Gly Val Asp Val Met Phe Cys Asn Ala Gly Thr Ala

85 90 95

Ser Ala Thr Ala Gln Thr Val Leu Asp Leu Asp Leu Ala Gln Phe Asp

100 105 110

Arg Val Met Arg Val Asn Ala Arg Gly Thr Ala Ala Cys Val Lys Gln

115 120 125

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

130 135 140

Thr Ala Ser Ala Thr Val His His Ala Gly Pro Asn Leu Thr Asp Tyr

145 150 155 160

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

165 170 175

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

180 185 190

Leu Ala Thr Pro Leu Thr Ala Thr Ile Gly Leu Arg Thr Ala Ala Asp

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Cys Leu Pro Phe Val Ala Val Ala Lys

260 265

<210> 7

<211> 314

<212> PRT

<213> （人工序列）

<400> 7

Met Ala Glu Val Gln Arg Tyr Ala Leu Val Thr Gly Ala Asn Lys Gly

1 5 10 15

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

20 25 30

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

35 40 45

Leu Leu Lys Glu Leu Asn Val Ser Glu Asn Arg Leu Val Phe His Gln

50 55 60

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

65 70 75 80

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

85 90 95

Ser Gly Val Glu Met Val Gly Asp Val Ser Val Phe Asn Glu Tyr Ile

100 105 110

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

115 120 125

Glu Pro Pro Phe Lys Pro Lys Ala Asn Gly Glu Met Ile Glu Lys Phe

130 135 140

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

145 150 155 160

Leu Thr Gln Ala Leu Ile Pro Leu Leu Gln Leu Ser Pro Ser Pro Arg

165 170 175

Ile Val Asn Val Ser Ser Ser Phe Gly Ser Leu Leu Leu Leu Trp Asn

180 185 190

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

195 200 205

Arg Val Asp Glu Val Val Glu Val Phe Leu Lys Asp Ile Lys Glu Gly

210 215 220

Lys Leu Glu Glu Ser Gln Trp Pro Pro His Phe Ala Ala Glu Arg Val

225 230 235 240

Ser Lys Ala Ala Leu Asn Ala Tyr Thr Lys Ile Ala Ala Lys Lys Tyr

245 250 255

Pro Ser Phe Arg Ile Asn Ala Ile Cys Pro Gly Tyr Ala Lys Thr Asp

260 265 270

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

275 280 285

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

290 295 300

Phe Pro Arg Asp Lys Ala Leu Ala Leu Tyr

305 310

<210> 8

<211> 131

<212> PRT

<213> （人工序列）

<400> 8

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

1 5 10 15

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

20 25 30

Ala Asp Asp Ala Thr Val Glu Asp Pro Phe Gly Gln Pro Pro Ile His

35 40 45

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

50 55 60

Lys Val Arg Ala Cys Leu Thr Gly Pro Val Arg Ala Ser His Asn Gly

65 70 75 80

Cys Gly Ala Met Pro Phe Arg Ile Glu Met Val Trp Asn Gly Gln Pro

85 90 95

Cys Ala Val Asp Ala Ile Ser Val Met Arg Phe Asp Glu His Gly Arg

100 105 110

Ile Gln Thr Met Gln Ala Tyr Trp Ser Glu Val Asn Leu Ser Val Arg

115 120 125

Glu Pro Gln

130

<210> 9

<211> 342

<212> PRT

<213> （人工序列）

<400> 9

Met Val Met Asn Lys Gln Ile Val Leu Asn Asn Tyr Ile Asn Gly Ser

1 5 10 15

Leu Lys Gln Ser Asp Leu Ala Leu Arg Thr Ser Thr Ile Cys Met Glu

20 25 30

Ile Pro Asp Gly Cys Asn Gly Ala Ile Leu Val Lys Asn Leu Tyr Leu

35 40 45

Ser Val Asn Pro Tyr Leu Ile Leu Arg Met Gly Lys Leu Asp Ile Pro

50 55 60

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

65 70 75 80

Ser Lys Val Leu Asp Ser Thr His Pro Ser Tyr Glu Lys Gly Glu Leu

85 90 95

Ile Trp Gly Ser Gln Ala Gly Trp Glu Glu Tyr Thr Leu Ile Gln Asn

100 105 110

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

115 120 125

Tyr Val Gly Ile Leu Gly Met Pro Gly Met Thr Ala Tyr Ala Gly Phe

130 135 140

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

145 150 155 160

Ala Ala Gly Ser Val Gly Gln Leu Val Gly Gln Phe Ala Lys Met Phe

165 170 175

Gly Cys Tyr Val Val Gly Ser Ala Gly Ser Lys Glu Lys Val Asp Leu

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Asn Met Arg Val His Gly Arg Ile Ala Val Cys Gly Met Val Ser Gln

