CN116121230A - 一种编码大根香叶烯a合成酶的基因的应用 - Google Patents
一种编码大根香叶烯a合成酶的基因的应用 Download PDFInfo
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Abstract
本发明公开一种编码大根香叶烯A合成酶的基因的应用,属于代谢工程技术领域。为了进一步提高大根香叶烯A异源微生物合成的产量。本发明提供一种大根香叶烯A合成酶氨基酸序列如SEQ ID NO.1所示,其编码基因序列如SEQ ID NO.2和SEQ IN NO.3所示。为微生物生产大根香叶烯A和β‑榄香烯提供了基础,将会提高大根香叶烯A和β‑榄香烯的产品质量、降低其生产成本、提高生产效率,为大根香叶烯A和β‑榄香烯的工业化生物合成提供技术前提。
Description
技术领域
本发明属于代谢工程技术领域,具体涉及一种编码大根香叶烯A合成酶的基因的应用。
背景技术
大根香叶烯A又称吉马烯A(germacrene A,CAS 28028-64-0)是生产β-榄香烯(β-elemene,CAS 33880-83-0)的前体。β-榄香烯是倍半萜类有效活性单体,为国家二类非细胞毒性抗肿瘤的药物,在临床上用于治疗肺癌、肝癌、乳腺癌等,由于在抗肿瘤方面具有广谱、安全、有效等突出优点,其应用前景十分广阔。大根香叶烯A合成酶催化法尼基焦磷酸生成大根香叶烯A,大根香叶烯A在高温下转化为β-榄香烯。目前,市场上的β-榄香烯主要来源于植物提取和化学合成。由于植物生长周期长,且提取工艺复杂,提取的产品纯度低,并不能满足人们对其日益增长的需求。而化学合成β-榄香烯工艺复杂,成本高。随着大根香叶烯A合成酶的发现,代谢改造微生物生产β-榄香烯具有巨大的潜力。目前为止,国内已报道了多个大根香叶烯A合成酶的基因序列。我国已对大根香叶烯A合成酶(germacrene Asynthase,GAS)的基因序列和表达做了比较深入的研究,并先后用大肠杆菌和酿酒酵母成功表达了大根香叶烯A合成酶基因,目前在摇瓶发酵水平上酿酒酵母中大根香叶烯A或β-榄香烯的最高产量仅为469mg/L,大肠杆菌中大根香叶烯A或β-榄香烯的最高产量仅为364.26mg/L(Hu et al.J Ind Microbiol Biotechnol,2017,44:1065-1072;Chen etal.frontiers in plant science,2017,13:932-966;Zhang et al.Microb Cell Fact,2021,20:7;CN 108060092 A)。但是目前公开的大根香叶烯A合成酶在微生物内的表达量和催化活性都很低,限制了大根香叶烯A的异源微生物合成。
发明内容
本发明的目的是为了进一步提高大根香叶烯A异源微生物合成的产量,亟需一种活性更高的大根香叶烯A合成酶,构建可更加高效合成大根香叶烯A的大肠杆菌或酿酒酵母重组菌,提高大根香叶烯A和β-榄香烯的产量,降低生产成本。
本发明提供一种SEQ ID NO.1所示的氨基酸序列在产大根香叶烯A合成酶中的应用。
进一步地限定,所述如SEQ ID NO.2或SEQ ID NO.3所示。
本发明提供一种含有上述的基因序列的重组载体。
进一步地限定,所述重组载体的出发载体为pET28a或pYES2。
本发明提供一种携带SEQ ID NO.1所示的氨基酸序列或过表达上述的基因序列的重组微生物细胞。
进一步地限定,重组微生物细胞为原核微生物细胞或真核微生物细胞。
本发明提供一种生产大根香叶烯A的方法,所述方法是将上述的重组微生物细胞,在含有法尼基焦磷酸的发酵培养基中,发酵获得大根香叶烯A。
本发明提供一种生产β-榄香烯的方法,其特征在于,所述方法是将上述的重组微生物细胞,在含有法尼基焦磷酸的发酵培养基中,发酵获得的大根香叶烯A,再加热转化获得β-榄香烯。
