CN114426929B - 一种发酵生产血根碱的酵母工程菌及其应用 - Google Patents
一种发酵生产血根碱的酵母工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种发酵生产血根碱的酵母工程菌及其应用,属于生物工程技术领域。本发明所述工程菌异源表达多个血根碱合成途径相关基因,引入酪氨酸脱羧酶突变体TYDCY350F重写上游途径,解决限速步骤牛心果碱氧化酶BBE的功能性表达,实现了以微生物发酵碳源从头合成血根碱。相较于传统的植物提取,本发明不受气候地域限制,环境友好污染小,产量可达628mg/L,接近产业化需求。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种发酵生产血根碱的酵母工程菌及其应用。
背景技术
血根碱(Sanguinarine)作为苄基异喹啉生物碱的代表产物,具有天然的抗炎、抗菌和促生长活性。自2006年起,为遏制畜牧业中抗生素滥用对公共卫生形成的巨大威胁,欧盟已全面禁止在饲料中添加抗生素。血根碱因作为植物天然抗菌活性成分,允许在畜牧饲料中添加,被广泛用于抗生素替代品,极大地降低了耐药菌的形成及传播。
市场上的血根碱主要依赖于从博落回等罂粟科植物中提取,但其天然含量低,提取工艺复杂且污染环境,植物种植也受季节气候限制,时间劳动力成本高。现阶段的化学合成法也因其结构复杂,制备过程繁琐收率低,不具有经济可行性。面对日益增加的市场需求,利用微生物细胞工厂生产血根碱成为一种更具潜力的选择。
目前,已有海量微生物生产目标化合物的报道,但多数并未能投入实际的生产应用中,关键原因在于下游分离纯化成本高,极大地降低了生物合成的经济效益。在没有革命性分离纯化手段出现之前,血根碱作为饲料添加剂,含量及纯度要求低,且酵母细胞无毒无害,开发高产血根碱的工程化菌株,能最大程度地简化下游分离纯化过程,为合成生物学的产业化推广提供新的思路。
虽然国内外不断尝试构建血根碱细胞工厂,但均未有成功实现微生物直接利用简单碳源(葡萄糖、半乳糖、甲醇、甘油等)发酵生产血根碱的报道。主要的技术壁垒包含以下三个方面:一是将微生物的中心代谢与植物次级代谢途径串联困难,关键结点去甲乌药碱合酶NCS的催化活性低。二是微生物自身的艾利希(Ehrlich)途径造成副产物4-羟基苯乙酸和酪醇的大量积累,使目标途径的代谢通量极大分流。三是植物中的血根碱合成途径虽已解析,但牛心果碱氧化酶BBE等关键步骤在微生物中的异源表达存在一定困难。上述问题的成功解决,将有望推动超过2500种苄基异喹啉类植物次级代谢产物的微生物细胞工厂合成。
授权公告号为CN109468351B的专利申请公开了一种高效酶催化合成血根碱和白屈菜红碱的方法,其分别从已知的普罗托品-6-羟基化酶基因、二氢苯并菲啶氧化酶基因及细胞色素P450还原酶基因中通过异源表达和结果比对分析,筛选出表达效率高的最优基因,然后对选出的最优基因进行密码子优化;再将优化基因序列构建到表达载体上,之后转入酵母工程菌中进行转化获得重组酵母工程菌株;最后用博落回的叶片原料液前体饲喂重组酵母工程菌进行发酵,即可获得血根碱和白屈菜红碱。该发明从基因水平、发酵工艺等多方面上提高血根碱和白屈菜红碱的酶催化效率,利用博落回的非传统药用部位叶片原料液直接与工程菌进行发酵,将叶片中生物碱含量高的原阿片碱和别隐品碱转化成高价值的血根碱和白屈菜红碱,以实现博落回资源的综合利用。但当转化率达到54.85%时,可得到910ng/mL的血根碱,可见,得到的血根碱含量低。
发明内容
本发明的目的在于克服现有的技术困难,实现微生物法从简单碳源出发定向生产血根碱,以绿色高效的方式合成苄基异喹啉类生物碱,提供了一种发酵生产血根碱的酵母工程菌及其应用。
本发明提供了一种发酵生产血根碱的酵母工程菌,以酵母作为出发菌株,通过基因敲除和导入基因构建获得所述酵母工程菌,其中,基因敲除的基因包括:酵母自身的氧化还原酶ARI1、ADH6、AAD3、HFD1、GRE2、YPR1和YDR541C的编码基因;导入基因包括:3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO7G141S、3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO4K229L_Q116K、酪氨酸脱羧酶编码基因突变体TYDCY350F、酪氨酸羟化酶编码基因CYP76AD5、多巴脱羧酶编码基因DODC、N端截短35个氨基酸的去甲乌药碱合酶编码基因NCS_N35、去甲基乌药碱6-O-甲基转移酶编码基因6OMT、乌药碱N-甲基转移酶编码基因CNMT、N-甲基去甲乌药碱羟化酶编码基因NMCH、3’羟基-N-甲基乌药碱4’-O-甲基酶编码基因4’OMT、牛心果碱氧化酶编码基因BBE、碎叶紫堇碱合酶编码基因CFS、刺罂粟碱合酶编码基因SPS、四氢原小檗碱-N-甲基转移酶编码基因TNMT、N-甲基刺罂粟碱14-羟化酶编码基因MSH、细胞色素P450还原酶编码基因CPR1、细胞色素b5编码基因CYB5、多药及毒性复合物挤出转运蛋白1编码基因DTX1、多药及毒性复合物挤出3样蛋白编码基因JAT1、多药及毒性复合物挤出转运蛋白2编码基因、原阿片碱6-羟化酶编码基因P6H和二氢苯并菲啶氧化酶编码基因DBOX。
优选的,牛心果碱氧化酶编码基因BBE为N端23个或24个氨基酸截短的牛心果碱氧化酶截短体编码基因。具体的,牛心果碱氧化酶截短体编码基因编码的牛心果碱氧化酶截短体还在C端融合有过氧化物酶体信号肽ePTS1、N端融合有线粒体信号肽MLS和液泡信号肽PRC1。
优选的,过氧化物酶体信号肽ePTS1的核苷酸序列如SEQ ID NO.6所示,N端融合线粒体信号肽MLS的核苷酸序列如SEQ ID NO.7所示,N端融合液泡信号肽PRC1的核苷酸序列如SEQ ID NO.8所示。
氧化还原酶ARI1编码基因的GeneBank号为NP_011358,氧化还原酶ADH6编码基因的GeneBank号为NP_014051,氧化还原酶AAD3编码基因的GeneBank号为NP_010032,氧化还原酶HFD1编码基因的GeneBank号为NP_013828,氧化还原酶GRE2编码基因的GeneBank号为NP_014490,氧化还原酶YPR1编码基因的GeneBank号为NP_010656,氧化还原酶YDR541C编码基因的GeneBank号为NP_010830;3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO7G141S的核苷酸序列如SEQ ID NO.14所示,3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO4K229L_Q116K的核苷酸序列如SEQ ID NO.15所示,所述酪氨酸脱羧酶编码基因突变体TYDCY350F的核苷酸序列如SEQ ID NO.1所示,酪氨酸羟化酶编码基因CYP76AD5的GeneBank号为AJD87473,多巴脱羧酶编码基因DODC的GeneBank号为AKH61506,N端截短35个氨基酸的去甲乌药碱合酶编码基因NCS_N35的核苷酸序列如SEQ ID NO.2所示,去甲基乌药碱6-O-甲基转移酶编码基因6OMT的GeneBank号为XP_026389064,乌药碱N-甲基转移酶编码基因CNMT的GeneBank号为Q7XB08,N-甲基去甲乌药碱羟化酶编码基因NMCH的GeneBank号为O64900,3’羟基-N-甲基乌药碱4’-O-甲基酶编码基因4’OMT的GeneBank号为XP_026440860;所述牛心果碱氧化酶编码基因BBE包括改造自罂粟(Papaver somniferum)的BBE基因PsBBE、改造自花菱草(Eschscholzia californica)的BBE基因EcBBE、改造自黄连(Coptisjaponica)的BBE基因CjBBE、改造自博落回(Macleaya cordata)的BBE基因McBBE、改造自白屈菜(Chelidonium majus)的BBE基因CmBBE和改造自血根草(Sanguinaria canadensis)的BBE基因ScBBE,其中,PsBBE的GeneBank号为XP_026421833,EcBBE的GeneBank号为P30986,CjBBE的GeneBank号为BAM44344,McBBE的核苷酸序列如SEQ ID NO.3所示,CmBBE的核苷酸序列如SEQ ID NO.4所示,ScBBE核苷酸序列如SEQ ID NO.5所示;所述碎叶紫堇碱合酶编码基因CFS的GeneBank号为B5UAQ8、刺罂粟碱合酶编码基因SPS的GeneBank号为EU882969;所述四氢原小檗碱-N-甲基转移酶编码基因TNMT包括改造自罂粟(Papaver somniferum)的TNMT基因PsTNMT、改造自花菱草(Eschscholzia californica)的TNMT基因EcTNMT、改造自蓟罂栗(Argemone mexicana)的TNMT基因AmTNMT、改造自虞美人(Papaver rhoeas)的TNMT基因PrTNMT和改造自大红罂粟(Papaver bracteatum)的TNMT基因PbTNMT,其中,PsTNMT的GeneBank号为Q108P1,EcTNMT的GeneBank号为C3SBS8,AmTNMT的核苷酸序列如SEQ ID NO.9所示,PrTNMT的核苷酸序列如SEQ ID NO.10所示,PbTNMT的核苷酸序列如SEQ ID NO.11所示;N-甲基刺罂粟碱14-羟化酶编码基因MSH的GeneBank号为L7X3S1;细胞色素P450还原酶编码基因CPR1的GeneBank号为NP_001190823,细胞色素b5编码基因CYB5的GeneBank号为KP411012;多药及毒性复合物挤出转运蛋白1编码基因DTX1的GeneBank号为Q9SIA5,多药及毒性复合物挤出3样蛋白JAT1编码基因的GeneBank号为NP_001313041,多药及毒性复合物挤出转运蛋白2编码基因的GeneBank号为7DQK_B;所述原阿片碱6-羟化酶编码基因P6H包括改造自罂粟(Papaver somniferum)的P6H基因PsP6H、改造自花菱草(Eschscholziacalifornica)的P6H基因EcP6H、改造自博落回(Macleaya cordata)的P6H基因McP6H、改造自变根紫堇(Corydalis linstowiana)的P6H基因ClP6H和改造自大红罂粟(Papaverbracteatum)的P6H基因PbP6H,其中,PsP6H的GeneBank号为L7X0L7,EcP6H的GeneBank号为F2Z9C1,McP6H的GeneBank号为OVA18597,ClP6H的核苷酸序列如SEQ ID NO.12所示,PbP6H的核苷酸序列如SEQ ID NO.13所示;二氢苯并菲啶氧化酶编码基因DBOX的GeneBank号为XP_026380178。
具体的,所述6OMT、CNMT、4’OMT、MSH、DBOX来源于罂粟Papaver somniferum;所述CYP76AD5来源于甜菜Beta vulgaris;所述DODC来源于恶臭假单胞菌Pseudomonas putida;所述NCS来源于黄连Coptis japonica;所述NMCH、CFS、SPS来源于花菱草Eschscholziacalifornica。
所述酪氨酸脱羧酶TYDCY350F突变体是指其第350位的酪氨酸突变为苯丙氨酸,其作用由催化酪氨酸脱羧形成酪胺转变为催化酪氨酸直接生成4-羟基苯乙醛。本发明实施例探究了酪氨酸脱羧酶TYDC和酪氨酸脱酸酶突变体TYDCY350F对关键中间体牛心果碱合成的影响。酪氨酸脱羧酶突变体TYDCY350F能极大程度地提高牛心果碱的积累,而酪氨酸脱羧酶TYDC是酪氨酸生成副产物酪胺,降低了牛心果碱的合成。
