CN115975831A - 一种高产5-氨基乙酰丙酸酿酒酵母工程菌株及其应用 - Google Patents
一种高产5-氨基乙酰丙酸酿酒酵母工程菌株及其应用 Download PDFInfo
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Abstract
本发明涉及一种高产5‑ALA酵母菌株及应用,该菌株可产3.53g/L的5‑ALA,为真菌合成最高水平。在菌株BY4741δ位表达ALAS酶,强化PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、KGD2、aceA、aceB、agxT、AOX、ACH1、pdxS、pdxT、GDH1、GLT1、IDP1、AGC1、gltX、hemL、pgr7、hemA、rhtA酶表达,引入蛋白支架序列,弱化HEM2表达,抑制SDH5、LSC1、LSC2、YOR131C、GDH2酶,通过融合获得菌株BY12‑MPCP。用于提高5‑ALA产量。
Description
技术领域
一种高产5-氨基乙酰丙酸酿酒酵母工程菌株及其应用,属于基因工程和微生物技术领域。本发明还包括上述菌株的菌剂,及在食品、饲料、医药及化妆品工业领域的应用。
背景技术
5-氨基乙酰丙酸(5-ALA)作为一种重要的四吡咯化合物,广泛应用于医药,农业,饲料等领域。目前,5-ALA的合成主要为化学合成法生产,但是化学合成方法成本高、污染重,限制了其在各领域的推广应用。随着生物技术的不断发展,通过构建微生物细胞工厂来发酵法合成5-ALA具有低成本和无污染的优势而逐渐成为研究的热点。生物体内合成5-ALA主要有两种途径,分别为C4途径和C5途径。其中C4途径经过5-ALA合成酶催化琥珀酰辅酶A和甘氨酸直接合成5-ALA;C4途径只涉及一步酶促反应,具有明显优势。虽然C4途径中的底物琥珀酰辅酶a和甘氨酸可以通过途径改造实现自我供给,但是对于高水平成合5-ALA,胞内琥珀酰辅酶a和甘氨酸合成通量不足。琥珀酰辅酶a和甘氨酸合成途径中关键酶的酶活力不高,现有利用C4途径合成5-ALA的菌株和工艺中通常需要外源添加琥珀酸和甘氨酸,以保证5-ALA的过量合成和积累。C5途径合成5-ALA仅需要谷氨酸作为前体,不需要像C4途径那样需要多种前体和辅因子参与。目前在国际期刊中尚未有在酿酒酵母细胞同时构建C4和C5途径合成5-ALA的报道。
在C5合成途径中,以三羧酸循环中的α-酮戊二酸合成的谷氨酸作为前体物,经过谷氨酸-tRNA合成酶(GluTS)催化生成L-谷氨酰胺-tRNA,之后由NADPH依赖的谷氨酰基-tRNA还原酶(GluTR)进一步甲基化生成L-谷氨酸1-半甲醛(GSA),最后L-谷氨酸1-半甲醛由谷氨酸-L-氨基醛氨基转移酶(GSA-AM)催化为5-ALA。已报到大肠杆菌C5途径中,分别由基因gltX、hemA和hemL编码GluTS、GluTR和GSA-AM三个关键酶。此外,有报道表示通过引入来源于拟南芥的pgr7编码的蛋白GBP能够刺激GluTR增强5-ALA的合成。
酿酒酵母较其他微生物,具有良好的食品安全性;具有低pH耐受性,有利于高效积累有机酸、降低下游提取纯化成本;具有高葡萄糖耐受性,为实现高密度发酵奠定基础。而且酿酒酵母具有生长周期短、发酵能力强、底物利用范围广、可进行大规模培养,作为食品安全菌株(GRAS),被广泛用于生物基化学品合成。Hara等人在酿酒酵母中过表达HEM1和ACO2基因,在培养基中添加甘氨酸和乙酰丙酸获得1.36mg/L的5-ALA(Hara et al.,MicrobCell Fact,2019,18(1):194.)。Mao等人以质粒的形式表达在酿酒酵母中HEM1基因,最后发酵获得65mg/L的5-ALA(Mao et al.,Journal of Biotechnology,2020,322:29-32.).
