CN117247881A - 一步法生产麦角硫因的谷氨酸棒杆菌的菌株构建和应用 - Google Patents
一步法生产麦角硫因的谷氨酸棒杆菌的菌株构建和应用 Download PDFInfo
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- CN117247881A CN117247881A CN202311000327.4A CN202311000327A CN117247881A CN 117247881 A CN117247881 A CN 117247881A CN 202311000327 A CN202311000327 A CN 202311000327A CN 117247881 A CN117247881 A CN 117247881A
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- corynebacterium glutamicum
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Abstract
本发明公开了一步法生产麦角硫因的谷氨酸棒杆菌的菌株构建和应用,属于合成生物学、酶工程、代谢工程技术领域。本发明构建的工程菌株可以一步法高产麦角硫因,极大降低了生产成本。该菌株的发酵过程不需要添加氨基酸,可以直接利用葡萄糖发酵,麦角硫因产量可达4.3g/L,是现在报道的最高产量。
Description
技术领域
本发明涉及一步法生产麦角硫因的谷氨酸棒杆菌的菌株构建和应用,属于合成生物学、酶工程、代谢工程技术领域。
背景技术
麦角硫因(Ergothioneine,EGT),又称麦角含硫碱,是一种来源于植物,并且可以在动物中积累的天然氨基酸,在人体内可以对细胞起到保护作用,是机体内的重要活性物质。麦角硫因作为一种天然抗氧化剂,安全,无毒,已经成为研究的热点,它具有清除自由基,解毒,维持DNA的生物合成,细胞的正常生长及细胞免疫等多种生理功能,在化妆品、保健品及医药等行业极具应用前景。
麦角硫因的制备方法包括天然生物提取法、化学合成法和微生物合成法。其中天然生物提取法是从食用菌的子实体、动物组织、猪血、谷物和麦角中提取麦角硫因;化学法合成主要是将巯基导入咪唑环;微生物合成法是以蕈菌菌丝体深层发酵技术生物合成制备麦角硫因,通过代谢调控等发酵过程控制手段提高麦角硫因的产率。近年来已经有一些专利报道了关于使用微生物来生产麦角硫因,通过基因工程手段改造宿主如大肠杆菌、酿酒酵母、枯草芽孢杆菌等,表达外源麦角硫因合成酶生产麦角硫因,是目前研究的热点。
传统的麦角硫因生产主要是通过化学合成或生物提取,目前化学方法合成麦角硫因困难,且存在产品安全性难以保证、合成原料及成本高昂等缺点;从富含麦角硫因的食用菌中提取存在提取效率低、耗费时间长、难以大规模制备等问题,不能从根源上解决麦角硫因产量不足,使得大规模生产难以满足不断增长的市场需求。在常用工业底盘菌株内,与大肠杆菌等相比,谷氨酸棒杆菌具有合成麦角硫因前体:组氨酸、半胱氨酸等氨基酸的能力,是大规模生产麦角硫因的潜力菌株。
发明内容
本发明提供一种谷氨酸棒杆菌底盘细胞,在谷氨酸棒杆菌出发菌株的基础上,包含了如下改进:
(1)降低磷酸烯醇丙酮酸羧化激酶、L-丝氨酸脱氨酶、转录激活因子glyR、磷酸乙酰转移酶、丙酮酸脱氢酶、乳酸脱氢酶中至少一种酶的表达;
(2)增强丝氨酸O-乙酰转移酶、磷酸甘油酸脱氢酶、磷酸丝氨酸转氨酶、6-磷酸果糖激酶、磷酸甘油酸激酶PGK、磷酸核糖-ATP环水解酶、O-乙酰丝氨酸裂解酶中至少一个酶的表达。