进一步地限定,产法尼基焦磷酸的重组菌的构建方法:构建过表达乙酰CoA酰基转移酶/HMG-CoA还原酶mvaE基因、HMG-CoA合成酶mvaS基因、甲羟戊酸激酶ERG12基因、甲羟戊酸-5-磷酸激酶ERG8基因、甲羟戊酸-5-二磷酸脱羧酶ERG19基因、异戊烯基二磷酸异构酶IDI基因、及法尼基焦磷酸合成酶ispA基因的大肠杆菌重组菌或过表达法尼基焦磷酸合成酶ERG20基因的酿酒酵母重组菌。
本发明提供一种SEQ ID NO.1所示的氨基酸序列、上述的基因序列、上的重组载体或上述的重组微生物细胞在产大根香叶烯A或/和产β-榄香烯中的应用。
有益效果:1)本发明所述大根香叶烯A合成酶分别在大肠杆菌和酿酒酵母中表达,分别获得8.2g/L和4.3g/L的β-榄香烯,均超过已报道的最高产量(专利公开号是CN108060092A的专利文件中公开的技术方案的效果,高密度发酵96h,产量2g/L的β-榄香烯)。
2)利用微生物合成的大根香叶烯A和β-榄香烯具有产量和纯度较高、无毒无害的优点,与植物提取和化学合成相比,是更为经济、环保、可持续的生产方式,更有利于推动生物法合成大根香叶烯A和β-榄香烯的工业化进程。
附图说明
图1大根香叶烯A合成途径。
图2大根香叶烯A加热产生β-榄香烯。
图3大肠杆菌作为出发菌的重组菌生产大根香叶烯A的NMR图。
图4大肠杆菌作为出发菌的重组菌生产的大根香叶烯A加热转化获得的β-榄香烯的NMR图。
图5质粒图谱,a为pACYC-mvaE-mvaS-ispA-AaFS质粒图谱;b为pTrcLower-idi1质粒图谱。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例所用试剂与培养基配方:EcoRI、BglII、NotI、SacI和SfiI内切酶购于TaKaRa;DNA聚合酶预混液PrimeSATR Max Premix购于TaKaRa;T4 DNA连接酶购于NEB;NEBuilder HiFi DNAAssembly Master Mix试剂盒购于NEB;质粒小提试剂盒及胶回收试剂盒购于Omega;上述分子生物学试剂用于基因克隆实验。
β-榄香烯标准品购自上海源叶公司,其他未做特殊说明的试剂和药品均为国产分析纯。
LB培养基:蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L;固体LB培养基添加1.5-2%的琼脂粉。
YPD培养基:蛋白胨20g/L,酵母提取物10g/L,葡萄糖20g/L;固体YPD培养基添加1.5-2%的琼脂粉。
发酵培养基:20g/L葡萄糖、9.8g/L K2HPO4、5g/L牛肉提取物、0.3g/L柠檬酸铁铵、2.1g/L一水合柠檬酸、0.06g/L MgSO4、1mL/L微量元素溶液,所述微量元素溶液含有(NH4)6Mo7O24·4H2O 0.37g/L、ZnSO4·7H2O 0.29g/L、H3BO3 2.47g/L、CuSO4·5H2O 0.25g/L和MnCl2·4H2O 1.58g/L,所述浓度为各成分在微量元素溶液中的终浓度(CN 111607546 A)。
本发明所使用的初始菌株是过表达乙酰CoA酰基转移酶/HMG-CoA还原酶mvaE基因、HMG-CoA合成酶mvaS基因、甲羟戊酸激酶ERG12基因、甲羟戊酸-5-磷酸激酶ERG8基因、甲羟戊酸-5-二磷酸脱羧酶ERG19基因、异戊烯基二磷酸异构酶IDI基因、法尼基焦磷酸合成酶ispA基因的重组菌,表达这些基因的大肠杆菌和酿酒酵母工程菌可产生法尼基焦磷酸。
CoA酰基转移酶/HMG-CoA还原酶mvaE基因、HMG-CoA合成酶mvaS基因、甲羟戊酸激酶ERG12基因、甲羟戊酸-5-磷酸激酶ERG8基因、甲羟戊酸-5-二磷酸脱羧酶ERG19基因、异戊烯基二磷酸异构酶IDI基因、法尼基焦磷酸合成酶ispA基因序列记载在公开号为CN111607546 A的专利中,其中IDI基因是CN 111607546 A中来自青蒿的AaIDI基因。
