CN118064287A - 一种高产淫羊藿素的重组工程菌及其制备方法和应用 - Google Patents
一种高产淫羊藿素的重组工程菌及其制备方法和应用 Download PDFInfo
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- CN118064287A CN118064287A CN202410136479.5A CN202410136479A CN118064287A CN 118064287 A CN118064287 A CN 118064287A CN 202410136479 A CN202410136479 A CN 202410136479A CN 118064287 A CN118064287 A CN 118064287A
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- icaritin
- yarrowia lipolytica
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Abstract
本发明公开了一种高产淫羊藿素的重组工程菌及其制备方法和应用,属于生物工程技术领域。本发明以能合成淫羊藿素的前体山柰酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶tEsPT*和氧甲基转移酶OsOMTm,成功在解脂耶氏酵母中构建了合成淫羊藿素的代谢通路。本发明利用多种代谢工程手段进一步地提高淫羊藿素的产量,目前最高可达1212mg/L。本发明实现了淫羊藿素在解脂耶氏酵母中的首次合成,构建的解脂耶氏酵母工程菌的淫羊藿素产量是迄今为止报道的通过微生物发酵生产的最高水平,为实现解脂耶氏酵母商业化生产淫羊藿素奠定了重要基础,具有重大的应用价值。
Description
技术领域
本发明涉及生物工程技术领域,特别是涉及一种高产淫羊藿素的重组工程菌及其制备方法和应用。
背景技术
淫羊藿素(Icaritin)是从传统中草药淫羊藿中提取的一种前炔类黄酮化合物。据报道,淫羊藿素对人类健康具有许多积极的药理和生物活性,包括神经保护、肺部保护、抗炎,或用于治疗各种病症,其中包括骨质疏松症、心血管疾病和类风湿性关节炎等。此外,淫羊藿素还能抑制肝细胞癌(HCC)的发生以及肿瘤的恶性生长,目前已被批准作为治疗肝癌的药物出售。
淫羊藿素可以从一些草本植物中提取,尤其是淫羊藿。然而,由于植物中的淫羊藿素浓度极低,要确保其纯度需要使用价格较为昂贵的色谱提取技术,因此不适于大规模生产。此外,过量采摘淫羊藿可能会对生态环境造成破坏,影响生态平衡。化学合成法生产淫羊藿素也存在一定弊端。具体而言,该过程需要添加强酸、强碱和有毒的有机试剂,还需要高温条件和大量水资源的浪费——所有这些都与当前政府所提倡的绿色环保背道而驰。由于合成生物学和代谢工程具有可持续、环境友好和成本效益高的优势,微生物生产已成为生产高附加值植物衍生化合物的可行替代方法。
作为一种非常规酵母,解脂耶氏酵母(Yarrowia lipolytica)是“一般认为安全”(GRAS)的食品级微生物。与大肠杆菌等原核生物相比,解脂耶氏酵母具有细胞器,能够翻译结构较为复杂的酶,且与酿酒酵母相比不会产生Crabtree效应。解脂耶氏酵母因其天然的细胞内疏水环境、富含脂质和过氧化物酶体的存在以及乙酰辅酶A、丙二酰辅酶A、NADPH、ATP和其他重要分子的较高通量而成为萜类化合物以及黄酮类化合物生物合成的极具吸引力的底盘。已成为生产高价值化学品和天然产品的新型有吸引力的微生物底盘。然而,目前利用微生物发酵法合成淫羊藿素的研究还处于起步阶段,离工业化生产尚且存在一定距离,因此以微生物为底盘细胞合成淫羊藿素还有很大发展潜力。
发明内容
本发明的目的是提供一种高产淫羊藿素的重组工程菌及其制备方法和应用,以解决上述现有技术存在的问题,本发明实现了在解脂耶氏酵母中首次合成淫羊藿素,所构建的重组解脂耶氏酵母工程菌的淫羊藿素产量是迄今为止报道的通过微生物发酵生产的最高水平,这为实现解脂耶氏酵母商业化生产淫羊藿素奠定了重要基础。
为实现上述目的,本发明提供了如下方案:
本发明提供一种高产淫羊藿素的重组工程菌,其是采用如下任一种方法制备:
(1)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶和氧甲基转移酶的编码基因获得;
(2)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶、氧甲基转移酶、透明颤菌血红蛋白、3-羟基-3-甲基戊二酰辅酶A还原酶、异戊烯二磷酸异构酶和己糖激酶的编码基因获得。
优选的是,所述改造后的异戊二烯基转移酶为在截断前55个氨基酸的异戊二烯基转移酶的基础上,对截断后的氨基酸序列中的第101位、第108位和第164位中至少一位的氨基酸残基进行替换,或者同时对第164位和第175位,或者第164位和第247位的氨基酸残基进行替换得到。
优选的是,所述改造后的异戊二烯基转移酶的氨基酸序列如SEQ ID NO.5-9所示,编码基因的序列如SEQ ID NO.13-17任一项所示的序列。
优选的是,所述氧甲基转移酶的编码基因的序列如SEQ ID NO.2所示,所述透明颤菌血红蛋白的编码基因如SEQ ID NO.3所示,所述3-羟基-3-甲基戊二酰辅酶A还原酶的编码基因的序列如SEQ ID NO.10所示,所述异戊烯二磷酸异构酶的编码基因的序列如SEQ IDNO.11所示,所述己糖激酶的编码基因的序列如SEQ ID NO.12所示。
本发明还提供一种所述的高产淫羊藿素的重组工程菌的制备方法,包括以下任一项所示的方法:
(1)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶和氧甲基转移酶的编码基因获得;
(2)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶、氧甲基转移酶、透明颤菌血红蛋白、3-羟基-3-甲基戊二酰辅酶A还原酶、异戊烯二磷酸异构酶和己糖激酶的编码基因获得。
优选的是,将改造后的异戊二烯基转移酶和氧甲基转移酶的编码基因共同导入至所述解脂耶氏酵母基因组的ACE位点和F17位点;
所述3-羟基-3-甲基戊二酰辅酶A还原酶和所述异戊烯二磷酸异构酶的编码基因导入至所述解脂耶氏酵母基因组的XPR2位点。
本发明还提供一种所述的重组工程菌在高效生产淫羊藿素方面的应用。
本发明还提供一种生产淫羊藿素的方法,包括从培养所述的重组工程菌的发酵液中获取淫羊藿素的步骤。
优选的是,所述发酵培养的条件为25-30℃、220rpm条件下发酵培养72小时。
优选的是,所述培养用的发酵培养基包括以下组分:20g/L蛋白胨、10g/L酵母提取物和20g/L葡萄糖。
本发明公开了以下技术效果:
本发明选择解脂耶氏酵母作为淫羊藿素合成的底盘菌株,利用多种代谢工程及酶工程手段对解脂耶氏酵母菌株进行改造,以实现解脂耶氏酵母商业化生产淫羊藿素,具体地,本发明通过异源表达截断前55个氨基酸的异戊二烯基转移酶(tEsPT)将山柰酚转化为8-异戊二烯山柰酚后经氧甲基转移酶OsOMTm的催化进而转化为淫羊藿素,从而实现了淫羊藿素的从头合成。通过对tEsPT蛋白关键氨基酸进行突变形成tEsPTM,进一步增加tEsPTM催化山柰酚合成8-异戊二烯山柰酚的酶活,增加合成淫羊藿素关键基因tEsPTM与OsOMTm在酵母细胞表达量,推动更多前体山柰酚转化为淫羊藿素。MVA途径中的DMAPP作为合成8-异戊二烯山柰酚的关键小分子基团,通过表达来自解脂耶氏酵母基因组的3-羟基-3-甲基戊二酰辅酶A还原酶HMGR基因、异戊烯二磷酸异构酶IDI1基因增加DMAPP含量;表达经密码子优化的透明颤菌血红蛋白VHB基因促进酵母氧气供应;同时表达来自解脂耶氏酵母基因组的己糖激酶HXK,加快葡萄糖消耗率,缩短发酵时间,得到了最终构建的菌株,生产淫羊藿素的产量可达1212mg/L。
本发明实现了在解脂耶氏酵母中首次合成淫羊藿素,其中,构建的重组解脂耶氏酵母工程菌的淫羊藿素产量是迄今为止报道的通过微生物发酵生产的最高水平,为实现解脂耶氏酵母商业化生产淫羊藿素奠定重要基础,具有重大的应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为重组解脂耶氏酵母菌株催化合成淫羊藿素的HPLC检测图;其中,A为淫羊藿素标准品的色谱图,B为产淫羊藿素菌株的色谱图;
