CN114107233A - 佛术烯的合成酶基因和高产菌株以及用途 - Google Patents

佛术烯的合成酶基因和高产菌株以及用途 Download PDF

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CN114107233A
CN114107233A CN202111252910.5A CN202111252910A CN114107233A CN 114107233 A CN114107233 A CN 114107233A CN 202111252910 A CN202111252910 A CN 202111252910A CN 114107233 A CN114107233 A CN 114107233A
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刘天罡
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Abstract

本发明公开了佛术烯的合成酶基因和高产菌株以及用途，属于生物技术领域。本发明首次发现了圣罗勒来源的氨基酸序列如SEQ ID NO.1所示的酶为佛术烯的合成酶，其或其编码基因或含有其编码基因的重组质粒、重组细胞可用于合成或生产佛术烯。一种佛术烯高产菌株为在过表达甲羟戊酸途径中的一个或多个基因以及法尼烯焦磷酸合酶基因的微生物中导入所述酶的编码基因获得，本发明的佛术烯高产菌株的发酵罐(15L钢罐)产量高达34.5g/L。佛术烯具有皮革、木质、草本香味，对害虫草地贪夜蛾有趋避效果，其可用于制备香料、香精或绿色拒食剂。

Description

佛术烯的合成酶基因和高产菌株以及用途
技术领域
本发明属于生物技术领域，具体涉及佛术烯((-)-Eremophilene)的合成酶基因、佛术烯的高产菌株和佛术烯的用途。
背景技术
圣罗勒是著名的印度阿育吠陀医学中的芳香药草，具备广泛的健康益处，具备治疗包括哮喘、发热、关节炎、被毒蛇咬伤和皮肤病等疾病的作用，该药草提取物还具备驱蚊效果，这与该植物富含的萜类化合物有关。
现有研究对圣罗勒的萜类基因的表征较少，对植物圣罗勒中潜在的萜类合酶进行表征，并进一步通过微生物高效合成所关注的目标产物，进一步进行产物结构鉴定、探索相关化合物的功能用途对于探索和拓展萜类化合物的用途具有重要的价值。
发明内容
本发明首要目的在于提供一种圣罗勒来源的酶及其编码基因在合成或生产佛术烯((-)-Eremophilene)中的应用。
本发明的另一目的在于提供一种(-)-Eremophilene高产菌株。
本发明的再一目的在于提供(-)-Eremophilene的用途。
本发明的目的通过下述技术方案实现：
一种酶在合成或生产(-)-Eremophilene中的应用，所述酶的氨基酸序列如SEQ IDNO.1所示。该酶为(-)-eremophilene的合成酶，本发明首次发现了(-)-eremophilene的合成酶。
一种上述酶的编码基因在合成或生产(-)-Eremophilene中的应用。在一些实施方案中，所述酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
一种含有上述酶的编码基因的重组质粒在合成或生产(-)-Eremophilene中的应用。
一种含有上述酶的编码基因的重组细胞在合成或生产(-)-Eremophilene中的应用。
一种(-)-Eremophilene高产菌株，通过在过表达甲羟戊酸途径中的一个或多个基因以及法尼烯焦磷酸合酶基因的微生物中导入上述酶的编码基因获得。所述的微生物优选为酿酒酵母。
在一些实施方案中，所述的(-)-Eremophilene高产菌株为重组酿酒酵母，所述重组酿酒酵母中甲羟戊酸途径基因、法尼烯焦磷酸合酶基因的拷贝数为ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20＝2、2、2-3、2、2、2、2、2，上述酶的编码基因的拷贝数为1-3。其中，ERG10为编码乙酰乙酰辅酶A硫解酶的基因，ERG13为编码HMG-CoA合酶的基因，tHMG1为编码HMG-CoA还原酶的基因，ERG12为编码甲羟戊酸激酶的基因，ERG8为编码甲羟戊酸-5-磷酸激酶的基因，MVD1为编码甲羟戊酸焦磷酸脱羧酶的基因，IDI1为编码异戊二烯焦磷酸异构酶的基因，ERG20为编码法尼烯焦磷酸合酶的基因。
本发明还发现了(-)-Eremophilene具有皮革、木质、草本香味，其还对害虫草地贪夜蛾有趋避效果，因此，(-)-Eremophilene可用于制备香料或香精，以及用于制备绿色拒食剂。
本发明相对于现有技术具有的优点和有益效果：