质粒pACYC-mvaE-mvaS-ispA-Sab1,构建时采用的原始空载体为pACYCDuet-1。所述质粒pACYC-mvaE-mvaS-ispA-Sab1记载在Zhang H,Liu Q,Cao Y,Feng X,Zheng Y,ZouH,Liu H,Yang J,Xian M.2014.Microbial production of sabinene-a new terpene-basedprecursor of advanced biofuel.Microbial Cell Factories 13:20。
质粒pACYC-mvaE-mvaS,构建时采用的原始空载体为pACYCDuet-1。所述质粒pACYC-mvaE-mvaS记载在Yang J,Xian M,Su S,Zhao G,Nie Q,Jiang X,Zheng Y,LiuW.2012.Enhancing production of bio-isoprene using hybrid MVApathwayandisoprene synthase in E.coli.PLOS ONE 7:e33509,该质粒中含有本发明所述的乙酰CoA酰基转移酶/HMG-CoA还原酶mvaE基因、HMG-CoA合成酶mvaS基因。
质粒pTrcLower,构建时采用的原始空载体为pTrcHis2B。所述质粒pTrcLower(即pTrc-ERG12-ERG8-ERG19-ScIDI)记载在Jiang X,Yang J,Zhang H,Zou H,Wang C,XianM.2012.In vitro assembly of multiple DNA fragments using successivehybridization.PLOS ONE7:e30267,该质粒中含有本发明所述的甲羟戊酸激酶ERG12基因、甲羟戊酸-5-磷酸激酶ERG8基因、甲羟戊酸-5-二磷酸脱羧酶ERG19基因和来自酿酒酵母的异戊烯基二磷酸异构酶ScIDI基因。
实施例所得产物采用以下检测方法进行表征:
400M核磁共振:仪器型号:布鲁克ASCEND 400兆核磁。
液相色谱质谱联用(LC-MS)条件:液相色谱-Q-TOF高分辨质谱,Bruke Maxis UHRTOF
对反应体系中的原料、中间体、产物进行定性和定量分析,产物通过反向色谱ZORBAX SB-C18(粒径为1.8μm;2.1×50mm)进行分离,流速为0.2mL/min,进样体积1μL,柱温箱40℃,检测模式正离子模式,检测电压1.56kV,雾化气(N2)流速1.5L/min,干燥气(N2)压力100kPa,离子收集时间30ms,碰撞能量50%,MS扫描范围100-600m/z,通过外标法对样品进行定量分析。
实施例1.构建大肠杆菌重组菌
制备一种高产法尼基焦磷酸的基因工程菌:为过表达乙酰CoA酰基转移酶/HMG-CoA还原酶mvaE基因、HMG-CoA合成酶mvaS基因、甲羟戊酸激酶ERG12基因、甲羟戊酸-5-磷酸激酶ERG8基因、甲羟戊酸-5-二磷酸脱羧酶ERG19基因、异戊烯基二磷酸异构酶IDI基因、法尼基焦磷酸合成酶ispA基因的重组菌,出发菌株为大肠杆菌。
1)质粒pACYC-mvaE-mvaS-ispA构建:
将质粒pACYC-mvaE-mvaS-ispA-AaFS(记载在专利号为CN 111607546 A的专利中,附图5中a所示)中的β-法尼烯合成酶AaFS基因去掉:质粒pACYC-mvaE-mvaS-ispA的构建采取Gibson Assembly的方法,首先以pACYC-mvaE-mvaS-ispA-AaFS质粒为模板扩增不含AaFS的载体部分即pACYC-mvaE-mvaS-ispA,上游引物pA-F(atatggcagatctcaattggatatc,SEQID NO.4),下游引物pA-F(atccagcgtaataaataattatttattacgctggatgatgt,SEQ ID NO.5)。
PCR扩增体系如下:模版1μL,PrimeSTAR Max Premix 25μL,上游引物2μL,下游引物2μL,水20μL。PCR反应程序:98℃2min;98℃10s,55℃15s,72℃1kb/15s,共35个循环;72℃5min。