所述去甲乌药碱合酶NCS为N端截短35个氨基酸的形式,相较于N端不截短或截短其他个数的氨基酸,NCS_N35具有更好的催化活性。
所述牛心果碱氧化酶BBE在截短膜定位序列后,进一步在其N端融合表达MBP标签,增强其在胞质中的可溶性表达。所述牛心果碱氧化酶BBE在截短膜定位序列后,进一步在其N端或C端加上过氧化物酶体、线粒体、液泡等细胞器定位的信号肽,将BBE靶向到酵母的各细胞器中,进一步增强BBE的催化活性。
在酵母中进一步转入上述所示核苷酸序列的TNMT基因或对应的酶,均能催化刺罂粟碱生成N-甲基刺罂粟碱。
在酵母中进一步转入表1和SEQ ID NO.12-13所示核苷酸序列的P6H基因或对应的酶,均能催化原阿片碱生成6-羟基原阿片碱。
优选的,所述出发菌株为酿酒酵母BY4741、酿酒酵母BY4742、酿酒酵母CEN.PK2-1C、酿酒酵母W303-1A或毕赤酵母GS115。
所述酵母工程菌构建时,先构建共有中间体牛心果碱的底盘菌株,所述共有中间体牛心果碱的底盘菌株的构建方法步骤:将酪氨酸羟化酶编码基因CYP76AD5、多巴脱羧酶编码基因DODC、N端截短35个氨基酸的去甲乌药碱合酶编码基因NCS_N35、去甲基乌药碱6-O-甲基转移酶编码基因6OMT、乌药碱N-甲基转移酶编码基因CNMT、N-甲基去甲乌药碱羟化酶编码基因NMCH和3’羟基-N-甲基乌药碱4’-O-甲基酶编码基因4’OMT整合到酿酒酵母BY4741基因组上的对应位置,并将酿酒酵母BY4741细胞中7个氧化还原酶ARI1、AAD3、ADH6、HFD1、GRE2、YPR1、YDR541C作为敲除位点,构建得到生产共有中间体牛心果碱的底盘菌株。通过敲除7个酵母自身功能冗余的氧化还原酶(ARI1、ADH6、AAD3、HFD1、GRE2、YPR1、YDR541C)减少艾利希(Ehrlich)途径造成副产物4-羟基苯乙酸和酪醇的积累。此外,在引入分支酸变位酶和3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸(DAHP)合酶解除负反馈抑制的突变体ARO7G141S和ARO4K229L_Q166K的同时,过表达酵母自身的苯丙酮酸脱羧酶ARO10、双功能分支酸合酶和黄素还原酶ARO2和苯甲酸脱氢酶TYR1以提高酪氨酸途径的代谢通量。
本发明还提供了上述的发酵生产血根碱的酵母工程菌在制备血根碱中的应用。
本发明还提供了一种制备血根碱的方法,发酵培养上述的发酵生产血根碱的酵母工程菌,并提取获得血根碱。
本发明的有益效果:(1)本发明的酵母工程菌可通过代谢葡萄糖、甲醇、半乳糖、甘油或海藻糖等直接合成血根碱,其产量高达到628mg/L,接近产业化需求。(2)本发明实现了利用微生物以简单碳源为底物发酵生产血根碱。通过突破目前从头合成血根碱的关键技术难点,强化宿主中心代谢与异源次级代谢途径的串联,为超2500种苄基异喹啉生物碱的生物合成提供参考。(3)本发明利用微生物一步法合成血根碱,与传统植物提取相比,不受气候地理等条件限制,环境友好,满足国家绿色生物制造的战略需求。且无潜在致病因子,产品的质量与安全性得到保证,适用于广大医药、畜牧业等领域。(4)本发明发酵生产的血根碱作为抗生素替代物添加到饲料中,其含量及纯度要求较低,本发明通过酵母细胞直接合成血根碱,发酵液整体无毒无害,简单处理后即可商用,最大程度地简化下游分离纯化过程,为合成生物学的产业化推广提供新的思路。
附图说明
图1为构建酵母工程菌株的模块图。
图2为酿酒酵母高效合成共有中间体牛心果碱的结果图。
图3为酿酒酵母表达不同BBE基因及其突变体生成中间体金黄紫堇碱的结果图。
图4为异源转运蛋白对血根碱产量影响的结果图。
图5为血根碱标准品与重组菌株生产的血根碱及途径中间体在同一条件下的高效液相色谱洗脱图。
图6为血根碱标准品与重组菌株生产血根碱(SAN028)在同一保留时间的LC-MS质谱图。
图7为血根碱的合成流程图。
具体实施方式
实施例1
克隆与表达血根碱生物合成所需的基因。
(1)血根碱合成途径中所需的植物或微生物来源的酶,大部分通过美国国家生物信息中心NCBI(https://www.ncbi.nlm.nih.gov/)找到对应的氨基酸序列或基因序列(见表1)。而针对筛选高效的牛心果碱氧化酶(BBE)、四氢原小檗碱-N-甲基转移酶(TNMT)、原阿片碱6-羟化酶(P6H),通过中国国家基因库(CNGB)开发的The 1000 Plants Project(https://db.cngb.org/blast/)的tBLASTn以及文献检索确定不同的植物来源和对应的基因序列。
获得的基因序列由金斯瑞生物科技有限公司开发的密码子优化算法、并依据相应的酵母宿主进行密码子优化,具体如序列表所示。并直接合成在带有半乳糖启动子(pGAL1或者pGAL10)的通用质粒pESC下,得到连有目的基因的重组质粒。
表1 NCBI查询到的途径基因
(2)利用带有40bp同源臂的基因组整合引物(详见表2)对重组质粒进行PCR,得到“启动子-目的基因-终止子”的基因组整合供体。再根据酿酒酵母中CRISPR/Cas9的基因组编辑工具(Lian,J.,HamediRad,M.,Hu,S.&Zhao,H.Combinatorial metabolicengineering using an orthogonal tri-functional CRISPR system.NatureCommunications,2017,8,1688-1696,doi:10.1038/s41467-017-01695-x),将整合供体连同对应的single guide RNA(sgRNA)编码序列的质粒和pRS41K-SpCas9的质粒(详见表3)利用化学法转化到酵母细胞中,涂布于相应氨基酸缺陷型的SED/G418平板上。
(3)对平板上的转化子,挑点破胞,并选取基因组上的两条引物(详见表2)利用PCR验证是否将目的基因整合到基因组上。若条带大小正确且测序结果无误,则说明基因组整合成功。
表2基因组整合异源基因所用引物
引物名称 | 引物序列(5’到3’) |
Int16-CPR1-CYB5-F | attctactaaaaacacatcagtagtcacagaagtcacagttcaggctgcgcaactgtt |
Int16-CPR1-CYB5-R | attttttcattaatacacctgtgttagttatgattgccagagtgagctgataccgctcg |
Int17-CYP76AD5-DODC-F | ttacgtgtcatttattatgggttcagaaattatgtgttaagagcgacctcatgctatac |
Int17-CYP76AD5-DODC-R | cacacaattttggtggcgttgaaattgatgccggaatttgcttcgagcgtcccaaaacc |
Int19-NCS_N35-F | ttacgtgtcatttattatgggttcagaaattatgtgttaagagcgacctcatgctatac |
Int19-NCS_N35-R | aaatcgacatgttaatgatcttacgacagagtagtttatgcttcgagcgtcccaaaacc |
Int14-6MOT-CNMT-F | tgaaaattgtgccggatattcaagactaagagatgtacaagagcgacctcatgctatac |
Int14-6MOT-CNMT-R | tatccgggtaacactcatcgtccggcctccgcccccttttcttcgagcgtcccaaaacc |
Int9-NMCH-4′OMT-F | gagagaaattaaacttggttggggttaattatttgatggggagcgacctcatgctatac |
Int9-NMCH-4′OMT-R | cactattgataaaggttttgtagaatatttattatcgatacttcgagcgtcccaaaacc |
Int11-BBE-F | gactagtatcatccgtcaagaagaacaagaacaagaacaagagcgacctcatgctatac |
Int11-BBE-R | tacaggcaatgagcgaaagcgactgaagccgagagatgtgcttcgagcgtcccaaaacc |
Int10-CFS-SPS-F | tgtacgctatacatttacgtgctgagctcctaggaaagctgagcgacctcatgctatac |
Int10-CFS-SPS-R | cctctatgtgacgctgtgtattctttgttgtagttatgctcttcgagcgtcccaaaacc |
Int4-TNMT-MSH-F | gttttcttatttctttctttttaaaaaactttcttaatatgagcgacctcatgctatac |
Int4-TNMT-MSH-R | gaattgagaaaaaaagtgtatatcattacattactttacacttcgagcgtcccaaaacc |
Int7-DBOX-P6H-F | tgtacggactattgctgtcttctcgtggtaaatgcgtgttgagcgacctcatgctatac |
Int7-DBOX-P6H-R | ccgaaaagacaacttaacaaatcggcaacacttttatgggcttcgagcgtcccaaaacc |
表3菌种构建所用质粒
实施例2
构建高产共有中间体牛心果碱的底盘菌株。
将实施例1中的CYP76AD5、DODC、NCS_N35(核苷酸序列如SEQ ID NO.2所示)、6OMT、CNMT、NMCH、4’OMT对应基因序列整合到酿酒酵母BY4741基因组上的对应位置,如图1所示,从而构建出生产共有中间体牛心果碱的底盘菌株。由于上游中心碳代谢的中间体4-羟基苯乙醛极容易被酵母自身的艾利希(Ehrlich)途径氧化还原生成副产物酪醇和4-羟基苯乙酸。为此,将酿酒酵母细胞中功能冗余的7个氧化还原酶ARI1、AAD3、ADH6、HFD1、GRE2、YPR1和YDR541C作为敲除位点,进行后续代谢途径的优化。
为进一步增强上游莽草酸(Shikimate)途径的代谢通量,在表2对应的位点整合分支酸变位酶和3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸(DAHP)合酶解除负反馈抑制的突变体ARO7G141S和ARO4K229L_Q116K对应的编码基因序列(突变体ARO7G141S和ARO4K229L_Q116K对应的编码基因序列如SEQ ID NO.14和SEQ ID NO.15所示)。与此同时,过表达酵母自身的苯丙酮酸脱羧酶ARO10(GeneBank号为NP_010668)、双功能分支酸合酶和黄素还原酶ARO2(GeneBank号为NP_011367)、苯甲酸脱氢酶TYR1(GeneBank号为NP_009725)以强化上游的酪氨酸供应。此外,过表达3’羟基-N-甲基乌药碱4’-O-甲基酶4’OMT以减少中间体的积累。如图2所示,RE001为构建牛心果碱合成途径的单拷贝菌株,经上述代谢途径优化得到RE006菌株,牛心果碱的滴度累计提高约43倍。各菌株摇瓶中牛心果碱的滴度如表4所示。
表4各菌株生产牛心果碱摇瓶中的滴度
为进一步提高前体供应,减少中间体的积累,过表达ARO2、ARO10、TYR1、4’OMT,同时,可能由于CYP76AD5活性过强,多巴胺仍过量积累,更多的4-羟基苯乙醛能极大地促进牛心果碱的生成。因此,在菌株RE006的基础上引入酪氨酸脱羧酶突变体TYDCY350F编码基因(核苷酸序列如SEQ ID NO.1所示)构建菌株RE007,以解决去甲乌药碱合酶底物平衡的问题。该酪氨酸脱羧酶突变体由其原本催化酪氨酸脱羧生成酪胺的活性,转变为更倾向于氧化脱氨作用,通过一步酶反应直接将酪氨酸转化为关键中间体4-羟基苯乙醛,极大地提高了去甲乌药碱合酶的前体供应,牛心果碱的滴度也因此提高约13倍。