245 250 255

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

260 265 270

Pro Lys Gln Ile Arg Met Gln Gly Phe Val Val Val Asp Tyr Tyr His

275 280 285

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

290 295 300

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

305 310 315 320

Ser Ala Leu Leu Gly Val Tyr Val Gly Arg Asn Val Gly Asn Gln Val

325 330 335

Val Ala Val Ser Arg Glu

340

<210> 10

<211> 311

<212> PRT

<213> （人工序列）

<400> 10

Met Ala Asp Thr Phe Thr Gln Arg Tyr Ala Leu Val Thr Gly Ala Asn

1 5 10 15

Lys Gly Ile Gly Phe Glu Ile Cys Arg Gln Leu Ala Ser Lys Gly Met

20 25 30

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

35 40 45

Glu Arg Leu Leu Lys Glu Ser Arg Ser Ile Ser Asp Asp Asp Val Val

50 55 60

Phe His Gln Leu Asp Val Ala Asp Pro Ala Ser Ala Val Ala Val Ala

65 70 75 80

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

85 90 95

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

100 105 110

Glu Cys Leu Glu Ala Asn Ile Ile Ala Ala Gln Gly Gly Gln Ala His

115 120 125

Pro Phe His Pro Lys Thr Thr Gly Arg Leu Ile Glu Thr Leu Glu Gly

130 135 140

Ser Lys Glu Cys Ile Glu Thr Asn Tyr Tyr Gly Thr Lys Arg Ile Thr

145 150 155 160

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

165 170 175

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

180 185 190

Ala Lys Gly Val Phe Ser Ser Asn Ser Leu Asn Glu Gly Lys Val Glu

195 200 205

Glu Val Leu His Glu Phe Leu Lys Asp Phe Ile Asp Gly Lys Leu Gln

210 215 220

Gln Asn His Trp Pro Pro Asn Phe Ala Ala Tyr Lys Val Ser Lys Ala

225 230 235 240

Ala Val Asn Ala Tyr Thr Arg Ile Ile Ala Arg Lys Tyr Pro Ser Phe

245 250 255

Cys Ile Asn Ser Val Cys Pro Gly Phe Val Arg Thr Asp Ile Cys Tyr

260 265 270

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

275 280 285

Leu Ala Leu Leu Pro Asp Gly Gly Pro Ser Gly Ser Phe Phe Ser Arg

290 295 300

Glu Glu Ala Leu Ser Leu Tyr

305 310

<210> 11

<211> 644

<212> DNA

<213> （人工序列）

<400> 11

tcattatcaa tactcgccat ttcaaagaat acgtaaataa ttaatagtag tgattttcct 60

aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac atgcccaaaa 120

tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac agtttattcc 180

tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa aaaaaaagaa 240

tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc attctcttag 300

cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct caatggagtg 360

atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat gtatctatct 420

cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag ctgaaaaaaa 480

aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat ataaagacgg 540

taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc tacttttata 600

gttagtcttt tttttagttt taaaacacca gaacttagtt tcga 644

<210> 12

<211> 166

<212> DNA

<213> （人工序列）

<400> 12

gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60

tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120

ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctc 166

<210> 13

<211> 401

<212> DNA

<213> （人工序列）

<400> 13

atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60

tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120

tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180

tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240

tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg 300

tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360

ttgctcatta gaaagaaagc atagcaatct aatctaagtt t 401

<210> 14

<211> 252

<212> DNA

<213> （人工序列）

<400> 14

tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 60

aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120

tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180

acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240

taatttgcgg cc 252

<210> 15

<211> 600

<212> DNA

<213> （人工序列）

<400> 15

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60

ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120

tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccta tggttgttaa 240

tttgattcgt taatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360

ctgcgtttca ggaacgcgac cggtgaagac gaggacgcac ggaggagagt cttccgtcgg 420

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540

catttccaca acatataagt aagattagat atggatatgt atatggtggt aatgccatgt 600

<210> 16

<211> 500

<212> DNA

<213> （人工序列）

<400> 16

ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60

atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120

tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180

taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240

acagccctcc gacggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg 300

aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360

tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaattaac 420

gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg 480

taattaatca gcgaagcgat 500

<210> 17

<211> 300

<212> DNA

<213> （人工序列）

<400> 17

ttttgaggga atattcaact gttttttttt atcatgttga tgctctgcat aataatgccc 60

ataaatattt ccgacctgct tttatatctt tgctagccaa actaactgaa catagctaca 120

cattattttc agcttggcta ttttgtgaac actgtatagc cagtccttcg gatcacggtc 180

aacagttgtc cgagcgcttt ttggaccctt tcccttattt ttgggttaag gaaaatgaca 240

gaaaatatat ctaatgagcc ttcgctcaac agtgctccga agtatagctt tccaaaagga 300

<210> 18

<211> 582

<212> DNA

<213> （人工序列）

<400> 18

aagggaaaga tatgagctat acagcggaat ttccatatca ctcagatttt gttatctaat 60

tttttccttc ccacgtccgc gggaatctgt gtatattact gcatctagat atatgttatc 120

ttatcttggc gcgtacattt aattttcaac gtattctata agaaattgcg ggagtttttt 180

tcatgtagat gatactgact gcacgcaaat ataggcatga tttataggca tgatttgatg 240

gctgtaccga taggaacgct aagagtaact tcagaatcgt tatcctggcg gaaaaaattc 300

atttgtaaac tttaaaaaaa aaagccaata tccccaaaat tattaagagc gcctccatta 360

ttaactaaaa tttcactcag catccacaat gtatcaggta tctactacag atattacatg 420

tggcgaaaaa gacaagaaca atgcaatagc gcatcaagaa aaaacacaaa gctttcaatc 480

aatgaatcga aaatgtcatt aaaatagtat ataaattgaa actaagtcat aaagctataa 540

aaagaaaatt tatttaaatg caagatttaa agtaaattca ct 582

Claims

1.一种产L-薄荷醇的基因工程菌，其特征在于，所述的基因工程菌是以酵母菌为宿主，过表达内源MVA合成途径中的甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1，并异源表达截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H、细胞色素P450还原酶基因CPR、反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR、类固醇异构酶基因KSI、长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR。

2.根据权利要求1所述的基因工程菌，其特征在于，所述的甲羟戊酸焦磷酸脱羧酶的氨基酸序列如SEQ ID NO.1所示，所述的3-羟基-3-甲基戊二酰辅酶A还原酶的氨基酸序列如SEQ ID NO.2所示。

3.根据权利要求1所述的基因工程菌，其特征在于，所述的甲羟戊酸焦磷酸脱羧酶基因IDI和截短的3-羟基-3-甲基戊二酰辅酶A还原酶基因tHMG1采用组成型启动子进行过表达。

4.根据权利要求3所述的基因工程菌，其特征在于，所述的截短的3-羟基-3-甲基戊二酰辅酶A还原酶是将3-羟基-3-甲基戊二酰辅酶A还原酶前530个氨基酸序列删除得到。

5.根据权利要求1所述的基因工程菌，其特征在于，所述的柠檬烯合成酶的氨基酸序列如SEQ ID NO.3所示，柠檬烯羟基化酶的氨基酸序列如SEQ ID NO.4所示，细胞色素P450还原酶的氨基酸序列如SEQ ID NO.5所示，反式异胡椒醇脱氢酶的氨基酸序列如SEQ ID NO.6所示，异薄荷二烯酮还原酶的氨基酸序列如SEQ ID NO.7所示，类固醇异构酶的氨基酸序列如SEQ ID NO.8所示，长叶薄荷酮还原酶的氨基酸序列如SEQ ID NO.9所示，薄荷醇还原酶的氨基酸序列如SEQ ID NO.10所示。

6.根据权利要求1所述的基因工程菌，其特征在于，所述的截短的柠檬烯合成酶基因LIS、柠檬烯羟基化酶基因L3H、细胞色素P450还原酶基因CPR、反式异胡椒醇脱氢酶基因IPDH、异薄荷二烯酮还原酶基因IPR、类固醇异构酶基因KSI、长叶薄荷酮还原酶基因PGR和薄荷醇还原酶基因MMR通过诱导型启动子进行表达。

7.根据权利要求6所述的基因工程菌，其特征在于，所述的截短的柠檬烯合成酶是将柠檬烯合成酶前56个氨基酸序列删除得到。

8.根据权利要求1所述的基因工程菌，其特征在于，所述的酵母菌为酿酒酵母、异常汉逊氏酵母、粟酒裂殖酵母、黏红酵母、热带假丝酵母、产朊假丝酵母、解脂假丝酵母或巴斯德毕赤酵母。

9.一种权利要求1-8任一项所述的产L-薄荷醇的基因工程菌的构建方法，包括如下步骤：强化酵母菌内源性MVA代谢途径的关键基因IDI与tHMG1，然后异源表达L-薄荷醇代谢途径的必需基因LIS、L3H、CPR、IPDH、IPR、KSI、PGR和MMR。

10.权利要求1-8任一项所述的产L-薄荷醇的基因工程菌在发酵生产L-薄荷醇中的应用。