PCR产物进行琼脂糖凝胶电泳,割胶回收pACYC-mvaE-mvaS-ispA载体片段,用NEBuilder试剂盒进行自连接反应,根据说明书计算片段的比例和各成分的量,连接反应50℃,60min,产物加等体积无菌水稀释,取5μL热激转化DH5α感受态细胞并涂布LB Cm平板,37℃培养过夜。第二天观察板子上的菌落情况,挑取单菌落到液体培养基,37℃培养至较浓,进行菌落PCR鉴定或提取质粒酶切鉴定,并送交测序,获得质粒pACYC-mvaE-mvaS-ispA。
2)质粒pTrcLower-ΔScIDI构建:
将质粒pTrcLower(附图5中b所示)中的异戊烯基焦磷酸异构酶ScIDI基因去掉:质粒pTrcLower-ΔScIDI的构建采取Gibson Assembly方法,以pTrcLower质粒为模板扩增不含ScIDI的载体部分即pTrc-ERG12-ERG8-ERG19,上游引物GA-Low-F(aaaggaataactgcagctggtaccatatgg,SEQ ID NO.6),下游引物GA-Low-R(ccagctgcagttattcctttggtagaccagtctttg,SEQ ID NO.7)。
PCR扩增体系如下:模版1μL,PrimeSTAR Max Premix 25μL,上游引物2μL,下游引物2μL,水20μL。PCR反应程序:98℃2min;98℃10s,55℃15s,72℃1kb/15s,共35个循环;72℃5min。
PCR产物进行琼脂糖凝胶电泳,割胶回收pTrc-ERG12-ERG8-ERG19载体片段,用NEBuilder试剂盒进行自连接反应,根据说明书计算片段的比例和各成分的量,连接反应50℃,60min,产物加等体积无菌水稀释,取5μL热激转化DH5α感受态细胞并涂布LB Amp平板,37℃培养过夜。第二天观察板子上的菌落情况,挑取单菌落到液体培养基,37℃培养至较浓,进行菌落PCR鉴定或提取质粒酶切鉴定,并送交测序,获得质粒pTrc-ERG12-ERG8-ERG-19即质粒pTrcLower-ΔScIDI。
3)质粒pTrcLower-IDI构建:
将异戊烯基二磷酸异构酶IDI基因序列经大肠杆菌密码子偏好性优化后,由华大基因进行序列合成,并克隆到pUC57-simple载体上得到质粒pUC57-IDI,质粒pTrcLower-IDI的构建采取酶切-连接的方法。首先以质粒pUC57-IDI和pTrcLower为模板,用带有SacI和PstI的引物分别扩增IDI基因片段和pTrc-ERG12-ERG8-ERG-19载体片段.。
PCR扩增体系如下:模版1μL,PrimeSTAR Max Premix 25μL,上游引物2μL,下游引物2μL,水20μL。PCR反应程序:98℃2min;98℃10s,55℃15s,72℃1kb/15s,共35个循环;72℃5min。
所述pTrc-ERG12-ERG8-ERG-19载体序列扩增用的上游引物low-Pst-F序列(aaaggaataactgcagctggtaccatatgg,SEQ ID NO.8),下游引物low-Sac-R序列(taggagctcttattcctttggtagaccagtctttg,SEQ ID NO.9);IDI基因扩增用的上游引物IDI-Sac-F序列(taggagctcgtaaggaggtatcaatatgaccattctgaccgatgc,SEQ ID NO.10),下游引物IDI-Pst-R序列(aactgcagttacagattatgaatggttttcatatcac,SEQ ID NO.11)。
PCR产物进行琼脂糖凝胶电泳,分别割胶回收IDI基因片段和pTrc-ERG12-ERG8-ERG19载体片段,用SacI和PstI进行双酶切。
酶切体系:胶回收产物20μL,10×限制性内切酶缓冲液5μL,SacⅠ2μL,PstⅠ2μL,水21μL,37℃反应30min。
酶切后产物进行琼脂糖凝胶电泳,分别割胶回收IDI基因片段和pTrc-ERG12-ERG8-ERG19载体片段,回收产物进行连接反应:
连接体系:载体4.5μL,插入片段4μL,10×连接酶缓冲液1μL,T4连接酶0.5μL。16℃连接过夜。
连接产物10μL热激转化DH5α感受态细胞并涂布LB Amp平板,37℃培养过夜。第二天观察板子上的菌落情况,挑取单菌落到液体培养基,37℃培养至较浓,进行菌落PCR鉴定或提取质粒酶切鉴定,并送交测序,获得质粒pTrc-ERG12-ERG8-ERG19-IDI即pTrcLower-IDI。
4)产法尼基焦磷酸工程菌的构建:
将步骤1)制备得到的pACYC-mvaE-mvaS-ispA和步骤3)制备得到的pTrcLower-IDI共同转化至E.coli BL21(DE3)感受态细胞,涂布相应的二抗(Cm和Amp)LB培养基平板,其中Cm在LB培养基中的终浓度为34mg/L,Amp在LB培养基中的终浓度为100mg/L,37℃培养至有单菌落长出,获得合成法尼基焦磷酸的大肠杆菌基因工程菌。
5)质粒pET28a-GAS构建:
将大根香叶烯A合成酶GAS基因序列经大肠杆菌密码子偏好性优化后,两段分别加限制性NdeI、XhoI酶切位点,由华大基因进行序列合成,合成后的序列如SEQ ID NO:2所示,并克隆到pET28a载体上得到质粒pET28a-GAS。
6)质粒转化:
将测序正确的质粒pET28a-GAS转化至步骤4)获得的合成法尼基焦磷酸的大肠杆菌工程菌的感受态细胞中,涂布相应的三抗(Cm、Amp和Kan)LB培养基平板,其中Cm在LB培养基中的终浓度为34mg/L,Amp在LB培养基中的终浓度为100mg/L,Kan在LB培养基中的终浓度为50mg/L,37℃培养至有单菌落长出,获得合成大根香叶烯A的大肠杆菌基因工程菌。
实施例2.构建酿酒酵母重组菌
1)质粒pUMRI-10-ERG20构建:
首先以酿酒酵母BY4741-C-04(Nat Commun.2016;7:12851)基因组为模板,以ERG20-F(gcggaattcatggcttcagaaaaagaaattagg,SEQ ID NO.12)和ERG20-R(ccttagatctctatttgcttctcttgtaaact,SEQ ID NO.13)为引物,PCR扩增获得法尼基焦磷酸合酶基因(ERG20,Gene ID:853272)。将得到的ERG20和酿酒酵母整合质粒pUMRI-10(MetabEng.2015;30:69-78)用EcoRI和BglII双酶切后,进行连接,转化至DH5α感受态中,获得质粒pUMRI-10-ERG20,具体如下:
双酶切体系:基因/质粒17μL,10×Buffer 2μL,EcoRI酶0.5μL,BglII酶0.5μL。
酶切条件:37℃放置酶切1.5-2h。
连接体系:基因5.5μL,质粒3μL,10×T4 DNA连接酶缓冲液1μL,350U/μL T4 DNA连接酶0.5μL。
连接产物10μL热激转化到DH5α感受态细胞并涂布LB Kan平板,37℃培养过夜。第二天观察板子上的菌落情况,挑取单菌落到液体培养基,37℃培养至较浓,进行菌落PCR鉴定或提取质粒酶切鉴定,并送交测序,获得质粒pUMRI-10-ERG20。
2)产法尼基焦磷酸工程菌的构建:
重组载体pUMRI-10-ERG20含有HO同源臂,同源臂处含有SfiI位点,通过SfiI酶切方法线性化。
酶切体系:总体系50μL,质粒43.5μL,10×G buffer 5μL,SfiI酶1.5μL。
酶切条件:放置50℃酶切2-3小时;
将线性化的pUMRI-10-ERG20整合到过表达甲羟戊酸途径的酿酒酵母BY4741-C-04中,从而构建得到高产法尼基焦磷酸的酿酒酵母生产菌株。
3)质粒pYES2-GAS构建:
将大根香叶烯A合成酶GAS基因序列经酿酒酵母密码子偏好性优化后,两段分别加限制性EcoRⅠ、XhoI酶切位点,由华大基因进行序列合成,合成后的序列如SEQ ID NO:3所示,并克隆到pYES2载体上得到质粒pYES2-GAS。
4)质粒转化:
将测序正确的质粒pYES2-GAS转化至步骤2)获得的合成法尼基焦磷酸的酿酒酵母工程菌的感受态细胞中,30℃培养至有单菌落长出,获得合成大根香叶烯A的酿酒酵母基因工程菌。
实施例3.大肠杆菌重组菌合成大根香叶烯A或β-榄香烯的应用
挑取实施例1中获得的大根香叶烯A的大肠杆菌基因工程菌在5L发酵罐中进行两相发酵:本实施例中所述一级种子培养基为LB培养基,其成分为:10g/L NaCl、10g/L蛋白胨、5g/L酵母提取物,其余为水。
所述摇瓶发酵培养基成分为:20g/L葡萄糖、9.8g/L K2HPO4、5g/L牛肉提取物、0.3g/L柠檬酸铁铵、2.1g/L一水合柠檬酸、0.06g/L MgSO4、1mL/L微量元素溶液,所述微量元素溶液含有(NH4)6Mo7O24·4H2O 0.37g/L、ZnSO4·7H2O 0.29g/L、H3BO3 2.47g/L、CuSO4·5H2O0.25g/L和MnCl2·4H2O 1.58g/L,所述浓度为各成分在微量元素溶液中的终浓度。
挑取实施例1获得基因工程菌的单菌落到5mL含有相应抗生素(Cm、Amp和Kan)的LB培养基中,37℃200rpm摇床培养8~12h,获得一级种子液。用体积250mL的锥形瓶进行摇瓶发酵,转接1mL一级种子液到100mL含有相应抗生素(Cm、Amp和Kan)的LB培养基中(2瓶),37℃200rpm摇床培养8~12h,获得二级种子液。最后经火焰接种环,将200mL种子液加入含2L发酵培养基的5L发酵罐(保兴生物,BIOTECH-5JG-7000A)中。当OD600达到25左右时,加入IPTG至终浓度为0.1mM和400mL的正十二烷30℃发酵培养120h后结束培养。
发酵过程中参数设定值分别为:温度30℃,pH 5.0,溶氧30%,空气流量3-20L/min,搅拌转速300-1000rpm,溶氧与搅拌转速、通气级联。当溶氧大于60%时,向发酵罐中加入补料培养基至发酵液中葡萄糖浓度为5g/L。
收集有机层,纯化后用NMR鉴定,发酵产物为大根香叶烯A,用液相色谱质谱联用检测产量为9.4g/L,纯度为97%。
将纯化的大根香叶烯A加热后获得的产物经NMR鉴定为β-榄香烯,β-榄香烯产量为8.2g/L,纯度为98%。
实施例4.酿酒酵母重组菌合成大根香叶烯A或β-榄香烯的应用
挑取实施例2中获得的大根香叶烯A的酿酒酵母基因工程菌在5L发酵罐中进行两相发酵,将实施例2中获得的获得基因工程菌在YPD平板上划线培养3天,然后挑取单菌落至5mL YPD试管中,220rpm 30℃进行过夜培养,获得一级种子液。用体积250mL的锥形瓶进行摇瓶发酵,转接1mL一级种子液到100mL YPD培养基中(2瓶),30℃220rpm摇床培养8~12h,获得二级种子液。最后经火焰接种环,将200mL种子液加入含2L发酵培养基的5L发酵罐中(保兴生物,BIOTECH-5JG-7000A)。当OD600达到25左右时,加入2%半乳糖和400mL的正十二烷,30℃发酵培养120h后结束培养。
发酵过程中参数设定值分别为:温度30℃,pH 5.0,溶氧30%,空气流量3-20L/min,搅拌转速300-1000rpm,溶氧与搅拌转速、通气级联。当溶氧大于60%时,向发酵罐中加入补料培养基至发酵液中葡萄糖浓度为5g/L。
收集有机层,纯化后用NMR鉴定,发酵产物为大根香叶烯A,用液相色谱质谱联用检测产量为5.1g/L,纯度为95%。
将纯化的大根香叶烯A加热后获得的产物经NMR鉴定为β-榄香烯,β-榄香烯产量为4.3g/L,纯度为97%。
如图3和图4所示,大根香叶烯A氢谱检测数据为:1H NMR(400MHz,CDCl3)δ4.66m2H,4.58m 2H,1.4~1.72m 18H,1.40s 1H,1.27m 1H.
β-榄香烯氢谱检测数据为:1H NMR(400MHz,CDCl3)δ5.84m 1H,4.93m 2H,4.84p1H,4.74m 2H,4.61dt 1H,2.00ddd 2H,1.75m 6H,1.66~1.41m 6H,1.03s 3H.