实施例3
N端截短信号肽、MBP标签融合表达和不同细胞器定位的牛心果碱氧化酶BBE。
5’端片段缺失的长度依据Discovery studio软件,在蛋白质数据库网站PDB(www.rcsb.org)以EcBBE(来源为花菱草(Eschscholzia californica)的BBE基因)的晶体结构(PDB号4EC3)为模板,对PsBBE(来源为罂粟(Papaver somniferum)的BBE基因)、CjBBE(来源为黄连(Coptis japonica)的BBE基因)、McBBE(来源为博落回(Macleaya cordata)的BBE基因)、CmBBE(来源为白屈菜(Chelidonium majus)的BBE基因)、ScBBE(来源为血根草(Sanguinaria canadensis)的BBE基因)进行同源建模。同时,对上述6种来源的牛心果碱氧化酶BBE利用Jaview软件进行蛋白质序列比对。结合同源建模和序列比对结果,大致确定出不同来源BBE的N端信号肽序列长度,并设计引物进行截短,如表5所示。
表5截短不同来源牛心果碱氧化酶BBE所用引物
引物名称 | 引物序列 |
PsBBE_N23-F | tttaacgtcaaggagaaaaaaccccggatccatgggtgatgttaatgataacttgt |
EcBBE_N23-F | ctttaacgtcaaggagaaaaaaccccggatccatgggtaacgacttgttatcttg |
CjBBE_N24-F | tttaacgtcaaggagaaaaaaccccggatccatggaggatgacttgccatcttgtttg |
McBBE_N23-F | tttaacgtcaaggagaaaaaaccccggatccatgggtgacgacttgttgtcttgtt |
CmBBE_N23-F | tttaacgtcaaggagaaaaaaccccggatccatgggtgacgatgacttattatc |
ScBBE_N24-F | tttaacgtcaaggagaaaaaaccccggatccatgggtgacgatgacttattatc |
为进一步提高牛心果碱氧化酶的催化活性,对不同来源截短信号肽的BBE突变体融合表达MBP标签(NCBI登录号为3MQ9_A)并用“GGGGSGGGGS”序列进行柔性连接,其中BBE突变体中,改造自PsBBE的GeneBank号为XP_026421833,改造自EcBBE的GeneBank号为P30986,改造自CjBBE的GeneBank号为BAM44344,SEQ ID NO.3所示核苷酸序列改造自McBBE,SEQ IDNO.4所示核苷酸序列改造自CmBBE,SEQ ID NO.5所示核苷酸序列改造自ScBBE。同时,尝试将各BBE突变体定位到酵母各细胞器中,各细胞器定位的信号肽如表6所示。由图3和表6所示,SAN006-SAN0011是在高产共有中间体牛心果碱的底盘菌株RE007上,整合不同来源的tBBE菌株。SAN012-SAN014是将效果最佳的BBE定位至不同的细胞器中。由图3和表7所示,SAN008、SAN009、SAN010菌株对应的BBE催化活性较高。
表6各细胞器定位的信号肽序列
英文缩写 | 细胞器定位区域 | 目的基因上的位置 | 信号肽序列 |
ePTS1 | 过氧化物酶体 | C端 | SEQ ID NO.6 |
MLS | 线粒体 | N端 | SEQ ID NO.7 |
PRC1 | 液泡 | N端 | SEQ ID NO.8 |
表7各菌株生产金黄紫堇碱摇瓶中的滴度
实施例4
构建以简单碳源合成血根碱的高产工程菌。
在上述菌株基础上,按照表2所示引物,以表3所示质粒为模板,PCR扩增出对应的异源基因表达盒的供体片段。利用醋酸锂高效转化法将余下的CFS、SPS、TNMT、MSH、P6H、DBOX对应编码基因序列依次整合到对应的基因组位点,从而得到以简单碳源为原料合成血根碱的酵母工程菌株SAN026,发酵罐中产量如表8和图4所示。
TNMT分别来源于6种不同的植物,其中:改造自罂粟(Papaver somniferum)的TNMT基因(PsTNMT)的GeneBank号为Q108P1;改造自花菱草(Eschscholzia californica)的TNMT基因(EcTNMT)的GeneBank号为C3SBS8;SEQ ID NO.9所示核苷酸序列改造自蓟罂栗(Argemone mexicana)的TNMT基因(AmTNMT);SEQ ID NO.10所示核苷酸序列改造自虞美人(Papaver rhoeas)的TNMT基因(PrTNMT);SEQ ID NO.11所示核苷酸序列改造自大红罂粟(Papaver bracteatum)的TNMT基因(PbTNMT)。
P6H分别来源于6种不同的植物,其中:改造自罂粟(Papaver somniferum)的P6H基因(PsP6H)的GeneBank号为L7X0L7;改造自花菱草(Eschscholzia californica)的P6H基因(EcP6H)的GeneBank号为F2Z9C1;SEQ ID NO.12所示核苷酸序列改造自变根紫堇(Corydalis linstowiana)的P6H基因(ClP6H);改造自博落回(Macleaya cordata)的P6H基因(McP6H)的GeneBank号为OVA18597;SEQ ID NO.13所示核苷酸序列改造自大红罂粟(Papaver bracteatum)的P6H基因(PbP6H)。
将从头合成血根碱的酿酒酵母工程菌进行分批补料发酵。首先在YPD的平板上划线得到单菌落,再接种于50mL YPD、SCD或SED液体培养基中,在30℃、250rpm条件下培养至菌体OD600达到6左右,再以初始OD600=0.4转接于250mL YPD、SCD或SED的液体培养基中。待OD600达到12左右,按照占发酵培养基的15%的体积比接种至含600mL发酵培养基的1L发酵罐中,控制发酵温度控制在25℃,pH在5.5左右,通气量设定为4.0vvm,搅拌转速与溶氧相关联,溶氧指数控制在30%,搅拌转速设置为300-1000rpm。每隔6h取样测定各类简单发酵碳源的含量(葡萄糖、甘油、乙醇、海藻糖、半乳糖等),并根据发酵碳源的消耗速率及时调整流加量,其中葡萄糖的浓度需稳定在1-2g/L,甘油则以20mL/(h·L)的速率恒速流加50%(v/v)甘油补料。整个发酵周期大概维持在96-120h之间。
表8各菌种生产血根碱发酵罐中的产量
取500μL发酵上清加入等体积的乙酸乙酯萃取3-4h。12000rpm两次离心10min后,将萃取出的上层液体用0.22μm的有机系滤头过滤。再用1290 Infinity II LC系统(Agilent Technologies),HyPURITYTM C18 HPLC(150mm×4.6mm,3μm,ThermoScientific)色谱柱,对2μL的样品进行分离。其中,流动相A相为0.1%的甲酸水溶液,流动相B相为0.1%的乙腈溶液。各代谢中间体及产物的详细梯度洗脱条件如下:保持5%B从0-2min(0.2mL/min),5%B到85%B从2-2.5min(0.2mL/min),保持85%B从2.5-27.5min(0.2mL/min),85%B到5%B从27.5-28min(0.2mL/min),保持5%B从28-30min(0.2mL/min)。
随后的洗脱液注入三重四级杆质谱MS/MS(Agilent Technologies)在正离子下使用100-400m/z的扫描范围。分辨率、毛细管电压、ESI离子源温度分别设置为100000、5kV、350℃。采用多反应监测模式(MRM)对血根碱及其途径中间体进行检测,血根碱的碰撞能(CE)为40eV,m/z跃迁从332.75到275.10;牛心果碱CE为20eV,m/z跃迁从330.15到192.05;金黄紫堇碱CE为24eV,m/z跃迁从328.10到178.05;碎叶紫堇碱CE为25eV,m/z跃迁从326.05到178.10;原阿片碱CE为30eV,m/z跃迁从354.10到189.10;其他中间体用正扫描模式m/z从50到800去溶剂化线(DL)的温度保持在200℃,喷雾电压为1.8kV,雾化气体流速为3L/min。血根碱的质谱图如图6所示。
通过对途径中间体去甲乌药碱、牛心果碱、金黄紫堇碱、碎叶紫堇碱、刺罂粟碱、原阿片碱的滴度分析,牛心果碱、金黄紫堇碱、原阿片碱有一定程度的积累,从而推测出牛心果碱氧化酶BBE、碎叶紫堇碱合酶CFS、原阿片碱6-羟化酶P6H是血根碱合成途径中的限速步骤。因此,本发明分别在血根碱合成菌株中,过表达BBE、CFS、P6H三个途径限速步骤,得到菌株SAN027、SAN028、SAN029,如图4和表8所示。其中,只有提高碎叶紫堇碱合酶CFS的拷贝数对血根碱的产量有正向作用,提高牛心果碱氧化酶BBE、原阿片碱6-羟化酶P6H的拷贝数反而降低了血根碱的产量,推测CFS基因的表达可能存在一定限制,而BBE、P6H可能是由于底物亲和力不高等原因导致酶本身的催化活性较低。
由于血根碱生物合成途径中存在4个S-腺苷甲硫氨酸依赖的甲基转移酶6OMT、CNMT、4’OMT和TNMT,分别整合了ZWF1基因(GeneBank号为NP_014158)、GAPN对应的编码基因、SAM2基因(GeneBank号为NP_010790)和SAH1基因(GeneBank号为NP_010961)以加速S-腺苷甲硫氨酸的循环利用。此外,血根碱合成途径中还包含6个细胞色素P450酶CYP76AD5、NMCH、CFS、SPS、MSH、P6H,本发明过表达了酵母自身的抑制共调节的磷脂生物合成酶所需正调节因子INO2和具有III型跨膜结构域的完整内质网膜蛋白ICE2以增大酵母细胞的内质网,同时引入细胞色素b5(CYB5)和细胞色素P450还原酶(CPR)对应的基因序列以提高细胞色素P450酶的活性。最后,考虑到血根碱的抗菌作用可能对细胞造成毒性,影响产量的进一步提高。本发明引入来自烟草的多药及毒性复合物挤出3样蛋白NtJAT1、多药及毒性复合物挤出转运蛋白NtMATE2和来自拟南芥的多药及毒性复合物挤出转运蛋白AtDTX1对应的编码基因序列,以加速胞质内外的物质运输,降低胞内过高的血根碱浓度对宿主细胞造成损害。
序列表
<110> 浙江大学
<120> 一种发酵生产血根碱的酵母工程菌及其应用
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> 罂粟(Papaver somniferum)
<400> 1
atgggttctt tgccaaccaa caaccttgaa tctatttcct tatgctctca aaacccatta 60
gacccagacg aattcagaag acaaggtcac atgatcattg atttcttggc tgattactac 120
aagaacgttg aaaagtaccc agtcagatcc caagtcgaac caggctactt aaagaagaga 180
ttgccagagt ctgctccata caacccagaa tccatcgaaa ccatcttgga agatgtcaca 240
aacgatatca tcccaggttt gacccactgg caatctccaa attactttgc ttacttccca 300
tcttctggtt ctatcgccgg ttttttaggt gaaatgttgt ctaccggttt caatgttgtc 360