在一种实施方式中,所述磷酸烯醇丙酮酸羧化激酶具有Genbank登录号CAF20888.1所示的氨基酸序列,或与所述氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
在一种实施方式中,所述L-丝氨酸脱氨酶具有Genbank登录号:CAF20029.1所示的氨基酸序列,或与所述氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
在一种实施方式中,所述转录激活因子glyR具有Genbank登录号:ASW13231.1所示的氨基酸序列,或与所述氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
在一种实施方式中,所述磷酸乙酰转移酶具有Genbank登录号:CAF20775.1所示的氨基酸序列,或与所述氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
在一种实施方式中,所述丙酮酸脱氢酶poxB具有Genbank登录号:CAF21272.1所示的氨基酸序列,或与所述氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
在一种实施方式中,所述乳酸脱氢酶LDH具有Genbank登录号:CAF20933.1所示的氨基酸序列,或与所述氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
在一种实施方式中,所述底盘细胞在谷氨酸棒杆菌出发菌株的基础上,包含了如下至少一种改进:
(1)敲除磷酸烯醇丙酮酸羧化激酶基因pck,增强丝氨酸O-乙酰转移酶cysE的表达:
(2)敲除L-丝氨酸脱氨酶基因sdaA,增强磷酸甘油酸脱氢酶突变体serAmut的表达;
(3)敲除转录激活因子glyR,并增强磷酸丝氨酸转氨酶serC的表达;
(4)敲除磷酸乙酰转移酶基因PTA,增强6-磷酸果糖激酶PFKA的表达;
(5)用启动子Ptuf替换磷酸丝氨酸磷酸酶serB的启动子区,并增强磷酸甘油酸激酶PGK的表达,
(6)用启动子Ptuf替换亚硫酸还原酶cysI的启动子区,并增强磷酸甘油酸脱氢酶突变体的表达;
(7)敲除丙酮酸脱氢酶基因poxB,并增强磷酸核糖-ATP环水解酶hisE的表达;
(8)敲除乳酸脱氢酶基因LDH,并增强O-乙酰丝氨酸裂解酶cysK的表达。
在一种实施方式中,所述底盘细胞敲除了磷酸烯醇丙酮酸羧化激酶(Genbank登录号CAF20888.1)基因pck,并在该位置上整合丝氨酸O-乙酰转移酶cysE(Genbank登录号:CAF21224.1)的编码基因。
在一种实施方式中,所述底盘细胞敲除了L-丝氨酸脱氨酶(Genbank登录号:CAF20029.1)基因sdaA,并在该位点整合了磷酸甘油酸脱氢酶突变体基因serAmut;所述基因serAmut具有SEQ ID NO.2所示的核苷酸序列。
在一种实施方式中,所述底盘细胞敲除了转录激活因子glyR(Genbank登录号为ASW13231.1)基因,并在该位点整合磷酸丝氨酸转氨酶(Genbank登录号为CAF19534.1)基因serC。
在一种实施方式中,所述底盘细胞敲除了磷酸乙酰转移酶(Genbank登录号:CAF20775.1)基因PTA,并在该位点整合6-磷酸果糖激酶(Genbank登录号:BAB98643.1)基因PFKA。
在一种实施方式中,所述底盘细胞敲除了磷酸丝氨酸磷酸酶serB(Genbank登录号:CAF21185.1)的启动子区,并在该位置整合了含Ptuf启动子的磷酸甘油酸激酶(Genbank登录号:CAF21595.1)基因PGK。