图2为重组解脂耶氏酵母菌株催化合成淫羊藿素的MS检测图;
图3为CRISPR-Cas9-ACE质粒图谱。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
下面实施例中所用的材料和试剂等,如无特殊说明,均可从商业途径得到。
以下实施例涉及的培养基:
SOC培养基:20g/L蛋白胨,5g/L酵母提取物,4g/L葡萄糖,0.5g/L氯化钠0.194g/L氯化钾2.0g/L氯化镁。
YPD固体培养基:20g/L蛋白胨,10g/L酵母提取物,20g/L葡萄糖,20g/L琼脂。
YNB固体培养基:6.7g/L无氨基酵母氮源(YNB),20g/L葡萄糖,20g/L琼脂。
5-FOA固体培养基:20g/L蛋白胨,10g/L酵母提取物,20g/L葡萄糖,1g/L 5-氟乳清酸,20g/L琼脂。
种子培养基:20g/L蛋白胨,10g/L酵母提取物,20g/L葡萄糖。
发酵培养基:20g/L蛋白胨,10g/L酵母提取物,20g/L葡萄糖。
实施例1在解脂耶氏酵母中初步建立淫羊藿素合成途径
首先使用PCR扩增目标基因。经过琼脂糖凝胶电泳确认PCR产物后,采用胶回收试剂盒对其进行纯化回收。接下来,将回收得到的DNA片段与质粒通过多片段同源重组酶连接。为了进行同源重组,使用Basic Seamless Cloning and Assembly Kit(全式金,中国北京)试剂,并按照其说明书来构建和转化质粒。一旦获得单克隆,使用测序引物进行菌落PCR验证,经琼脂糖凝胶电泳,选择条带大小与目的条带相似的PCR产物进行测序验证,以确认质粒构建成功。具体操作如下:
1、构建tEsPT-OsOMTm重组质粒
利用引物P1/P2,以经密码子优化的EsPT基因(如SEQ ID NO.1所示)为合成模板扩增tEsPT基因;利用引物P3/P4,以经过密码子优化好的OsOMTm基因(如SEQ ID NO.2所示)为合成模板中扩增OsOMTm基因。将上述带有同源臂的片段通过同源重组的方式与线性化的质粒pINA1269连接获得tEsPT-OsOMTm重组质粒。
P1:5′-cacatacaaccacacacatccacgtgATGACCCCTTTCACCCACACCCACGA-3′;
P2:5′-tttgaaaaaatttatttctagacagttatataTTAACCAATGAGGGGGATGAGCAG-3′;
P3:5′-ttttgcagtactaaccgcagGCTCCCGAGGAGGACTCTCTG-3′;
P4:5′-aacgtggggacaggccatggaggtaccGGGGTAAGCCTCAATGACAGACTC-3′。
2、构建突变质粒
以步骤(1)构建的tEsPT-OsOMTm重组质粒为模板,将tEsPT进行点突变,后续以同源重组的方式进行连接。所用引物序列见表1,分别构建经截断的异戊二烯基转移酶(截断前55个氨基酸的异戊二烯基转移酶EsPT基因核苷酸序列如SEQ ID NO.4所示)的第101位、108位或164位的突变质粒pINA1269-N101K、pINA1269-L108R、pINA1269-Y164D。对应的三种突变蛋白的序列如SEQ ID NO.5-7所示。
表1构建突变质粒表达载体所用引物
随后,以重组质粒pINA1269-Y164D为模板,将tEsPT进行点突变,后续以同源重组的方式进行连接。所用引物序列见表2,分别构建异戊烯基转移酶的175位或247位氨基酸的突变质粒pINA1269-N175T、pINA1269-N247Y。对应的两种突变蛋白的序列如SEQ ID NO.8-9所示。
表2构建突变质粒表达载体所用引物