ggtttcaact ggatgtctag tccagctgcc actgaattgg aatctattgt tatgaactgg 420
ttgggtcaaa tgttgacttt gccaaaatct ttcttattct cttctgatgg ttcctccggt 480
ggtggtggtg tcttgcaagg tactacttgt gaagctatct tgtgtacttt gaccgctgct 540
agagacaaga tgctgaataa aattggtaga gaaaacatta acaagttggt tgtctacgct 600
tccgatcaaa ctcattgtgc tttgcaaaag gccgctcaaa ttgctggtat taaccctaaa 660
aatgttagag ctatcaagac ctctaaggct actaacttcg gtttgagccc gaactctttg 720
caatccgcca ttctagccga tattgaatcc ggattggttc ctttgttctt gtgcgctact 780
gtcggtacca cttcaagcac tgctgtcgat ccaatcggtc cattgtgtgc tgttgccaag 840
ttgtacggta tctgggttca catagatgct gcttatgccg gttccgcttg tatctgtcca 900
gaattccgtc actttattga cggtgttgaa gacgctgact ctttctcctt gaacgctcac 960
aagtggttct tcaccacctt ggactgttgt tgtttgtggg tcaaggattc cgactccttg 1020
gtcaaagctt tatctacctc tccagaattt ttgaagaaca aggctaccga atccaagcaa 1080
gttattgact acaaggactg gcaaatcgct ttgtcacgta gattcagatc catgaagcta 1140
tggttagttt tgagatctta cggtgttgct aacttgagaa ccttcttgcg ttctcatgtc 1200
aagatggcta agcatttcca aggtttgatt ggtatggaca acagattcga aatcgttgtc 1260
ccaagaactt tcgctatggt ttgtttcaga ttaaagccaa ctgccatctt caagcaaaag 1320
attgttgaca acgactacat tgaagaccaa acgaacgaag tcaacgtcaa attgttggaa 1380
tctgttaacg cttctggtaa gatctatatg actcacgctg tcgttggtgg tgtttacatg 1440
atcagatttg ccgtaggtgc caccctcact gaagaaagac acgttactgg tgcttggaag 1500
gttgttcaag aacacactga tgcaatcttg ggtgcttga 1539
<210> 2
<211> 489
<212> DNA
<213> 黄连(Coptis japonica)
<400> 2
atggaagaaa ccgttatgtt gtatcacgaa ttggaagttg ctgcttctgc tgatgaagtc 60
tggtctgtcg aaggttctcc agaattaggt ttgcacttgc ctgacttgtt gccagctggt 120
atctttgcta aattcgaaat taccggtgac ggtggtgaag gttccatctt ggacatgact 180
ttcccaccag gtcaattccc acatcactac cgtgaaaagt tcgttttctt tgatcataag 240
aacagataca agttggttga acaaatcgat ggtgatttct tcgatttagg cgtgacttac 300
tacatggaca ctatcagagt tgttgccact ggtccagact cttgtgtcat caagtcaacc 360
actgaatacc acgttaagcc agaattcgct aagattgtca aaccattgat tgacaccgtc 420
ccactagcca ttatgtccga agctattgcc aaggttgtct tggaaaacaa gcacaagtcc 480
tctgagtga 489
<210> 3
<211> 1611
<212> DNA
<213> 博落回(Macleaya cordata)
<400> 3
atggacacca aaatcagaaa cttgtcttct tctttgttca tcttcattag tgtcttaacc 60
tgcgctttgg gtgacgactt gttgtcttgt ttgacttctc acggtgttca caactacacc 120
accccatctt ctgattctaa ctctgactac ttgagattct tgcatgtttc cattcaaaac 180
ccattattcg aaaatccagc tactccaaag ccagctgcca ttgttatgcc aagaaccaag 240
gaagaactgg cgtccactgt tagatgttgt accagaggtt cctggactgt ccgtttgaga 300
tctggtggtc actcttacga aggtttgtcc tacacagctg acactccgtt cgttttgatt 360
gacttgatga acatgaacag aatttctatc gatgtcgaat ccgaaaccgc ttgggttgaa 420
tctggtgcca cgttgggtga attgtattac gccatcactg aatctactga ctctttcggt 480
ttcactgctg gttggtgtcc aaccgtcggt tccggtggtc acatctctgg tggaggtttc 540
ggtatgatgt ctagaaagta cggtttagcc gctgacaacg ttgttgacgc tatcctaata 600
gatggtaatg gtgtcatctt ggacagaaac tccatgggtg aagatgtttt ttgggctatt 660
cgtggtggtg gtggcggtgt ctggggtgct atctacgctt ggaagatcaa gttgttgcca 720
gttccaaaga aggttactgt cttcagacta atgaagcacg ttaagattga agaagcttcc 780
aacttgttgc acaagtggca attcgtcgcc gatgaattag acgatgattt cactttgtca 840
gtcttgggag gtgctgatga aaacgaagtt tggttaatgt tccttggttt gtacttgggt 900
ccaaagaccg ttgctaaatc taccatcgac cctaaattcc cagaattggg tttgattgag 960
gaagaattct ctgaaatgtc ctggggtgaa tcctttgctt acctagccgg tttgaaaact 1020
gtcggtgaat tgaacaaccg ttttttgaag ttcgatgaca gagctttcaa gactaaggtc 1080
gacttcgcta aggaaccaat cccattgaag gttatcaacg gtgctttgga aattttatcc 1140
aaggaaccaa gaggtttcgt cgccctcaat ggtctgggtg gtatgatgtc aaaagtttct 1200
tccgatttta ctccattccc acaccgttcc ggtaccagat tgatggttga atacatcatt 1260
gcttggaaca aggacgaaga ctccaagtct gaagaattca ccaactggtt gcaaagattc 1320
tacgattata tggaaccttt tgtctctaag aacccaagag ttggttacgt taaccacatt 1380
gacttggatt tgggtggtat tgactggaag aacaagacct ccacaagcaa cgctattgaa 1440
atcgccagaa cttggggtga aaaatacttc ttgactaact acgaaagatt ggtcaaggct 1500
aagactctta tcgatccaaa gaacgttttc aaccatccac aatctattcc accaatggaa 1560
tttgatttgg aacaaaactg gggtgtcaat gtcgaaggta tcgttcaatg a 1611
<210> 4
<211> 1596
<212> DNA
<213> 白屈菜(Chelidonium majus)
<400> 4
atggacgctc aaatccgtaa attgttgtca tatttcttca tctttctatc tgtcttgtct 60
tgtgctttag gtgacgatga cttattatcc tgtttgactt ctcacggtgt tcacaactac 120
acctctccaa cctccgactc caactccgac tacttaagat ttttgcatgt ctccatccaa 180
aacccattgt tcgaaacccc tgctactcca aagccagcta ccattgtttt gccacgtaca 240
aaagaagaat tggcttctac tgttagatgt tgtacttccg gttcctggac tatcagattg 300
agatctggtg gtcactctta cgaaggttta agttacactg ctgacactcc atttgttttg 360
attgacttga tgaacatgaa cagaatctct atagatgttg actctgaaac tgcttgggtt 420
gaatctggtg ccactttggg tgaattatac tacgctatct ccgttgccac cgattctttc 480
ggtttcactg ccggttggtg cccaactgtg ggctctggtg gtcatatttc tggagggggt 540
tacggtatga tgtctagaag atatggtcta gctgctgaca acgttgtcga tgccattttg 600
attgattcca atggtgctat cttggacaga gccaccatgg gtgaagacgt tttctgggct 660
attagaggtg gtggtggtgg tgtctggggt gctatctacg cctggaagat caagttgttg 720
ccagtaccaa agcaagttac cgtcttcaga ttgatgaaga atgtcaagat tgaagaagct 780
tctaacttgt tgcacaagtg gcaattcgtc gcagctgaat tggacgacca cttcaccctc 840