在一种实施方式中,所述底盘细胞敲除了亚硫酸还原酶cysI(Genbank登录号:CAF20840.1)的启动子区,并在该位置整合了含Ptuf启动子的磷酸甘油酸脱氢酶突变体基因serAmut。
在一种实施方式中,所述底盘细胞敲除了丙酮酸脱氢酶(Genbank登录号:CAF21272.1)基因poxB,并在该位置整合了磷酸核糖-ATP环水解酶(Genbank登录号:CAF21513.1)基因hisE。
在一种实施方式中,所述底盘细胞敲除了乳酸脱氢酶(Genbank登录号:CAF20933.1)基因LDH,并在该位置整合了O-乙酰丝氨酸裂解酶(Genbank登录号:CAF21223.1)基因cysK。
在一种实施方式中,所述出发菌株包括但不限于谷氨酸棒杆菌ATCC 13032。
在一种实施方式中,所述启动子Ptuf的核苷酸序列如SEQ ID NO.12所示。
本发明提供了磷酸甘油酸脱氢酶突变体,在野生型磷酸甘油酸脱氢酶的基础上,含有D240N突变、D313N突变和/或C端197个氨基酸截短。
在一种实施方式中,所述突变体是在磷酸甘油酸脱氢酶的基础上将第240位天冬氨酸突变成天冬酰胺,和/或将第313位天冬氨酸突变成天冬酰胺,和/或切除C端197个氨基酸。
在一种实施方式中,所述磷酸甘油酸脱氢酶突变体含有SEQ ID NO.3所示的氨基酸序列,或具有与SEQ ID NO.3所示氨基酸序列具有至少90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%同源性的氨基酸序列。
本发明还提供一种麦角硫因的重组谷氨酸棒杆菌,在所述底盘细胞上,表达了麦角硫因合成酶。
在一种实施方式中,所述麦角硫因合成酶的编码基因序列如SEQ ID NO.10~SEQID NO.11所示。
在一种实施方式中,所述麦角硫因合成酶由SEQ ID NO.12所示的启动子Ptuf调控表达。
本发明还提供了一种生产麦角硫因的方法,所述方法是将所述重组谷氨酸棒杆菌在含葡萄糖的培养基中培养。
在一种实施方式中,所述培养基还含有(NH4)2SO4、尿素、KH2PO4、K2HPO4、MgSO4·7H2O、CaCl2·2H2O、MOPS、生物素和微量元素。
在一种实施方式中,所述微量元素包括FeSO4·7L-天冬氨酸半醛H2O、MnSO4·1H2O、ZnSO4·7H2O、CuSO4·5H2O、NiCl·6H2O。
本发明还请求保护所述谷氨酸棒杆菌底盘细胞在生产以L-半胱氨酸、组氨酸和/或甲硫氨酸为前体的生物产品中的应用。
有益效果:
(1)本发明构建了一种谷氨酸棒杆菌底盘细胞,可实现从葡萄糖到L-半胱氨酸、组氨酸及甲硫氨酸的高效合成,有助于构建以L-半胱氨酸、组氨酸及甲硫氨酸为前体代谢合成相关生物产品的基因工程菌株。
(2)本发明构建的谷氨酸棒杆菌工程菌株是GRAS安全食用菌株,可以直接利用葡萄糖一步法生产麦角硫因,无需添加氨基酸,极大降低了生产成本,相比添加氨基酸的发酵工艺成本可降低至其二十分之一以下。该工程菌株发酵96h生产的麦角硫因产量可达4.3g/L,是现有技术中报道的最高产量。
附图说明
图1为麦角硫因的合成途径以及参与的基因;
图2为麦角硫因的液相检测结果;
图3为麦角硫因生产重组菌的摇瓶发酵产量;
图4为菌株Erg8/pCES-Egt12在10升罐发酵生产麦角硫因的发酵过程曲线。
图5为表达不同serA突变体的麦角硫因生产菌株的摇瓶发酵结果。
具体实施方式
技术术语:
本发明所述的“底盘细胞”是指经过人工改造的被置入某些功能化生物系统模块,具备特定功能的细胞。在一些实施方式中,特指经过基因改造的谷氨酸棒杆菌细胞,可实现从葡萄糖到L-半胱氨酸、组氨酸及甲硫氨酸的高产量的合成。
本发明所述的“表达”包括涉及酶产生的任何步骤,包括但不限于转录、转录后修饰、翻译、翻译后修饰、以及分泌。