(1)发现了圣罗勒来源的(-)-Eremophilene合成酶及其编码基因，该酶能够高效地合成产物(-)-Eremophilene。
(2)构建的(-)-Eremophilene高产菌株，摇瓶产量达到708mg/L，发酵罐(15L钢罐)产量达到34.5g/L，为目前报道的最高产量，为(-)-Eremophilene的工业化应用奠定坚实的基础。
(3)发现了(-)-Eremophilene新的用途。
附图说明
图1是JVA46菌株产物的GC-MS检测结果。
图2是JHM1-JHM5菌株摇瓶发酵结果。
图3是倍半萜产物的1H NMR谱(600MHz,CDCl3)。
图4是倍半萜产物的13C NMR谱(150MHz,CDCl3)。
图5是(-)-eremophilene对三龄草地夜蛾幼虫的拒食活性，横坐标为(-)-eremophilene浓度，纵坐标为拒食率，不同字母表示差异显著(P<0.05)。
具体实施方式
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。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1
(1)酵母表达载体构建
质粒pZY900相关特征：△LEU2：LEU2(URA3)_TCYC1_LacZ_pGAL10pGAL1_ERG20_tERG20，用启动子GAL1、GAL10分别控制表达基因ERG20、LacZ，筛选标记为Leu2，插入的染色体位点为Leu2。
质粒pZY900具体构建过程：以酿酒酵母S288c基因组为模板，用引物900-1F/1R、900-2F/2R、900-6F/6R、900-7F/7R分别扩增获得片段9001(Leu2的左同源臂)、9002(终止子tTDH2)、9006(基因ERG20与终止子tERG20)、9007(Leu2右同源臂)；以酿酒酵母CEN.PK2-1D的基因组为模板，用引物900-3F/3R、900-5F/5R分别扩增获得片段9003(终止子tCYC1)和9005(启动子pGAL1和Pgal10)；用引物900-4F/4R以pCAS为模板扩增获得片段9004(无义基因，用于目标基因的替换)；以pRS426为模板，用引物900-8F/8R扩增获得质粒骨架(引入MssI酶切位点，筛选标记)。通过DNA assemble(酵母组装)的方法将以上片段在酿酒酵母体内重组构建pZY900，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY900。(pCAS的构建见文献Zhang,Yueping et al.“A gRNA-tRNA array for CRISPR-Cas9 basedrapid multiplexed genome editing in Saccharomyces cerevisiae.”Naturecommunications vol.10,1 1053.5Mar.2019,doi:10.1038/s41467-019-09005-3)。
构建质粒pZY900所用引物的序列见下表1：
表1
Figure BDA0003322872580000031
Figure BDA0003322872580000041
(2)倍半萜合成载体构建
文献中(Kumar,Yogesh et al.“Genome-wide detection of terpene synthasegenes in holy basil(Ocimum sanctum L.).”PloS one vol.13,11e0207097.16Nov.2018,doi:10.1371/journal.pone.0207097)对圣罗勒的基因组进行了分析，其中OsaTPS7被预测为是化合物Germacrene-A的合成酶。OsaTPS7的氨基酸序列如SEQID NO.1所示，我们对这个酶的编码基因按照酿酒酵母密码子优化后合成，其核苷酸序列如SEQ ID NO.2所示。
设计特异基因引物对P1/P2，以合成的基因(SEQ ID NO.2)为模板，利用Takara公司的Prime STAR高保真酶通过PCR扩增获得OsaTPS7基因片段，利用天根胶回收试剂盒进行胶回收后，通过翊圣公司同源重组试剂盒，采用同源重组的方法连接到BsaI切后的酵母表达载体pZY900中，经过测序确认无误后，获得含有该基因的酵母表达载体，命名为pKZ747。