tccgtcctgg gtggtgccgc tggtaacgaa gtttggttaa tcttcttggg tttgtacttg 900
ggtccaaaga ccgttgctaa atctaccatc gaccaaaagt tcccagaatt gggtttgatc 960
gaagaagaaa tcttggaaat gtcctggggt gaatccatgg cttacttggc tgatttgaag 1020
actgtcgctg aattgaacaa cagatttttg aaattcgatg atagagcttt caagaccaag 1080
gttgatttcg ctaaggaacc agtcccattg ccagtcatca acggtgttct acaaatgttg 1140
ttgaaggaac caagaggttt cattgctttg aacggtttgg gtggtatgat gtccaagtct 1200
agttctgatt ttaccccttt cccacacaga aagggcaccc gtttaatgtt ggaatacatt 1260
attgcttgga acaaggatga aagttctaag tctgacgaat tcactcactg gttgcaaaga 1320
ttctacgact tcatggaacc atacgtctcc aagaacccaa gagttggtta cgttaaccat 1380
attgatctcg atttgggtgg tatcaactgg aacaacaaga cttcttcgtc taatgccatt 1440
gaaattgcca gaacctgggg tgaaagatat ttcttgtcta actacgaacg tttggttaag 1500
gctaagactt tgattgaccc aaagaacgtc ttcaaccacc cacaatccat tccaccaatg 1560
ggtttcgact tggaacaaaa atggtcatac atgtga 1596
<210> 5
<211> 1554
<212> DNA
<213> 血根草(Sanguinaria canadensis)
<400> 5
atgatcaagc accataaata ctttgctgat gtcttgtaca ttgatttctt cactatggac 60
ccaaaaatca gaaacttgtt cagttccttg ttcatttttt tgtctgcctt gacttgtgct 120
ttgggtgacg atttgttatc ttgtttgacc tcccacggtg ttcacaacta caccacccca 180
tcttctgatt ctgacagcga ctatttgcgt ctattgcacg tttccatcca aaacccactc 240
ttcttgaatc ctaccacacc aaagccagtc gccatcgtta tgccaagaac caaggaagaa 300
ttggcttcca ctgtcagatg ttgtactaga ggttcctgga ctgttagatt gagatccggt 360
ggtcactcct acgaaggtct ctcttacact gctgacactc ctttcgtttt ggtcgacttg 420
atgaacttga acagaatctc catcgacatt gaatctgaaa ctgcttgggt tgagtccggt 480
gctaccttgg gtgaattgta cttcgccatt gctgaatcca ccaactcttt cggtttcacc 540
gctggttggt gcccaacagt cggttctggc ggccatattt ctggtggtgg tttcggtatg 600
atgtccagaa agtacggttt ggcctctgac aacgttgtcg atgctatttt gattgactct 660
aacggtgcta ttttggacag aaactccatg ggtgaagatg ttttctgggc tatccgtggt 720
ggtggtggtg gagtctgggg tgccatctac gcttggaaaa tcaagttgtt gccagttcca 780
aagaaggtta ccgtcttcag gttgatgaag aacattaaga ttgaagaagc ttctttctta 840
ctgcataagt ggcaattcgt tgccgatgaa ttggatgacg acttcacttt gtctatcttg 900
ggtggtgctg acgaaaacga agtctggtta atctttctgg gtctatactt gggtccaaag 960
accgttgcca agtctaccat ggaccgtaag ttcccagaat tgggtttgat cgaagaagaa 1020
gttttagaaa tgtcttgggg tgaatctttt gcttacctag ccggtttaaa gactgtcgat 1080
gaattgaata acagattttt gaaattcgat gacagagctt tcaagactaa ggtcgatttc 1140
gctaacgaac caattccatt gaaggttatt aacggtgctt tagaaatctt gtccaaggaa 1200
ccaagaggtt tcgtcgcatt gaatgggttg ggtggtatga tgcacaaaat ctcatctgat 1260
ttcactccat tcccacacag aaacggtacc agattaatgg ttgaatacat cattgcttgg 1320
aacaaggatg aagaatctaa gaacgacgaa ttcactgact ggttggaaag attctacgac 1380
tacatggaac cattcgtctc caaggaccca agagttggtt acgttaacca catcgatttg 1440
gacttgggtg gtatcgactg gaacaacaag acttcttcta gaaacgctat agaaattgct 1500
agaacctggg gtgaaagata ctttctctcc aactatgaac gtttggttgt ttga 1554
<210> 6
<211> 39
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 6
ggttccggtt ctttgggtag aggtagacgt tctaagttg 39
<210> 7
<211> 75
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 7
ctttcactac gtcaatctat aagatttttc aagccagcca caagaacttt gtgtagctct 60
agatatctgc ttcag 75
<210> 8
<211> 48
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 8
aaggccatct cattgcaaag accgttgggt ctagataagg acgttttg 48
<210> 9
<211> 1092
<212> DNA
<213> 蓟罂栗(Argemone mexicana)
<400> 9
atggacaact gtaaggttga aggcatgaaa aaagaaaccc gtgctgctga agaaatcttg 60
ggtagattat tgaagggtga aatcaaggat gaagaattga gaaagttgat taagttccaa 120
ttccaaaaga gattggaatg gggttacaag tctacacatc accaacaatt gtctttcaac 180
ttggacttca tcaagtcctt gaagaacatg gacatgtccg gtgaaatcga aactatgaac 240
aaggaaacct acgaactgcc aactgaattc ttggaagctg ctttcggtaa gaccatgaag 300
caatctggtt gttactttaa gcacgaatct actaccattg acgaagccga agaagcctct 360
cacgaactat attgtgaaag agcccaaatc aaagacggtc aaaccgtctt ggacatcggt 420
tgcggtcaag gtggtctagt tttatacatt gctgaaaagt ataagaagtg tcacgttact 480
ggtttgacta actccaaggc tcaagtcaac tacatcgaaa tgcaagctaa gaagctcgga 540
ttatccaacg ttgatgtcat tttggctgat gtcaccaagt acgattccga caagacttac 600
gacagattgt tgatgattga agccattgaa cacatgaaga atattcaatt gttcatgaag 660
aaattaagta cctggatgac taaagattct ttgttgtttg tcgatcacat ctgtcacaag 720
accttttccc acccattcga agctgtcgat gaggacgact ggtactctgg tttcattttc 780
ccaccaggtt gtgttaccat cttgtcttct gacgctttgt tgtacttcca agatgacgtt 840
agcgtcatcg accactgggt tgttaatggt atgcacatgg ctcgttccgt tgatgcttgg 900
agaaagaagt tggataagaa catggaagct gccaaggaaa ttattttgtt gcctggtctg 960
ggtggttcac atgaggccgt caaccaaatc atcactcata tcagaacttt ctgtatgggt 1020
ggttacgaac aattctctta caacaacggt gaagaatgga tggtttctca aattttattc 1080
aagaagaaat ga 1092
<210> 10
<211> 897
<212> DNA
<213> 虞美人(Papaver rhoeas)
<400> 10
atgggtagta ttgacgaagt caagaaggaa tctgctggtg aaaccctcgg tcgtttgttg 60
aagggtgaaa tcaaggatga agaattaaag aagttgatta aatctcaatt cgaaaagaga 120
ttgcaatggg gttacaaatc atctcaccaa gaacaattgt ctttcaactt ggctttcatc 180
aagtctttga agaagatgga catgtctggt gaaattgaaa agatgaacaa ggaaacttac 240
gaattgccat ctgaattctt ggaagccgtt ttcggtaaga ctgccaagca atccatgtgt 300
tacttcaagc acaagtccgc taccattgat gaagctgaag aggctgccca cgaattgtac 360
tgtgaaagag cccaaatcaa ggacggtcaa actgttttgg acattggttg tggtcaaggt 420