本发明所述的“突变体”意指具有酶活性的、在一个或多个(例如若干个)位置包含改变(即取代、插入和/或缺失)的多肽。取代意指用不同的氨基酸替代占据某一位置的氨基酸;缺失意指去除占据某一位置的氨基酸;而插入意指在邻接并且紧随占据某一位置的氨基酸之后添加氨基酸。
在描述本发明的突变体中,为了便于参考,对以下所述的命名法进行了改编,采用了已接受的IUPAC单字母或三字母的氨基酸缩写。对于氨基酸取代,使用以下命名法:原始氨基酸、位置、被取代的氨基酸。例如突变体D240N表示在磷酸甘油酸脱氢酶野生酶序列的基础上将第240位天冬氨酸由天冬酰胺取代。
本发明所述“同源性”,是指在氨基酸序列或核苷酸序列的方面,本领域技术人员可以根据实际工作需要对序列进行调整,使使用的序列与现有技术获得的序列相比,具有(包括但不限于)1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11%,12%,13%,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,70%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,99.9%的同一性。
本发明所述的“包含”或“包括”是开放式的描述,含有所描述的指定成分或步骤,以及不会实质上影响的其他指定成分或步骤。然而在用于描述蛋白质或核酸的序列时,所述蛋白质或核酸可以是由所述序列组成,或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸,但仍然具有本发明所述的活性。
培养基:
CGXII培养基:葡萄糖50g/L,(NH4)2SO4 20g/L,尿素5g/L,KH2PO4 1g/L,K2HPO4 1g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O 13.3mg/L,3-吗啉丙磺酸(MOPS)42g/L,生物素0.2mg/L,微量元素溶液1ml/L,运用KOH将pH调节到7.0;其中,微量元素溶液:FeSO4·7H2O10g/L,MnSO4·1H2O 10g/L,ZnSO4·7H2O 1g/L,CuSO4·5H2O 313mg/L,NiCl·6H2O 20mg/L。
BHIS培养基:脑心浸液肉汤BHI 37g/L,山梨醇91g/L。
发酵培养基:葡萄糖30g/L,硫酸铵12g/L,硫酸镁0.87g/L,玉米浆3ml/L,磷酸0.4ml/L,氯化钾0.53g/L,硫酸亚铁120mg/L,硫酸锰120mg/L,烟酰胺42mg/L,泛酸钙6.3mg/L,维生素B1 6.3mg/L,生物素0.5mg/L。
实施例1高产麦角硫因的谷氨酸棒杆菌工程菌株底盘细胞的构建
图1为麦角硫因的合成途径,敲除其代谢途径和一些支路途径,增强合成途径中一些关键基因表达,构建能够合成麦角硫因的谷氨酸棒杆菌。具体如下:
1)构建重组质粒pK18-Δpck::cysE,敲除磷酸烯醇丙酮酸羧化激酶pck,增强丝氨酸O-乙酰转移酶cysE的合成:以谷氨酸棒杆菌ATCC13032基因组为模板分别克隆磷酸烯醇丙酮酸羧化激酶pck(Genbank登录号CAF20888.1)基因上下游1000bp的同源臂,克隆丝氨酸O-乙酰转移酶cysE(Genbank登录号:CAF21224.1)的编码基因(SEQ ID NO.1所示),构建在pck基因上下游同源臂之间,与Pk18mobsacB骨架通过Gibson方法相连,构建重组质粒pK18-Δpck::cysE,将获得的重组质粒pK18-Δpck::cysE转化至谷氨酸棒杆菌ATCC13032感受态细胞中,重组得到菌株WT/Δpck::cysE,命名为Erg1。