大根香叶烯A合成酶(Artificial Sequence)氨基酸序列SEQ ID NO.1:MVPCSETSDLVEISRFDTRGLGAGYKLRRHKFEHLADAGCHKARSDWIKHVGPLNEFGGCNHVNGNFSAVVLPLCRPDRLELVAYVLEYAFLHDSVLEAEDISPESQIQAEAGLRFLYERCISRLLQTDEVCAKRIAKAWKDAIDTTIRDKRIDFQSVEDYLEFRMIDTGAPFVEAIMLFGMAMTLTSQEDAELARVIRPCSAALALTNDYFSFDREMKEADTSTLINSVSIVMRLQNLDIATAKEVIKETIQSYEREFLRRIDEYKHQRGPVSEKIHQYLEAMAYQVSGNLVWSLNCPRYHPDFRCGLKACQQKE;
大根香叶烯A合成酶(Artificial Sequence)的编码基因SEQ ID NO.2:ATGGTGCCGTGCAGCGAAACCAGCGATCTGGTGGAAATTAGCCGCTTTGATACCCGCGGCCTGGGCGCGGGTTATAAATTACGCCGTCATAAATTTGAGCACCTGGCGGATGCGGGCTGCCATAAAGCGCGTAGCGATTGGATTAAACATGTGGGCCCGCTGAATGAATTTGGCGGCTGCAATCATGTGAATGGCAATTTTAGCGCGGTGGTGCTGCCGCTGTGTCGCCCAGATCGTTTAGAATTGGTTGCGTATGTGCTGGAATATGCGTTTCTGCATGATAGCGTGCTGGAAGCGGAAGATATTAGCCCGGAAAGCCAGATTCAGGCGGAAGCGGGTTTGCGCTTTCTGTATGAACGCTGCATTAGCCGCCTGCTGCAGACCGATGAAGTGTGCGCGAAACGCATTGCGAAAGCGTGGAAAGATGCGATTGATACCACCATTCGCGATAAACGCATTGATTTTCAGAGCGTGGAAGATTATCTGGAGTTTCGCATGATTGACACCGGCGCGCCGTTTGTGGAAGCGATTATGCTGTTTGGCATGGCGATGACCCTGACCAGCCAGGAAGATGCGGAACTGGCGCGTGTTATTCGCCCGTGTAGCGCGGCGTTAGCGTTAACCAATGATTATTTTAGCTTCGACCGCGAGATGAAGGAGGCGGATACCAGCACTCTGATTAATAGCGTGAGCATTGTGATGCGCCTGCAGAATCTGGATATTGCGACCGCGAAAGAAGTGATTAAAGAAACCATTCAGAGCTACGAGCGCGAATTTCTGCGCCGCATTGATGAATATAAACACCAGCGCGGCCCGGTGAGCGAAAAAATTCATCAGTATCTGGAAGCGATGGCGTATCAGGTGAGCGGCAATCTGGTGTGGAGCCTGAATTGCCCGCGCTATCATCCGGATTTTCGCTGCGGCTTAAAAGCGTGCCAGCAGAAAGAATAA;
大根香叶烯A合成酶(Artificial Sequence)编码基因SEQ ID NO.3:ATGGTTCCTTGCTCAGAAACCTCAGATCTTGTCGAAATTTCAAGATTCGATACTAGAGGTTTAGGTGCAGGTTACAAGCTGAGGAGGCACAAGTTTGAACATTTAGCCGATGCAGGCTGCCACAAAGCAAGATCCGACTGGATCAAACACGTTGGTCCTCTTAATGAATTTGGTGGTTGCAACCATGTTAACGGCAACTTTTCCGCAGTAGTTCTGCCTTTGTGCAGACCTGATAGATTAGAATTAGTAGCATACGTTCTGGAATACGCATTTCTACATGATAGTGTACTTGAGGCTGAAGATATTTCCCCGGAATCTCAAATCCAAGCCGAAGCAGGTTTGAGATTTTTATATGAAAGATGTATTTCTCGTTTGTTACAGACAGACGAAGTTTGTGCGAAAAGAATAGCCAAAGCTTGGAAGGACGCCATAGACACAACAATTCGTGATAAAAGAATAGATTTTCAATCTGTGGAAGATTATTTAGAATTCAGAATGATAGATACAGGCGCTCCCTTCGTCGAAGCCATCATGCTATTTGGTATGGCTATGACATTAACTTCTCAAGAAGATGCTGAATTGGCAAGAGTGATACGTCCCTGTTCTGCTGCTTTGGCACTAACTAATGATTATTTTTCTTTTGATCGTGAAATGAAAGAAGCAGACACCAGTACTTTGATAAACTCCGTTTCTATAGTGATGAGATTGCAAAATCTAGATATCGCGACTGCTAAGGAAGTCATAAAAGAAACGATTCAAAGCTATGAAAGAGAATTCTTAAGAAGAATCGACGAGTATAAACATCAAAGAGGTCCAGTTTCAGAAAAGATTCACCAGTATCTTGAGGCTATGGCTTACCAAGTCTCTGGTAATTTGGTATGGTCTCTGAATTGTCCAAGATATCACCCAGACTTTAGATGTGGATT
AAAGGCTTGCCAACAGAAGGAG;
pA-F序列SEQ ID NO.4:
ATATGGCAGATCTCAATTGGATATC;
pA-F序列SEQ ID NO.5:
ATCCAGCGTAATAAATAATTATTTATTACGCTGGATGATGT;GA-Low-F序列SEQ ID NO.6:
AAAGGAATAACTGCAGCTGGTACCATATGG;
GA-Low-R序列SEQ ID NO.7:
CCAGCTGCAGTTATTCCTTT GGTAGACCAGTCTTTG;
low-Pst-F序列SEQ ID NO.8:
AAAGGAATAACTGCAGCTGGTACCATATGG;
low-Sac-R序列SEQ ID NO.9:
TAGGAGCTCTTATTCCTTTGGTAGACCAGTCTTTG;
IDI-Sac-F序列SEQ ID NO.10:
TAGGAGCTCGTAAGGAGGTATCAATATGACCATTCTGACCGATGC
IDI-Pst-R序列SEQ ID NO.11:
AACTGCAGTTACAGATTATGAATGGTTTTCATATCAC;
ERG20-F序列SEQ ID NO.12:
GCGGAATTCATGGCTTCAGAAAAAGAAATTAGG;
ERG20-R序列SEQ ID NO.13:
CCTTAGATCTCTATTTGCTTCTCTTGTAAACT。
Claims (10)
1.SEQ ID NO.1所示的氨基酸序列在产大根香叶烯A合成酶中的应用。
2.根据权利要求1所述的基因序列,其特征在于,所述如SEQ ID NO.2或SEQ ID NO.3所示。
3.含有权利要求2所述的基因序列的重组载体。
4.根据权利要求3所述的重组载体,其特征在于,所述重组载体的出发载体为pET28a或pYES2。
5.携带SEQ ID NO.1所示的氨基酸序列或过表达权利要求2所述的基因序列的重组微生物细胞。
6.根据权利要求5所述的重组微生物细胞,其特征在于,重组微生物细胞为原核微生物细胞或真核微生物细胞。
7.一种生产大根香叶烯A的方法,其特征在于,所述方法是将权利要求5或6所述的重组微生物细胞,在含有法尼基焦磷酸的发酵培养基中,发酵获得大根香叶烯A。
8.一种生产β-榄香烯的方法,其特征在于,所述方法是将权利要求5或6所述的重组微生物细胞,在含有法尼基焦磷酸的发酵培养基中,发酵获得的大根香叶烯A,再加热转化获得β-榄香烯。
9.根据权利要求7或8所述的方法,其特征在于,产法尼基焦磷酸的重组菌的构建方法:构建过表达乙酰CoA酰基转移酶/HMG-CoA还原酶mvaE基因、HMG-CoA合成酶mvaS基因、甲羟戊酸激酶ERG12基因、甲羟戊酸-5-磷酸激酶ERG8基因、甲羟戊酸-5-二磷酸脱羧酶ERG19基因、异戊烯基二磷酸异构酶IDI基因及法尼基焦磷酸合成酶ispA基因的大肠杆菌重组菌或过表达法尼基焦磷酸合成酶ERG20基因的酿酒酵母重组菌。
10.SEQ ID NO.1所示的氨基酸序列、权利要求2所述的基因序列、权利要求3或4所述的重组载体或权利要求5或6所述的重组微生物细胞在产大根香叶烯A或/和产β-榄香烯中的应用。
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