使用引物P11/P12以合成的OsaTPS7基因片段为模板扩增含OsaTPS7基因的片段，使用引物P13/P14以质粒pZY184为模板扩增含Tpgk1和URA3右侧同源臂的片段，使用引物P17/P18以质粒pZY184为模板扩增含URA3左侧同源臂、His3标签、Tcyc1的片段，使用引物P15/P16以质粒pZY141为模板扩增含Thmg1和pGAL1-pGAL10的片段，使用引物LL0016-F/R以商用质粒pRS426为模板扩增含质粒骨架的片段，通过同源重组的方式将以上片段连接，获得的阳性质粒命名为pHM001。(pZY184、pZY141的构建见文献Shi,Bin et al.“Systemat icMetabolic Engineering of Saccharomyces cerevisiae for LycopeneOverproduction.”Journa l of agricultural and food chemistry vol.67,40(2019):11148-11157.doi:10.1021/acs.jafc.9b04519)。
使用引物P19/P20以pHM001为模板扩增含pGAL1-pGAL10、OsaTPS7基因、Tpgk1的片段，使用引物LL0033-F/P22以酿酒酵母S288C基因组为模板扩增含Tgpm1的片段，使用引物P21/LL0032-R以pTM36为模板扩增含潮霉素筛选标记的片段，使用引物P25/P26以酿酒酵母CEN.PK2-1D基因组为模板扩增含GAL80右侧同源臂的片段，使用引物P23/P24以酿酒酵母CEN.PK2-1D基因组为模板扩增含GAL80左侧同源臂的片段，使用引物0038-F/R以商用质粒pRS426为模板扩增含质粒骨架的片段，通过同源重组的方式将以上片段连接，获得的阳性质粒命名为pHM003。pTM36的构建见论文【马田.虾青素生物合成的元件挖掘及代谢工程改造〔D〕.武汉：武汉大学，2016.】。
所用引物序列见下表2：
表2
Figure BDA0003322872580000051
Figure BDA0003322872580000061
(3)菌株构建
通过PEG/LiAC方法将pKZ747质粒转化到酵母菌株YZL141感受态中，涂布于SD-URA筛选平板上，培养3d后，利用P1和P2引物，使用诺唯赞PCR试剂进行菌落PCR验证，将阳性菌命名为JVA46。(酵母菌株YZL141的构建见文献Shi,Bin et al.“Systematic MetabolicEngineering of Saccharomyces cerevisiae for Lycopene Overproduction.”Journalof agricultural and food chemistry vol.67,40(2019):11148-11157.doi:10.1021/acs.jafc.9b04519)。菌株JVA46是在酿酒酵母CEN.PK2-1D基础上，首先加强了MVA途径，过表达了MVA途径的限速酶tHMG1，并转入了含有法尼基焦磷酸合酶基因以及OsaTPS7的质粒。
同时利用PmeI内切酶酶切pKZ747回收含有OsaTPS7基因片段，通过PEG/LiAC方法将该片段导入到酵母菌株JCR27感受态中，进行酵母菌落PCR验证后，将阳性菌命名为JHM1。(酵母菌株JCR27的构建见文献Siemon,Thomas et al.“Semisynthesis of Plant-DerivedEnglerin AEnabled by Microbe Engineering of Guaia-6,10(14)-diene as BuildingBlock.”Journal of the American Chemical Society vol.142,6(2020):2760-2765.doi:10.1021/jacs.9b12940)。菌株JHM1相较于CEN.PK2-1D菌株，加强了MVA途径，过表达整个MVA途径，在此基础上，又过表达了法尼基焦磷酸合酶基因以及OsaTPS7基因，菌株JHM1中各基因的拷贝数为ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20、OsaTPS7＝2、2、2、2、2、2、2、2、1。
同理，利用PmeI内切酶酶切pHM001回收含有OsaTPS7基因片段，通过PEG/LiAC方法将该片段导入到酵母菌株JHM1感受态中，进行酵母菌落PCR验证后，将阳性菌命名为JHM2(菌株JHM2中各基因的拷贝数为ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20、OsaTPS7＝2、2、3、2、2、2、2、2、2)。对菌株JHM2利用cre酶重组将基因组中的Hyg基因移去后，获得菌株JHM3(菌株JHM3关键基因的拷贝数和菌株JHM2一样，只是移除了筛选标记，以便进行下一步改造)。