将pINA1269-N247Y中突变所得tEsPT命名为tEsPT*,同时以解脂耶氏酵母基因组为模板扩增不同启动子。具体地,经密码子优化和酶工程手段改造的异戊二烯基转移酶tEsPT*、氧甲基转移酶OsOMTm基因共同导入至解脂耶氏酵母基因组;tEsPT*通过启动子hp4d(如SEQ ID NO.18所示)和FBAin(如SEQ ID NO.19所示)进行表达;氧甲基转移酶OsOMTm基因通过启动子TEFin(如SEQ ID NO.20所示)进行表达。3-羟基-3-甲基戊二酰CoA还原酶HMGR基因和异戊烯二磷酸异构酶IDI1基因的核苷酸序列导入至解脂耶氏酵母基因组。HMG R基因通过启动子hp4d和FBAin进行表达;异戊烯二磷酸异构酶IDI1基因通过启动子TEF in进行表达。密码子优化的透明颤菌血红蛋白VHB基因和己糖激酶HXK基因的核苷酸序列导入至解脂耶氏酵母基因组;VHB基因通过启动子TEFin进行表达;己糖激酶HXK基因通过启动子FBAin进行表达。
以tEsPT*通过启动子FBAin进行表达为例,首先使用引物P5/P6扩增FBAin启动子;同时tEsPT*和OsOMTm与pINA1269通过同源重组的方式构建pINA1269-tEsPT*-OsOMTm,并以此为模板使用引物P7/P8进行PCR,扩增得到的DNA片段回收后通过与启动子FBAin融合PCR获得tEsPT*-OsOMTm基因表达盒,其他表达模块参考此构建方式。
将线性化的质粒载体CRISPR-Cas9-ACE(图3)上下1000bp同源臂供体模块(同源臂分别使用PaF/PaR和PbF/PbR引物对扩增)用于基因的定向整合,其他位点的整合质粒参考此构建方式。再将tEsPT*-OsOMTm基因表达盒用P5/P8进行融合PCR得到质粒CRISPR-Cas9-ACE-tEsPT*OsOMTm。
通过相似的方法,以解脂耶氏酵母cDNA为模板,利用引物对P9/P10扩增目的基因tH MGR(核苷酸序列如SEQ ID NO.10所示),利用引物对P11/P12扩增目的基因IDI1(核苷酸序列如SEQ ID NO.11所示),其中tHMGR是通过截断HMGR前500个氨基酸所得。将片段与基因组整合质粒载体CRISPR-Cas9-XPR2连接得到质粒CRISPR-Cas9-XPR2-tHMG R;
PaF:5′-ttttttctgtacagacgcgtGCTATTCTTACGGTGTACAGTTACGAGC-3′;
PaR:5′-AGACGCCGTTAGCGGCTTTactagtTGTGTGAGATGGGGTAGTACGGAAG-3′;
PbF:5′CATCTCACACAactagtAAAGCCGCTAACGGCGTCTACCC-3′;
PbF:5′agagttgtgtcaacttttgcaactgggGACGCGAGTAGGTTGACTCATACATGCTTTCC-3′;
PcF:5′TGTATGAGTCAACCTACTCGCGTCcccagttgcaaaagttgacacaactc-3′;
PcR:5′cttgctatttctagctctaaaacCTCGGTATCTCTTGCATTGTacgtcaacctgcgccg-3′;
PdF:5′ACAATGCAAGAGATACCGAGgttttagagctagaaatagcaagttaaaataaggctagt-3′;
PdR:5′gaaaagtgccacctgacgtctttaGCGGCCGCattcTtcgactctagaggatctgggcc-3′;
P5:5′-GTACTACCCCATCTCACACAactagtCAGTGTACGCAGTACTATAGAGGAACAATT G-3′;
P6:5′-GGTTCTGCTGCAGCTTGTAAGGGATTCGCATggAGAGCTGGGTTAGTTTGTGTA GA-3′;
P7:5′-CACTCTCTACACAAACTAACCCAGCTCTccATGCGAATCCCTTACAAGCTGC AGC-3′;
P8:5′-GGGTAGACGCCGTTAGCGGCTTTctatttacaacaatatctggtcaaatttcagtttcg-3′;
P9:5′-gcagaggtgataccgttggacgagtACTAGTCAGTGTACGCAGTACTATAGAGGAACA A-3′;
P10:5′-atccagaagctggactctctggctatttacaacaatatctggtcaaatttcagtttcgt-3′;
P11:5′-accagcactttttgcagtactaaccgcagACGACGTCTTACAGCGACAAAATCAAGAGT-3′