2)按照上述相同策略构建重组质粒pK18-ΔsdaA::serAmut,敲除L-丝氨酸脱氨酶sdaA(Genbank登录号:CAF20029.1),并在该位置整合携带Ptuf启动子的磷酸甘油酸脱氢酶突变体基因serAmut(SEQ ID NO.2所示),以增强磷酸甘油酸脱氢酶突变体serAmut(第240位天冬氨酸突变成天冬酰胺,第313位天冬氨酸突变成天冬酰胺,同时切除serA的C端197个氨基酸)的表达。将构建的重组质粒pK18-ΔsdaA::serAmut转化至Erg1感受态细胞中,重组得到菌株Erg1/ΔsdaA::serAmut,命名为Erg2。
3)按照上述相同策略构建重组质粒pK18-ΔglyR::serC,敲除转录激活因子glyR(Genbank登录号为ASW13231.1),并在该位置整合磷酸丝氨酸转氨酶(Genbank登录号为CAF19534.1)基因serC(SEQ ID NO.5所示),以增强磷酸丝氨酸转氨酶(Genbank登录号为CAF19534.1)的表达。将构建的重组质粒pK18-ΔglyR::serC转化至Erg2感受态细胞中,得到重组菌株Erg2/ΔglyR::serC,命名为Erg3。
4)按照上述相同策略构建重组质粒pK18-Δpta::pfka,敲除磷酸乙酰转移酶PTA(Genbank登录号:CAF20775.1),并在该位置整合6-磷酸果糖激酶基因PFKA(SEQ ID NO.6所示),以增强6-磷酸果糖激酶(Genbank登录号:BAB98643.1)的表达。将构建的重组质粒pK18-Δpta::pfka转化至Erg3感受态细胞中,重组得到菌株Erg3/Δpta::pfka,命名为Erg4。
5)按照上述相同策略构建重组质粒pK18-ΔpserB::pgk,敲除磷酸丝氨酸磷酸酶serB(Genbank登录号:CAF21185.1)启动子区,将携带启动子Ptuf(SEQ ID NO.12所示)的磷酸甘油酸激酶基因(SEQ ID NO.7所示)整合在该位置,以增强磷酸甘油酸激酶(Genbank登录号:CAF21595.1)的表达,将构建的重组质粒pK18-ΔpserB::pgk转化至Erg4感受态细胞中,重组得到菌株Erg4/ΔpserB::pgk,命名为Erg5。
6)按照上述相同策略构建重组质粒pK18-ΔpcysI::serAmut,敲除亚硫酸还原酶cysI(Genbank登录号:CAF20840.1)启动子区,将启动子Ptuf-磷酸甘油酸脱氢酶突变体基因serAmut-启动子Ptuf所示连接的片段整合在该位置,以增强磷酸甘油酸脱氢酶突变体serAmut(SEQ ID NO.3所示)的表达,其中,启动子Ptuf的核苷酸序列如SEQ ID NO.12所示;磷酸甘油酸脱氢酶突变体基因serAmut的核苷酸序列如SEQ ID NO.2所示。将构建的重组质粒pK18-ΔpcysI::serAmut转化至Erg5感受态细胞中,重组得到菌株Erg5/ΔpcysI::serAmut,命名为Erg6。
7)按照上述相同策略构建重组质粒pK18-ΔpoxB::hisE,敲除丙酮酸脱氢酶poxB(Genbank登录号:CAF21272.1),并在该位置整合磷酸核糖-ATP环水解酶基因hisE(SEQ IDNO.8所示),以增强磷酸核糖-ATP环水解酶(Genbank登录号:CAF21513.1)的表达。将构建的重组质粒pK18-ΔpoxB::hisE转化至Erg6感受态细胞中,重组得到菌株Erg6/ΔpoxB::hisE,命名为Erg7。