进一步利用PmeI内切酶酶切pHM003回收含有OsaTPS7基因片段，类似地转化酵母菌株JHM3感受态细胞后，进行酵母菌落PCR验证，获得阳性菌JHM4(菌株JHM4中各基因的拷贝数为ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20、OsaTPS7＝2、2、3、2、2、2、2、2、3)。进一步将含有URA和TRP1基因与YPRCdelta15基因的同源臂序列的表达框(使用引物P27/P28以酵母CEN.PK2-1D基因组为模板扩增片段YPRCdelta15左侧同源臂，使用引物P29/P30以商用质粒pRS426为模板扩增含ura3筛选标记的片段，使用引物P31/P32以商用质粒pRS424为模板扩增含his3筛选标记的片段，使用引物P33/P34以酵母CEN.PK2-1D基因组为模板扩增含YPRCdelta15右侧同源臂的片段，利用重叠延伸PCR将上述片段连接成一个片段得到所述表达框)，直接导入菌株JHM4感受态细胞后，进行酵母菌落PCR验证，获得阳性菌JHM5(菌株JHM5关键基因的拷贝数和菌株JHM4一样，只是补足了营养缺陷基因，以便于后续发酵)。
(4)摇瓶发酵
将JVA46菌株接种于50mL补充1％半乳糖的YPD培养基中，30℃培养3d后，8000rpm离心10min收集菌体，菌体经10mL萃取剂正己烷萃取后制样送GC-MS检测。JVA46菌株检测到特异性倍半萜产物，如图1所示。
JHM1-JHM5菌株采用类似的方法，添加10％肉豆蔻酸异丙酯(IPM)覆盖后发酵72h，离心后取油相进行GC-MS检测。倍半萜产物的产量从菌株JHM1的231mg/L增加至菌株JHM5的708mg/L，产量增加约3倍(图2)。
参照文献(van Hoek,P.；de Hulster,E.；van Di jken,J.P.；Pronk,J.T.Fermentative capacity in high-cell-density fed-batch cultures of baker’syeast.Biotechnol.Bioeng.2000,68,517-523.)中所记载的发酵培养基，对所构建的菌株JHM5进行分批补料发酵，在发酵过程中添加覆盖剂以实现原位萃取，覆盖剂为肉豆蔻酸异丙酯。发酵过程控制溶氧在20％以上，pH为5，葡萄糖浓度为1-2g/L，乙醇浓度为5g/L以下。最终在发酵罐(15L钢罐)上，倍半萜产物产量达到了34.6g/L。
(5)产物鉴定
为了确定倍半萜产物的结构，在不覆盖有机溶剂IPM的发酵条件下对菌株JHM5发酵，发酵控制和覆盖时相同，区别仅为不加覆盖剂。待发酵结束后，使用甲醇等体积萃取菌液，随后再用正己烷萃取，静止分离，获得上层有机相进行减压浓缩，减压浓缩后使用半制备HPLC进一步纯化，纯化的产物经过1H和13CNMR核磁鉴定，鉴定结果见图3和4，确定该倍半萜的化学结构为
Figure BDA0003322872580000081
其为(-)-eremophilene(佛术烯)。从结果可以知道，OsaTPS7所对应的产物并不是文献(enome-wide detection of terpene synthase genesin holy basil(Ocimum sanctum L.))中预测的Germacrene-A，而是(-)-eremophilene，本发明也是首次发现了(-)-eremophilene的合成酶。
实施例2
(1)为了评价(-)-eremophilene作为调味剂的潜在价值，分析了该分子中的香味，其混合了皮革、木头和药草的香味。当(-)-eremophilene被加热时，伴随着增强的气味产生白烟，这表明它有潜力用作香料成分和食品调味剂。
(2)采用叶片浸渍法研究(-)-eremophilene的拒食活性，选择农业害虫草地贪夜蛾作为研究对象。使用丙酮将(-)-eremophilene稀释成5个浓度，分别为50、100、200、500、800μg/mL。将新鲜花生(Arachis hypogaea)叶盘用直径15mm的钻孔器切割，分别在上述5种不同浓度的溶液中浸泡5s，对照叶盘单独用丙酮处理。然后，将两个对照叶盘和两个试验叶盘交替排列放置在培养皿中(直径为150毫米，直径)。然后将饥饿6h的3龄幼虫放在培养皿上方的中心。每个处理有5个重复。饲养24h后，测定叶片圆盘的取食面积。结果发现处理后的叶片取食面积显著低于对照叶片。当浓度为50μg/mL时，(-)-eremophilene的驱避效果高达90％左右(图5)。这些数据表明，该植物源化合物(-)-eremophilene具有驱虫活性，可作为一种绿色拒食剂。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 武汉臻智生物科技有限公司
<120> 佛术烯的合成酶基因和高产菌株以及用途
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 545
<212> PRT
<213> 圣罗勒(Ocimum sanctum L.)