P12:5′-accggcaacgtggggacaggccatggaggtaccCTACTTGATCCACCGCCGAATCTCGT-3′
P13:5′-cacactctctacacaaactaacccagctctccATGGTTCATCTTGGTCCCCGAAAACCC-3′
P14:5′-tttgaaaaaatttatttctagacagttatataCTAAATATCGTACTTGACACCGGGCTT-3′
P15:5′-tccgaccagcactttttgcagtactaaccgcagCTGGACCAGCAGACCATTAACATCAT-3′
P16:5′-aggcaagaccggcaacgtggggacaggccatggaTTACTCCACAGCCTGGGCGTACAGG-3′
利用引物对P13/P14扩增目的基因HXK(核苷酸序列如SEQ ID NO.12所示),利用引物对P15/P16扩增目的基因VHB(核苷酸序列如SEQ ID NO.3所示)基因。
3、重组质粒的酵母转化
将含有正确构建质粒的大肠杆菌DH5α培养12-16小时后,使用质粒提取小型试剂盒(全式金,中国北京)提取质粒。将质粒pINA1312与pINA1269线性化以利于其转入进酵母基因组。酵母转化的步骤可参考ZYMO Frozen EZ Yeast TransformationII Kit说明书。具体操作如下:
首先,将pINA1269-N101K、pINA1269-L108R、pINA1269-Y164D分别转化到解脂耶氏酵母菌株Yl-kae-01得到解脂耶氏酵母菌株Yl-ict-01-1、Yl-ict-01-2、Yl-ict-01-3。
待转化后,将菌液涂布在YNB平板上,该平板缺失相应的氨基酸,如LEU或URA。培养2-3天后,由于营养缺陷型筛选标记进行筛选容易出现假阳性,因此可直接挑取板上的白色菌泥(12~24个或更多)进行发酵,待培养72h时取样500μL菌液进行HPLC检测。发现Yl-ict-01-3的突变株产量更高,因此用于后续突变。同理将质粒pINA1269-N175T、pINA 1269-D247Y转化,并命名为Yl-ict-01-4、Yl-ict-01-5,其中Yl-ict-01-5的产量最高,即tEsPT-D247Y的催化效率最高。
将质粒CRISPR-Cas9-ACE-tEsPT*转化到解脂耶氏酵母菌株Yl-kae-01得到解脂耶氏酵母菌株Yl-ict-01。
接下来,依次将基因tHMGR、IDI1、tEsPT*-OsOMT2、VHB转化到解脂耶氏酵母菌株Yl-ict-01得到Yl-ict-02。
转化后,使用LEU或URA营养缺陷进行克隆筛选。随后使用PCR进行目标基因的扩增并通过测序验证是否成功转入酵母基因组。随后,为了去除游离未整合至基因组的质粒,将单菌落涂布至含有5-氟乳清酸的YPD平板上,从而导致含有URA3的酵母菌的死亡。随后,挑选的菌落被涂布在含有LEU的YNB平板上,以验证质粒是否已经丢失。如果酵母细胞在YNB-LEU平板上没有生长,表明质粒已经被成功丢弃,可以进行下一步的整合。
4、解脂耶氏酵母的发酵培养
以Yl-ict-02菌株的培养为例,为了评估重组菌株合成淫羊藿素的生产潜力,首先使用24深孔板进行单菌落的初筛。对于筛选出的合成淫羊藿素水平较高的单菌落,使用50%甘油进行保种,同时在YPD固体培养基上进行标记。在培养基上生长1-2天后,从中挑选单菌落,并将其培养在含有5mL YPD的25mL摇瓶中。在30℃下培养24小时,直到达到酵母对数生长期。然后,测定种子液的细胞密度,并以初始OD600为0.1的细胞密度接种到含有50mLYPD的250mL摇瓶中。培养在220rpm、30℃的摇床上,并每隔24小时取样,以测试不同的重组解脂耶氏酵母菌株的淫羊藿素生产能力。
淫羊藿素的检测通过HPLC进行。首先,取500μL的发酵液,然后加入相同体积的乙醇。之后,在漩涡震荡器上充分混匀,12000rpm的离心5分钟,吸取上清液。最后,通过0.22μm的有机滤膜过滤,将过滤后的样品装入液相瓶中,以进行HPLC检测。