ggtttagtct tgttcattgc tcaaaagtac aagaactgtc acgttactgg tttgactaat 480
tccaaggctc aagttaacta cttgttggaa caagctaaga aattgggctt gaccaacgtc 540
gatgctatcc ttgccgatgt cactcaatac gaatccgaca agacctacga cagattgttg 600
atgattgaag ccatcgaaca catgaagaac ttgcaattat tcatgaaaaa attgtccacc 660
tggatgactg aagactcttt actatttgtt gaccacatct gtcataagac ctttgctcac 720
ttctttgaag ctgtcgacga agatgactgg tactccggtt tcatcttccc accaggttgc 780
gtcacaatct tggctgctaa ctctttgtta tatttccaag atgatgtttc tgttgttgat 840
cattgggttg tcaacggtat gcacatggct agatccgttg acatctggag aaagtga 897
<210> 11
<211> 1077
<212> DNA
<213> 大红罂粟(Papaver bracteatum)
<400> 11
atgggttcca ttgacgaagt taagaaagaa tctgctggtg aaactttggg tagattgttg 60
aagggtgaaa tcaaggacga agaattaaag aaattaatca agttccaatt cgaaaagaga 120
ctacaatggg gttacaagtc ctctcaccaa gaacaattgt ctttcaactt ggatttcatt 180
aagtctttga agaagatgga aatgtcaggt gaaattgaaa ccatgaacaa ggaaacctac 240
gaattgccat ctgaattctt ggaagctgtt ttcggtaaga ccgtcaagca atccatgtgt 300
tactttaagc acgaatccgc taccattgat gaagctgaag aagctgctca cgaattgtac 360
tgtgaaagag cccaaatcaa agatggtcaa actgtcttgg acattggttg tggtcaaggt 420
ggtttggttt tgtatattgc cagaaagtac aagaaatgtc acgttactgg tttgactaac 480
tccaaggccc aagttaacta cttgttgaag caagctgaaa agttgggctt aaccaacgtt 540
gatgctattt tggctgatgt cactcaatac gagtctgaca aaacatacga tagattattg 600
atgattgaag ccatcgaaca catgaagaac ttgcaattgt tcatgaaaaa gttgtccacc 660
tggatgactg aagaaagcct tctattcgtt gatcatgtct gtcacaagac ttttgcacat 720
ttcttcgaag ccgtcgatga agacgactgg tactctggtt tcatcttccc accaggttgc 780
gctactatcc tagccgctaa ctctctgtta tacttccaag atgacgtctc tgttgttgac 840
cactgggttg tcaatggtat gcacatggct cgttctgtcg acatctggag aaaggctttg 900
gacaagaaca tggaagccgc taaggaaatc ttgttgccag gtttgggtgg ttctcatgaa 960
gctgtcaacg gtgttgttac ccacatcaga accttctgta tgggtggtta cgaacaattt 1020
tccatgaacg acggtgacga atggatggtt gctcaattgt tgttcaagaa gaagtga 1077
<210> 12
<211> 1632
<212> DNA
<213> 变根紫堇(Corydalis linstowiana)
<400> 12
atggccatct tcccattatc cttgttgatt ctaaagttgc catctccaaa gcacatggac 60
tcttctttaa tccaatggta cactgcttcc attgctgcct tattggctct tgcatttttg 120
tacaagctct ggtctaagcc aagaaccttc aagaacggta agagcacctt gcaagcccca 180
gttgccgctg gtgcttggcc agtcttaggt catttgcact tgttctccgg cggtgaatta 240
cctcacaaga tgttggctgt tatggctgac aagtacggtc cagctttcac catgaaattc 300
ggaacccaca gaactcttgt cgtctctgac actaagatcg ttaaggaatg tttcaccact 360
aacgatacct tatttgctaa cagaccatcc actactgctt tccacttaat gacctatgat 420
aacgaatctg tcactttcac tccatacggt cctttttggc gtgaattgag aaagatttct 480
actttaaaat tgttgagtaa taacagattg caagctatca aagatgttag agcttctgaa 540
gtcaacttgt gtttcagaac attatacaac caatggagaa tcaacaagtc tgaaactgtc 600
gaaactggtg accaccaagg tccaatcttg gttgacatga agaaatggtt cgaagaagtt 660
tctaacaacg ttgttatcag ggtcattatt ggtaagcaca acttcggttc caagatcgcc 720
agaggtgaag gtgaagcttt gcactacaag aaggttatgg atgaagtttt gagattagct 780
gctgtctcca tgttgtccga tgttgctcca ttgctgggtt ggtttgatca tttccaaggt 840
cacgcttctg ctatgaagag aaacgccaag gaattggaca tcttgttgtg ttcttggatg 900
gaagaacaca gaaagaaaag aacttccgac tccaacaatg gtgttgaaca agatttcatg 960
gacgccatgc tttctatcat ggaagaaaac aagttctctg gtcacgactc tgatacggtc 1020
atcaaggcta ccgttttggc catgattatg ggtggcactg acaccaccgc ggtgtctttg 1080
acctggattg tctctttgtt gatgaacaac cgtcatgttt tgaagcgtgc tagagaagag 1140
ttggacactc acgttggtaa ggacagacaa gttgatgact cagacttgaa gaacttagtt 1200
tacttgaacg ctattgttaa ggaaaccatg agattatacc cattgggtgc tctattggaa 1260
agagaaacca aagaagactg tgaagttggt ggtttccatg tcaaagctgg taccagattg 1320
ttggtctgga ttgatccaac tgaattcaag ccagaaagat tcttgactga taacgccaac 1380
attgatgtcg gtggtcaaca cttcgaattg ttgccattcg gtgccggtag aagagtttgc 1440
ccaggtgtct ccttcgcttt gcaattcatg catttggtct tggctcgttt gattcacggt 1500
tttgaattgg acaccccaat gaatgctaac gtcgacatga ccgaatccac cgaaggtcac 1560
gttaaccaca aggcttccgc tttggatctg ttgatcactc caagattgcg ttcaactttg 1620
tacgactact ga 1632
<210> 13
<211> 1620
<212> DNA
<213> 大红罂粟(Papaver bracteatum)
<400> 13
atggacttct cttctttgtt attattgctt ttgaacaact ggatctctgc ttactccatg 60
gctgctttgt tggccttggt tttggtctac aacttgagaa tgacctcgtc ttcttcttcc 120
aagaccaatt ctttgaaggg taagaagatc atcactagac ctccagccgt cactggtgct 180
tggccagttt tcggtcattt gcacttattt ggttctggtg aacacccaca cgaaatgttg 240
tctaaattag ccgaaaagta cggtccagct ttcaccatga agttcggtaa gcacactact 300
ttagttgtat ccgacaccag agttgtcaag gaatgtttca ccaccaacga taccttgttt 360
tccaacagac catccaccat cgcctttgac ttgatgactt atgctaccga ttctattgct 420
ttcacaccat actctccata ctggagagaa ttaagaaaga tttctacctt gaagctcctt 480
tcaaataacc gtttggaatc tatcaagcaa ttgagaactt ctgaagtctc tgtttgtttc 540
aaggaattgt acgatttgac ctctaaggaa aacgacaacg gcgctccagt cccaattgac 600
ttaaagagat ggttcgacga agtttctaac aacgttatca tgagagtcat cttcggtaag 660
caaaactttg gttccaaaat tgtctccggt gaagaccaag aagctgtcca ctacaagaaa 720
atcatggatg aattgtccag attgtcctct ttgactatgt tgtctgacat ggttccatct 780
ctgggttggt tagactactt caagggtgac ctaagagcta tgaagagaaa cggtaaggaa 840
ttgaactcca ttttgcaaaa gtggttggaa gaacacaagt ccaagaaatc ctccgatgac 900