<400> 1
Met Ala Pro Ala Gln Ala Glu Ile Gln Arg Pro Val Ala Asn Phe Ser
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Pro Ser Leu Trp Gly Glu Gln Phe Ile Lys His His Ser Ala Ser His
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Gln Val Glu Gly Lys His Ser Arg Ile Val Glu Val Leu Lys Asn Glu
35 40 45
Val Arg Ser Met Ile Asp Ala Gly Asn Met Val Asp Thr Met Ser Leu
50 55 60
Ile Asp Thr Leu Glu Arg Leu Gly Val Ser Tyr His Phe Glu Asn Gln
65 70 75 80
Ile Glu Gln Lys Leu His Gln Tyr Phe Asp Leu Asn Thr Asp Tyr Asn
85 90 95
Asn Glu Asp Tyr Asp Leu Tyr Thr Val Ala Leu His Phe Arg Leu Phe
100 105 110
Arg Gln His Gly Tyr Arg Leu Ser Ala Asp Val Phe Gly Arg Trp Arg
115 120 125
Asp Glu Asn Gly Lys Phe Gln Glu Gly Leu Lys Asn Asp Ala Lys Gly
130 135 140
Leu Leu Ser Leu Tyr Glu Ala Ser Tyr Leu Arg Thr Arg Gly Glu Pro
145 150 155 160
Leu Leu Asp Asp Ala Leu Gly Phe Ala Thr Ala Thr Leu Lys Ser Ile
165 170 175
Ala Pro Arg Leu Glu Ser Pro Leu Arg Glu Gln Ile Glu His Ala Leu
180 185 190
Ile Gln Ser Leu His Phe Gly Asn Pro Arg Ile Glu Ala Arg Lys Phe
195 200 205
Ile Ser Ile Tyr Glu Gln Tyr Glu Asp Lys Asn Glu Ser Leu Leu Arg
210 215 220
Phe Ala Lys Leu Asp Tyr Asn Gln Leu Gln Met Leu His Lys Glu Glu
225 230 235 240
Leu Asp Val Val Ser Arg Trp Trp Lys Asp Leu Asp Leu Val Ala Lys
245 250 255
Leu Pro Tyr Ala Arg Asp Arg Val Val Glu Cys Phe Phe Trp Ala Met
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Gly Val Tyr His Glu Pro Gln Tyr Ser Arg Ala Arg Ile Met Leu Thr
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Lys Thr Ile Ala Met Thr Ser Ile Ile Asp Asp Thr Tyr Asp Ala Tyr
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Gly Thr Ile Glu Glu Leu Asp Val Phe Thr Glu Ala Ile Glu Arg Trp
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Asn Ile Glu Glu Met Lys Arg Leu Pro Asp Tyr Val Lys Pro Phe Tyr
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Lys Ala Leu Leu Glu Leu Tyr Asp Gln Phe Glu Glu Glu Leu Ala Lys
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Glu Gly Arg Ser Tyr Ala Ala His Tyr Ala Ile Glu Ser Leu Lys Glu
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Leu Val Arg Ser Tyr His Leu Glu Ala Lys Trp Phe Ile Gln Gly Tyr
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Tyr Cys Tyr His Thr Thr Thr Ser Leu Leu Gly Val Glu Ser Ala Met
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Asn
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<210> 2
<211> 1638
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggctccag cacaagctga aattcaaaga ccagttgcaa atttttctcc atcattgtgg 60