产物的定性与定量检测方法:使用高效液相色谱(Prominence-ILC2030,Shimadzu)测定发酵液中山柰酚和淫羊藿素的产量,具体采用反相色谱柱DiamonsilC18(2)(5μm,150×4.6mm)和紫外-可见光检测器进行检测,并在360nm处检测紫外吸收。柱温箱设定为35℃,流速设置为0.8mL/min,进样体积为10μL。A相为0.01%的乙酸,C相为乙腈。25分钟的梯度洗脱程序为0-2min保持15%C;2-16min,C相从15%增加到70%;16-18min,C相从70%增加到95%;18-25min,C相从95%减少到15%。采用C18色谱柱(2.1×100mm,内径1.7μm),液相色谱-串联质谱(LC/MS)对样品进行鉴定。以正离子监测模式进行检测。
结果发现在发酵72h,葡萄糖浓度为20g/L时,检测到淫羊藿素产量最高达到1212mg/L(见图1和图2)。
表3不同菌株的产量
菌株 | 淫羊藿素产量(mg/L) |
YL-ICT-01-1 | 2.8 |
YL-ICT-01 | 22.4 |
YL-ICT-02 | 1212 |
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (10)
1.一种高产淫羊藿素的重组工程菌,其特征在于,其是采用如下任一种方法制备:
(1)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶和氧甲基转移酶的编码基因获得;
(2)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶、氧甲基转移酶、透明颤菌血红蛋白、3-羟基-3-甲基戊二酰辅酶A还原酶、异戊烯二磷酸异构酶和己糖激酶的编码基因获得。
2.如权利要求1所述的高产淫羊藿素的重组工程菌,其特征在于,所述改造后的异戊二烯基转移酶为在截断前55个氨基酸的异戊二烯基转移酶的基础上,对截断后的氨基酸序列中的第101位、第108位和第164位中至少一位的氨基酸残基进行替换,或者同时对第164位和第175位,或者第164位和第247位的氨基酸残基进行替换得到。
3.如权利要求2所述的高产淫羊藿素的重组工程菌,其特征在于,所述改造后的异戊二烯基转移酶的氨基酸序列如SEQ ID NO.5-9所示,编码基因的序列如SEQ ID NO.13-17任一项所示的序列。
4.如权利要求1所述的高产淫羊藿素的重组工程菌,其特征在于,所述氧甲基转移酶的编码基因的序列如SEQ ID NO:2所示,所述透明颤菌血红蛋白的编码基因如SEQ ID NO.3所示,所述3-羟基-3-甲基戊二酰辅酶A还原酶的编码基因的序列如SEQ ID NO.10所示,所述异戊烯二磷酸异构酶的编码基因的序列如SEQ ID NO.11所示,所述己糖激酶的编码基因的序列如SEQ ID NO.12所示。
5.一种如权利要求1-4任一项所述的高产淫羊藿素的重组工程菌的制备方法,其特征在于,包括以下任一项所示的方法:
(1)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶和氧甲基转移酶的编码基因获得;
(2)以能合成淫羊藿素前体山奈酚的解脂耶氏酵母为出发菌株,导入改造后的异戊二烯基转移酶、氧甲基转移酶、透明颤菌血红蛋白、3-羟基-3-甲基戊二酰辅酶A还原酶、异戊烯二磷酸异构酶和己糖激酶的编码基因获得。
6.如权利要求5所述的制备方法,其特征在于,将改造后的异戊二烯基转移酶和氧甲基转移酶的编码基因共同导入至所述解脂耶氏酵母基因组的ACE位点和F17位点;
所述3-羟基-3-甲基戊二酰辅酶A还原酶和所述异戊烯二磷酸异构酶的编码基因导入至所述解脂耶氏酵母基因组的XPR2位点。
7.一种如权利要求1-4任一项所述的重组工程菌在高效生产淫羊藿素方面的应用。
8.一种生产淫羊藿素的方法,其特征在于,包括从培养权利要求1-4任一项所述的重组工程菌的发酵液中获取淫羊藿素的步骤。
9.如权利要求8所述的方法,其特征在于,所述发酵培养的条件为25-30℃、220rpm条件下发酵培养72小时。
10.如权利要求8所述的方法,其特征在于,所述培养用的发酵培养基包括以下组分:20g/L蛋白胨、10g/L酵母提取物和20g/L葡萄糖。
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