agacaagatt tcatggatgt tatgttgtcc atctccaagg aaactcaatt atacggtcac 960
gatcaagaca ctttcatcaa ggctacttgt ttggctatga ttatgggtgg cactaactct 1020
accgaagttg ctttgacctg gattctttct ttgttgatga acaacagata cgcattgcat 1080
aaggccaggg aagaaattga tttgttggtt ggtaaggacc gtcaagttga agactctgat 1140
gttaagaacc taacttacat gaatgctatc attaaagaaa ccatgagatt atacccattg 1200
ggtttcttgt tggaacgtga cagcaaagaa gattgtgaag tcggtggttt caacattgaa 1260
ggtggtactc gtttattgat caacgtatgg aagctgcaaa gagatccaaa cgtctggact 1320
gacccaatgg aattcaagcc agaaagattc ttgactgaaa acgctgatat cgatgtcggt 1380
ggtcaacact tcgaattgtt gccattcggt gccggtagaa gagtctgtcc aggtgtctct 1440
ttcgctttgc aattcatgca cttggttttg gctagattga ttcacggtta cgacatggag 1500
actttgaatg gtgaagatgt tgacttgtcc gtttcttctg gtggtcacgt taacatcaag 1560
agtactcctc tcgaattgat tttgacccca agattgcatc cagaattgta tgactgctga 1620
<210> 14
<211> 771
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 14
atggatttca caaaaccaga aactgtttta aatctacaaa atattagaga tgaattagtt 60
agaatggagg attcgatcat cttcaaattt attgagaggt cgcatttcgc cacatgtcct 120
tcagtttatg aggcaaacca tccaggttta gaaattccga attttaaagg atctttcttg 180
gattgggctc tttcaaatct tgaaattgcg cattctcgca tcagaagatt cgaatcacct 240
gatgaaactc ccttctttcc tgacaagatt cagaaatcat tcttaccgag cattaactac 300
ccacaaattt tggcgcctta tgccccagaa gttaattaca atgataaaat aaaaaaagtt 360
tatattgaaa agattatacc attaatttcg aaaagagatg gtgatgataa gaataacttc 420
tcttctgttg ccactagaga tatagaatgt ttgcaaagct tgagtaggag aatccacttt 480
ggcaagtttg ttgctgaagc caagttccaa tcggatatcc cgctatacac aaagctgatc 540
aaaagtaaag atgtcgaggg gataatgaag aatatcacca attctgccgt tgaagaaaag 600
attctagaaa gattaactaa gaaggctgaa gtctatggtg tggaccctac caacgagtca 660
ggtgaaagaa ggattactcc agaatatttg gtaaaaattt ataaggaaat tgttatacct 720
atcactaagg aagttgaggt ggaatacttg ctaagaaggt tggaagagta a 771
<210> 15
<211> 1113
<212> DNA
<213> 酿酒酵母(Saccharomyces cerevisiae)
<400> 15
atgagtgaat ctccaatgtt cgctgccaac ggcatgccaa aggtaaatca aggtgctgaa 60
gaagatgtca gaattttagg ttacgaccca ttagcttctc cagctctcct tcaagtgcaa 120
atcccagcca caccaacttc tttggaaact gccaagagag gtagaagaga agctatagat 180
attattaccg gtaaagacga cagagttctt gtcattgtcg gtccttgttc catccatgat 240
ctagaagccg ctcaagaata cgctttgaga ttaaagaaat tgtcagatga attaaaaggt 300
gatttatcca tcattatgag agcatacttg gagaagccaa gaacaaccgt cggctggaaa 360
ggtctaatta atgaccctga tgttaacaac actttcaaca tcaacaaggg tttgcaatcc 420
gctagacaat tgtttgtcaa cttgacaaat atcggtttgc caattggttc tgaaatgctt 480
gataccattt ctcctaaata cttggctgat ttggtatcct tcggtgccat tggtgccaga 540
accaccgaat ctcaactgca cagagaattg gcctccggtt tgtctttccc agttggtttc 600
aagaacggta ccgatggtac cttaaatgtt gctgtggatg cttgtcaagc cgctgctcat 660
tctcaccatt tcatgggtgt tactttacat ggtgttgctg ctatcaccac tactaagggt 720
aacgaacact gcttcgttat tctaagaggt ggtaaaaagg gtaccaacta cgacgctaag 780
tccgttgcag aagctaaggc tcaattgcct gccggttcca acggtctaat gattgactac 840
tctcacggta actccaataa ggatttcaga aaccaaccaa aggtcaatga cgttgtttgt 900
gagcaaatcg ctaacggtga aaacgccatt accggtgtca tgattgaatc aaacatcaac 960
gaaggtaacc aaggcatccc agccgaaggt aaagccggct tgaaatatgg tgtttccatc 1020
actgatgctt gtataggttg ggaaactact gaagacgtct tgaggaaatt ggctgctgct 1080
gtcagacaaa gaagagaagt taacaagaaa tag 1113
<210> 16
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
attctactaa aaacacatca gtagtcacag aagtcacagt tcaggctgcg caactgtt 58
<210> 17
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
attttttcat taatacacct gtgttagtta tgattgccag agtgagctga taccgctcg 59
<210> 18
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ttacgtgtca tttattatgg gttcagaaat tatgtgttaa gagcgacctc atgctatac 59
<210> 19
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cacacaattt tggtggcgtt gaaattgatg ccggaatttg cttcgagcgt cccaaaacc 59
<210> 20
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ttacgtgtca tttattatgg gttcagaaat tatgtgttaa gagcgacctc atgctatac 59
<210> 21
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aaatcgacat gttaatgatc ttacgacaga gtagtttatg cttcgagcgt cccaaaacc 59
<210> 22
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tgaaaattgt gccggatatt caagactaag agatgtacaa gagcgacctc atgctatac 59
<210> 23
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tatccgggta acactcatcg tccggcctcc gccccctttt cttcgagcgt cccaaaacc 59
<210> 24
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gagagaaatt aaacttggtt ggggttaatt atttgatggg gagcgacctc atgctatac 59
<210> 25
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cactattgat aaaggttttg tagaatattt attatcgata cttcgagcgt cccaaaacc 59
<210> 26
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gactagtatc atccgtcaag aagaacaaga acaagaacaa gagcgacctc atgctatac 59
<210> 27
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tacaggcaat gagcgaaagc gactgaagcc gagagatgtg cttcgagcgt cccaaaac 58
<210> 28
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tgtacgctat acatttacgt gctgagctcc taggaaagct gagcgacctc atgctatac 59
<210> 29
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
cctctatgtg acgctgtgta ttctttgttg tagttatgct cttcgagcgt cccaaaacc 59
<210> 30