8)按照上述相同策略构建重组质粒pK18-Δldh::cysK,敲除乳酸脱氢酶(Genbank登录号:CAF20933.1)基因LDH,并在该位置整合O-乙酰丝氨酸裂解酶基因cysK(SEQ IDNO.9所示),以增强O-乙酰丝氨酸裂解酶(Genbank登录号:CAF21223.1)的表达。将构建的重组质粒pK18-Δldh::cysK转化至Erg7感受态细胞中,重组得到菌株Erg7/Δldh::cysK,命名为Erg8。
实施例2麦角硫因合成酶重组质粒和重组菌的构建
麦角硫因合成酶(Egt12)由Egt1和Egt2组成,Egt1将三个甲基从s-腺苷甲硫氨酸(SAM)转移到组氨酸中形成组氨酸三甲内盐,并利用氧和半胱氨酸进一步催化组氨酸三甲内盐生成海西烯半胱氨酸亚砜,然后通过Egt2催化生成麦角硫因,是麦角硫因生产菌中的关键性酶。选择来自Neurospora crassa菌株的egt1和Claviceps purpurea菌株的egt2基因作针对谷氨酸棒状杆菌的密码子优化后,得到核苷酸序列如SEQ ID NO.10和SEQ IDNO.11所示,并设计扩增引物:
Egt12F:CTTGGTTGGTAGGAGTAGCATGGGATCCATGCCATCCGCAGAATCCATGAC CCCATC;
Egt12R:
CGCGCTACTGCCGCCAGGCAGCGGCCGTTATTCCACTGGCTGTGGCACCAGGTATTC,
Egt1和Egt2基因都使用Ptuf启动子(SEQ ID NO.12所示)调控表达,以合成的基因作为模板为模板,通过PCR进行扩增得到目的片段,经DNA纯化试剂盒纯化后,通过Gibson将PCR扩增产物和质粒pCES(质粒公开于论文《Development of a high-copy-numberplasmid via adaptive laboratory evolution of Corynebacterium glutamicum》)的主干片段相连,转化至E.coli DH5α中,测序验证其构建成功。将携带Egt12编码序列的表达载体pCES-Egt12分别转化到实施例1构建的谷氨酸棒杆菌工程菌株Erg1-Erg8中,得到麦角硫因生产重组菌Erg1/pCES-Egt12~Erg8/pCES-Egt12。
实施例3利用谷氨酸棒杆菌工程菌株发酵生产麦角硫因
将实施例2中构建的麦角硫因生产重组菌,用于一步法从葡萄糖发酵生产麦角硫因。将菌株Erg1/pCES-Egt12~Erg8/pCES-Egt12在BHIS培养基中,用试管于30℃培养12小时,获得种子液;将50mL CGXII培养基加到500mL摇瓶中,按10%的接种率接种,发酵温度为30℃,摇床转速200rpm,发酵72小时。
利用液相色谱检测麦角硫因产量:安捷伦液相色谱仪1290,配备光电二极管阵列检测器,色谱柱:Kinetex F5(2.6um 2.1mm×100mm,Phenomenex);流动相采用纯水(含0.1%甲酸),运行时间为5.1min,0-5min梯度流速0.1-0.3mL/min,5.1min时流速为0.1mL/min,检测波长254nm;柱温20℃;进样量1uL;如图所示,对照麦角硫因的标准品,在样品中检测到了麦角硫因的峰,显示构建的菌株能生产麦角硫因。图3显示了摇瓶发酵72小时后,各麦角硫因生产重组菌所产的麦角硫因产量。随着底盘菌株的不断改造,产量逐步提升,Erg8的麦角硫因产量达到103.73mg/L。
实施例4谷氨酸棒杆菌工程菌株在发酵罐水平发酵生产麦角硫因