ggtgaacaat tcattaaaca tcattctgct tcacatcaag ttgaaggtaa acattctaga 120
atcgttgaag ttttgaaaaa tgaagttaga tcaatgatcg atgctggtaa catggttgat 180
acaatgtctt tgatcgatac tttggaaaga ttgggtgttt cataccattt cgaaaaccaa 240
atcgaacaaa aattgcatca atattttgat ttgaatactg attacaataa tgaagattat 300
gatttgtaca cagttgcatt gcatttcaga ttgtttagac aacatggtta cagattatct 360
gctgatgttt ttggtagatg gagagatgaa aacggtaaat tccaagaagg tttgaaaaat 420
gatgcaaagg gtttgttgtc tttgtacgaa gcttcatact taagaacaag aggtgaacca 480
ttgttagatg atgcattggg ttttgcaaca gctactttaa aatctattgc tccaagattg 540
gaatcaccat taagagaaca aatcgaacat gcattgatcc aatctttgca tttcggtaac 600
ccaagaatcg aagctagaaa gtttatttct atctatgaac aatacgaaga taaaaatgaa 660
tcattgttaa gatttgcaaa attggattat aatcaattgc aaatgttaca taaagaagaa 720
ttagatgttg tttcaagatg gtggaaagat ttggatttgg ttgcaaaatt gccatacgct 780
agagatagag ttgttgaatg tttcttttgg gctatgggtg tttatcatga accacaatac 840
tctagagcaa gaatcatgtt gacaaagact atcgctatga catcaatcat cgatgatact 900
tacgatgcat acggtacaat cgaagaattg gatgttttta ctgaagctat cgaaagatgg 960
aacatcgaag aaatgaagag attgccagat tacgttaagc cattctacaa ggctttgtta 1020
gaattgtatg atcaattcga agaagaattg gcaaaggaag gtagatctta tgctgcacat 1080
tacgctatcg aatctttgaa ggaattagtt agatcatacc atttggaagc aaaatggttt 1140
attcaaggtt atttgccacc attcgaagaa tacttgaaaa atgctttgat tacatgtact 1200
tactgttacc atactacaac ttctttgtta ggtgttgaat cagctatgaa ggaagatttc 1260
gaatggttgt ctaagaaacc aaagatgttg gttgcatcat tgttgatctg tagagttatt 1320
gatgatatcg ctacttacga agttgaaaag gatagaggtc aaattgcaac aggtatcgaa 1380
tcttacatga aggaaaactg tgctactaag gaagaagcag ttgctaagtt tttcgaaatt 1440
gcaacagatg cttggaagga tatcaacgaa gaatgtatga gaccatctcc atactcaaga 1500
gatgttttga tgagaatctt gaatttggaa agaatcatcg atgttactta caagggtaac 1560
gaagatggtt acacacaacc agaaaaggtt ttgaagccac atatcatcga tttgtttgtt 1620
gatccaattg aaaattaa 1638

Claims (10)

1.一种酶在合成或生产佛术烯中的应用，其特征在于：所述酶的氨基酸序列如SEQ IDNO.1所示。
2.权利要求1中所述酶的编码基因在合成或生产佛术烯中的应用。
3.一种含有权利要求1中所述酶的编码基因的重组质粒在合成或生产佛术烯中的应用。
4.一种含有权利要求1中所述酶的编码基因的重组细胞在合成或生产佛术烯中的应用。
5.根据权利要求2-4任一项所述的应用，其特征在于：所述酶的编码基因的核苷酸序列如SEQ ID NO.2所示。
6.一种佛术烯高产菌株，其特征在于：通过在过表达甲羟戊酸途径中的一个或多个基因以及法尼烯焦磷酸合酶基因的微生物中导入权利要求1中所述酶的编码基因获得。
7.根据权利要求6所述的佛术烯高产菌株，其特征在于：所述的佛术烯高产菌株为为重组酿酒酵母，所述重组酿酒酵母中甲羟戊酸途径基因、法尼烯焦磷酸合酶基因的拷贝数为ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20＝2、2、2-3、2、2、2、2、2，权利要求1中所述酶的编码基因的拷贝数为1-3。
8.佛术烯在制备香料或香精中的应用。
9.佛术烯在制备害虫拒食剂中的应用。
10.根据权利要求9所述的应用，其特征在于：所述的害虫为草地贪夜蛾。
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