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gttttcttat ttctttcttt ttaaaaaact ttcttaatat gagcgacctc atgctatac 59
<210> 31
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gaattgagaa aaaaagtgta tatcattaca ttactttaca cttcgagcgt cccaaaacc 59
<210> 32
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tgtacggact attgctgtct tctcgtggta aatgcgtgtt gagcgacctc atgctatac 59
<210> 33
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ccgaaaagac aacttaacaa atcggcaaca cttttatggg cttcgagcgt cccaaaacc 59
<210> 34
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tttaacgtca aggagaaaaa accccggatc catgggtgat gttaatgata acttgt 56
<210> 35
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ctttaacgtc aaggagaaaa aaccccggat ccatgggtaa cgacttgtta tcttg 55
<210> 36
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
tttaacgtca aggagaaaaa accccggatc catggaggat gacttgccat cttgtttg 58
<210> 37
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tttaacgtca aggagaaaaa accccggatc catgggtgac gacttgttgt cttgtt 56
<210> 38
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
tttaacgtca aggagaaaaa accccggatc catgggtgac gatgacttat tatc 54
<210> 39
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tttaacgtca aggagaaaaa accccggatc catgggtgac gatgacttat tatc 54
<210> 40
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10
Claims (7)
1.一种发酵生产血根碱的酵母工程菌,其特征在于,以酵母作为出发菌株,通过基因敲除和导入基因构建获得所述酵母工程菌,其中,基因敲除的基因为:酵母自身的氧化还原酶ARI1、ADH6、AAD3、HFD1、GRE2、YPR1和YDR541C的编码基因;导入基因为:3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO7G141S、3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO4K229L_Q116K、酪氨酸脱羧酶编码基因突变体TYDCY350F、酪氨酸羟化酶编码基因CYP76AD5、多巴脱羧酶编码基因DODC、N端截短35个氨基酸的去甲乌药碱合酶编码基因NCS_N35、去甲基乌药碱6-O-甲基转移酶编码基因6OMT、乌药碱N-甲基转移酶编码基因CNMT、N-甲基去甲乌药碱羟化酶编码基因NMCH、3’羟基-N-甲基乌药碱4’-O-甲基酶编码基因4’OMT、牛心果碱氧化酶编码基因BBE、碎叶紫堇碱合酶编码基因CFS、刺罂粟碱合酶编码基因SPS、四氢原小檗碱-N-甲基转移酶编码基因TNMT、N-甲基刺罂粟碱14-羟化酶编码基因MSH、细胞色素P450还原酶编码基因CPR1、细胞色素b5编码基因CYB5、多药及毒性复合物挤出转运蛋白1编码基因DTX1、多药及毒性复合物挤出3样蛋白编码基因JAT1、多药及毒性复合物挤出转运蛋白2编码基因、原阿片碱6-羟化酶编码基因P6H和二氢苯并菲啶氧化酶编码基因DBOX;氧化还原酶ARI1编码基因的GeneBank号为NP_011358,氧化还原酶ADH6编码基因的GeneBank号为NP_014051,氧化还原酶AAD3编码基因的GeneBank号为NP_010032,氧化还原酶HFD1编码基因的GeneBank号为NP_013828,氧化还原酶GRE2编码基因的GeneBank号为NP_014490,氧化还原酶YPR1编码基因的GeneBank号为NP_010656,氧化还原酶YDR541C编码基因的GeneBank号为NP_010830;3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO7G141S的核苷酸序列如SEQ ID NO.14所示,3-脱氧-D-阿拉伯-庚酮糖酸酯-7-磷酸合酶编码基因突变体ARO4K229L_Q116K的核苷酸序列如SEQ ID NO.15所示,所述酪氨酸脱羧酶编码基因突变体TYDCY350F的核苷酸序列如SEQ ID NO.1所示,酪氨酸羟化酶编码基因CYP76AD5的GeneBank号为AJD87473,多巴脱羧酶编码基因DODC的GeneBank号为AKH61506,N端截短35个氨基酸的去甲乌药碱合酶编码基因NCS_N35的核苷酸序列如SEQ ID NO.2所示,去甲基乌药碱6-O-甲基转移酶编码基因6OMT的GeneBank号为XP_026389064,乌药碱N-甲基转移酶编码基因CNMT的GeneBank号为Q7XB08,N-甲基去甲乌药碱羟化酶编码基因NMCH的GeneBank号为O64900,3’羟基-N-甲基乌药碱4’-O-甲基酶编码基因4’OMT的GeneBank号为XP_026440860;所述牛心果碱氧化酶编码基因BBE为改造自罂粟(Papaver somniferum)的BBE基因PsBBE、改造自花菱草(Eschscholzia californica)的BBE基因EcBBE、改造自黄连(Coptis japonica)的BBE基因CjBBE、改造自博落回(Macleaya cordata)的BBE基因McBBE、改造自白屈菜(Chelidonium majus)的BBE基因CmBBE和改造自血根草(Sanguinaria canadensis)的BBE基因ScBBE,其中,PsBBE的GeneBank号为XP_026421833,EcBBE的GeneBank号为P30986,CjBBE的GeneBank号为BAM44344,McBBE的核苷酸序列如SEQ ID NO.3所示,CmBBE的核苷酸序列如SEQ ID NO.4所示,ScBBE核苷酸序列如SEQ ID NO.5所示;所述碎叶紫堇碱合酶编码基因CFS的GeneBank号为B5UAQ8、刺罂粟碱合酶编码基因SPS的GeneBank号为EU882969;所述四氢原小檗碱-N-甲基转移酶编码基因TNMT为改造自罂粟(Papaver somniferum)的TNMT基因PsTNMT、改造自花菱草(Eschscholzia californica)的TNMT基因EcTNMT、改造自蓟罂栗(Argemone mexicana)的TNMT基因AmTNMT、改造自虞美人(Papaver rhoeas)的TNMT基因PrTNMT和改造自大红罂粟(Papaver bracteatum)的TNMT基因PbTNMT,其中,PsTNMT的GeneBank号为Q108P1,EcTNMT的GeneBank号为C3SBS8,AmTNMT的核苷酸序列如SEQ ID NO.9所示,PrTNMT的核苷酸序列如SEQ ID NO.10所示,PbTNMT的核苷酸序列如SEQ ID NO.11所示;N-甲基刺罂粟碱14-羟化酶编码基因MSH的GeneBank号为L7X3S1;细胞色素P450还原酶编码基因CPR1的GeneBank号为NP_001190823,细胞色素b5编码基因CYB5的GeneBank号为KP411012;多药及毒性复合物挤出转运蛋白1编码基因DTX1的GeneBank号为Q9SIA5,多药及毒性复合物挤出3样蛋白JAT1编码基因的GeneBank号为NP_001313041,多药及毒性复合物挤出转运蛋白2编码基因的GeneBank号为7DQK_B;所述原阿片碱6-羟化酶编码基因P6H为改造自罂粟(Papaver somniferum)的P6H基因PsP6H、改造自花菱草(Eschscholzia californica)的P6H基因EcP6H、改造自博落回(Macleaya cordata)的P6H基因McP6H、改造自变根紫堇(Corydalis linstowiana)的P6H基因ClP6H和改造自大红罂粟(Papaver bracteatum)的P6H基因PbP6H,其中,PsP6H的GeneBank号为L7X0L7,EcP6H的GeneBank号为F2Z9C1,McP6H的GeneBank号为OVA18597,ClP6H的核苷酸序列如SEQ ID NO.12所示,PbP6H的核苷酸序列如SEQ ID NO.13所示;二氢苯并菲啶氧化酶编码基因DBOX的GeneBank号为XP_026380178。
2.根据权利要求1所述发酵生产血根碱的酵母工程菌,其特征在于,牛心果碱氧化酶编码基因BBE为N端23个或24个氨基酸截短的牛心果碱氧化酶截短体编码基因。
3. 根据权利要求2所述发酵生产血根碱的酵母工程菌,其特征在于,牛心果碱氧化酶截短体编码基因编码的牛心果碱氧化酶截短体还在C端融合有过氧化物酶体信号肽ePTS1、N端融合有线粒体信号肽MLS和液泡信号肽PRC1;过氧化物酶体信号肽ePTS1的核苷酸序列如SEQ ID NO.6所示,N端融合线粒体信号肽MLS的核苷酸序列如SEQ ID NO.7所示,N端融合液泡信号肽PRC1的核苷酸序列如SEQ ID NO.8所示。
4.根据权利要求1所述发酵生产血根碱的酵母工程菌,其特征在于,所述出发菌株为酿酒酵母BY4741、酿酒酵母BY4742、酿酒酵母CEN.PK2-1C、酿酒酵母W303-1A或毕赤酵母GS115。
5.根据权利要求1所述发酵生产血根碱的酵母工程菌,其特征在于,所述酵母工程菌构建时,先构建共有中间体牛心果碱的底盘菌株,所述共有中间体牛心果碱的底盘菌株的构建方法步骤:
将酪氨酸羟化酶编码基因CYP76AD5、多巴脱羧酶编码基因DODC、N端截短35个氨基酸的去甲乌药碱合酶编码基因NCS_N35、去甲基乌药碱6-O-甲基转移酶编码基因6OMT、乌药碱N-甲基转移酶编码基因CNMT、N-甲基去甲乌药碱羟化酶编码基因NMCH和3’羟基-N-甲基乌药碱4’-O-甲基酶编码基因4’OMT整合到酿酒酵母BY4741基因组上的对应位置,并将酿酒酵母BY4741细胞中7个氧化还原酶ARI1、AAD3、ADH6、HFD1、GRE2、YPR1、YDR541C作为敲除位点,构建得到生产共有中间体牛心果碱的底盘菌株。
6.根据权利要求1-5任一项所述的发酵生产血根碱的酵母工程菌在制备血根碱中的应用。
7.一种制备血根碱的方法,其特征在于,发酵培养权利要求1-5任一项所述的发酵生产血根碱的酵母工程菌,并提取获得血根碱。
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