将菌株Erg8/pCES-Egt12在BHIS培养基中,于30℃培养24小时至OD为8±0.2,获得种子液;将4.5L发酵培养基加到10L发酵罐中,按10%的接种率将种子液接种到发酵罐,使初始OD为0.8,发酵温度为30℃,溶氧30%,利用氨水来调节pH 7.0±0.5,当葡萄糖消耗到10g/L以下开始流加葡萄糖,葡萄糖浓度控制在10g/L以下。图4显示了Erg8/pCES-Egt12摇瓶发酵过程中OD以及麦角硫因产量的变化。发酵23小时后,30g/L的葡萄糖接近耗光,OD在47小时达到最高值158。最终在96小时,摇瓶发酵可以生产4.3g/L麦角硫因,是利用谷氨酸棒杆菌发酵且发酵过程中不添加氨基酸获得的最高产量。
对比例:
具体实施方式同实施例1,区别在于,将serA的突变体分别替换为野生型serA(SEQID NO.4所示),在野生型serA的基础上将第240位天冬氨酸突变成天冬酰胺的突变体serAD240N,在野生型serA的基础上将第313位天冬氨酸突变成天冬酰胺的突变体serA D313N,在野生型serA的基础上将第240位天冬氨酸突变成天冬酰胺并切除serA的C端197个氨基酸的突变体serA D240 Dc179AA。按照实施例2的方法发酵,结果如图5所示。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种谷氨酸棒杆菌底盘细胞,其特征在于,在谷氨酸棒杆菌出发菌株的基础上,包含了如下改进:
(1)降低磷酸烯醇丙酮酸羧化激酶、L-丝氨酸脱氨酶、转录激活因子glyR、磷酸乙酰转移酶、丙酮酸脱氢酶、乳酸脱氢酶中至少一种酶的表达;
(2)增强丝氨酸O-乙酰转移酶、磷酸甘油酸脱氢酶、磷酸丝氨酸转氨酶、6-磷酸果糖激酶、磷酸甘油酸激酶、磷酸核糖-ATP环水解酶、O-乙酰丝氨酸裂解酶中至少一个酶的表达。
2.根据权利要求1所述的谷氨酸棒杆菌底盘细胞,其特征在于,在谷氨酸棒杆菌出发菌株的基础上,包含了如下至少一种改进:
(1)敲除磷酸烯醇丙酮酸羧化激酶基因pck,增强丝氨酸O-乙酰转移酶cysE的表达:
(2)敲除L-丝氨酸脱氨酶基因sdaA,增强磷酸甘油酸脱氢酶的表达;
(3)敲除转录激活因子glyR,增强磷酸丝氨酸转氨酶的表达;
(4)敲除磷酸乙酰转移酶基因PTA,增强6-磷酸果糖激酶的表达;
(5)用启动子Ptuf替换磷酸丝氨酸磷酸酶serB的启动子区,增强磷酸甘油酸激酶的表达,
(6)用启动子Ptuf替换亚硫酸还原酶cysI的启动子区,增强磷酸甘油酸脱氢酶的表达;
(7)敲除丙酮酸脱氢酶基因poxB,增强磷酸核糖-ATP环水解酶hisE的表达;
(8)敲除乳酸脱氢酶基因LDH,增强O-乙酰丝氨酸裂解酶cysK的表达。
3.根据权利要求1或2所述的谷氨酸棒杆菌底盘细胞,其特征在于,所述磷酸甘油酸脱氢酶的氨基酸序列如SEQ ID NO.3所示。
4.根据权利要求1~3任一所述的谷氨酸棒杆菌底盘细胞,其特征在于,所述出发菌株包括但不限于谷氨酸棒杆菌ATCC 13032。
5.一种磷酸甘油酸脱氢酶突变体,其特征在于,在野生型磷酸甘油酸脱氢酶的基础上,含有D240N突变、D313N突变和/或C端197个氨基酸截短的氨基酸序列。
6.一种麦角硫因的重组谷氨酸棒杆菌,其特征在于,在权利要求1~4任一所述的底盘细胞上,表达了麦角硫因合成酶。
7.根据权利要求6所述的重组谷氨酸棒杆菌,其特征在于,所述麦角硫因合成酶的编码基因序列如SEQ ID NO.10~SEQ ID NO.11所示。
8.一种生产麦角硫因的方法,其特征在于,将权利要求1~4任一所述的重组谷氨酸棒杆菌在含葡萄糖的培养基中培养。
9.根据权利要求8所述的方法,其特征在于,当葡萄糖降低至10g/L以下,补料葡萄糖。
10.权利要求1~4任一所述的谷氨酸棒杆菌底盘细胞在生产以L-半胱氨酸、组氨酸和/或甲硫氨酸为前体的生物产品中的应用。
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