虽然上述工作已经取得一定成效,但其5-ALA最终的产量依然很低,无法满足工业化规模的生产应用。质粒的形式表达方式不适合在丰富培养基中进行高密度发酵。发酵过程中添加前体物或抗生素也会增加5-ALA合成成本。因此,提供一株易于发酵培养的高产5-ALA菌株具有重要的现实意义。
发明内容
本发明提供了一株高产5-氨基乙酰丙酸酿酒酵母工程菌株及构建及发酵方法。
技术方案如下:
一种5-ALA高产工程菌,以BY4741为出发菌株,经过基因修饰后具有增强的ALAS、PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、aceA、aceB、AGXT、AOX1、ACH1、pdxS、pdxT、GDH1、GLT1、IDP1、AGC1,gltX、hemL、pgr7、hemA和rhtA相关酶活性,同时抑制了SDH5、LSC1、LSC2、YOR131C、GDH2酶活性,强化KGD2活性和弱化HEM2活性,以及携带蛋白支架序列GBD1SH32PDZ2,通过酵母融合从而获得酿酒酵母菌株,简称BY12-MPCP。
所述5-ALA合酶ALAS来源于Pichia pastoris;
所述PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、GDH1、GLT1、IDP1、AGC1 SDH5、LSC1、LSC2、YOR131、KGD2、HEM2、GDH2为酿酒酵母内源酶;
所述异柠檬酸裂合酶aceA和苹果酸合酶aceB来自E.coli;
所述丙氨酸-乙醛酸转氨酶AGXT来自Homo sapiens;
所述烟酰胺腺嘌呤二核苷酸替代氧化酶AOX1来自Hansenula anomala;
所述乙酰辅酶A水解酶ACH1来源于Yarrowia lipolytica;
所述吡哆醛生物合成裂解酶pdxS和谷氨酰胺氨基转移酶pdxT来源于Bacillussubtilis;
所述L-苏氨酸/高丝氨酸转运蛋白RhtA、谷氨酸转运核糖核酸连接酶gltX、谷氨酸转运核糖核酸还原酶HemA、谷氨酸L-氨基醛氨基转移酶HemL来自E.coli;
所述谷氨酸转运核糖核酸还原酶激活蛋白PGR7来自Arabidopsis thaliana。
发明的5-ALA高产工程菌应用酿酒酵母工程菌。
所述5-ALA高产工程菌应用酿酒酵母工程菌的发酵方法,包括如下步骤过程:
(1)将BY12-MPCP菌株接种于种子培养基,培养基包括葡萄糖1-3%、蛋白胨1-3%、酵母提取物0.5-2%,接入菌株后在30℃、220rpm条件下摇瓶培养获得种子液培养;
(2)将(1)中种子培养基按照体积比为1%的比例转接至发酵优化培养基中,在pH5-6、在30℃、300rpm条件培养。发酵优化培养基包括5-10g/L甘氨酸、1.44-1.87mM硫酸亚铁、30-34mM乙酰丙酸、0.01-0.05mM硫酸镁、3-4g/L硫酸铵、5-10g/L谷氨酸、2-4%葡萄糖、2-6%蛋白胨、1-4%酵母提取物。
(3)待培养基中的葡萄糖消耗完时,补加葡萄糖阶段的补料液:补料液包括5-10g/L甘氨酸、1.44-1.87mM硫酸亚铁、30-34mM乙酰丙酸、0.01-0.05mM硫酸镁、3-4g/L硫酸铵,维持葡萄浓度在1-2g/L,pH在5-6、温度在30℃、通气量为1-2VVM,溶氧设置为35-45%,;
(4)当菌株生长至OD600为38-40,开始补加半乳糖阶段的补料液:补料液包括1.44-1.87mM的硫酸亚铁、30-34mM乙酰丙酸、0.01-0.05mM硫酸镁、3-4g/L硫酸铵、5-10g/L谷氨酸,维持半乳糖浓度在1-2g/L、pH在5-6、温度在30℃、通气量为1-2VVM、溶氧设置为35-45%,培养至5-ALA的产量不增加时停止发酵。
所述5-ALA高产工程菌应用在食品、饲料、医药、农业及化妆品工业领域。
具体说明如下:
本发明中用到的5-ALA合酶ALAS来源于Pichia pastoris,异柠檬酸裂合酶aceA,苹果酸合酶aceB,L-苏氨酸/高丝氨酸转运蛋白RhtA,谷氨酸转运核糖核酸连接酶gltX、谷氨酸转运核糖核酸还原酶hemA、谷氨酸L-氨基醛氨基转移酶的编码基因hemL来自E.coli,谷氨酸转运核糖核酸还原酶激活蛋白PGR7来自Arabidopsis thaliana,丙氨酸-乙醛酸转氨酶AGXT来自Homo sapiens,烟酰胺腺嘌呤二核苷酸替代氧化酶AOX1来自Hansenulaanomala,乙酰辅酶A水解酶ACH1来源于Yarrowia lipolytica,吡哆醛生物合成裂解酶pdxS和谷氨酰胺氨基转移酶pdxT来源于Bacillus subtilis。PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、GDH1、GLT1、IDP1和AGC1为酿酒酵母内源酶,其核酸序列根据引物表1中的引物从Saccharomyces cerevisiae基因组中PCR获得。
本发明中一种高产5-氨基乙酰丙酸的工程菌,所述工程菌被修饰为与内源活性相比,具有增强的ALAS、PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、aceA、aceB、AGXT、AOX、ACH1、pdxS、pdxT相关酶活性,同时抑制了SDH5、LSC1、LSC2、YOR131C活性。其构建方法为:根据如图1所述的酿酒酵母CRISPR/Cas9基因编辑操作系统质粒,在delta位点多轮整合来源于Pichia pastoris的ALAS编码基因,其次过表达PYC2、PDA1、CIT1和ACO2基因,同时引入大肠杆菌来源的aceA以及aceB基因。然后敲除SDH5、LCS1、LCS2基因,并在LCS1、LCS2基因位点表达ACH1基因以及过表达AOX1和NDI1基因。之后过表达AGXT、GLY1、HEM25和YMC1基因,敲除YOR131C基因同时过表达来源枯草芽孢杆菌的pdxS、pdxT基因以及酿酒酵母自身的GND1、PDX3、MTM1、MCX1、GDH1、GLT1、IDP1和AGC1基因过表达,并引入外源基因gltX、hemL、pgr7、hemA和rhtA以及支架蛋白结合域序列GBD1SH32PDZ2,强化KGD2和弱化HEM2蛋白表达,通过不同配型之间的融合获得高产菌株BY12-MPCP。
本发明中一种高产5-氨基乙酰丙酸的工程菌BY12-MPCP的发酵培养基和发酵方法为;
(1)所述菌株接种于种子培养基(YPD),包括葡萄糖1-3%;蛋白胨1-3%;酵母提取物0.5-2%,在30℃、220rpm条件下摇瓶培养获得种子液培养;
(2)将种子液培养转接至发酵优化培养基中(OPYPD),所述OPYPD培养基包括甘氨酸(5-10g/L)、硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L)、谷氨酸(5-10g/L)、葡萄糖(2-4%)、蛋白胨(2-6%)、酵母提取物(1-4%)、pH(5-6),温度在30℃、300rpm条件培养。
(3)待培养基中的糖消耗完时,补加葡萄糖阶段的补料液:甘氨酸(5-10g/L)、硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L),维持葡萄浓度在1-2g/L。
(4)当菌株生长OD600至为38-40时,开始补加半乳糖阶段的补料液:硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L)、谷氨酸(5-10g/L),维持半乳糖浓度在1-2g/L、pH在5-6、温度在30℃、通气量为1-2VVM,溶氧设置为35-45%,培养至5-ALA的产量不增加时停止发酵。
本发明提供了上述携带上述基因修饰的酿酒酵母工程菌BY12-MPCP及其发酵培养基和发酵方法。
本发明提供了上述酿酒酵母工程菌BY12-MPCP在制备5-氨基乙酰丙酸方面的应用。
以上一个或多个技术方案的有益效果是:
1.本发明提供了一种新的酿酒酵母菌株,可用于5-氨基乙酰丙酸生产,该菌株具有增强的ALAS、PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、aceA、aceB、AGXT、AOX1、ACH1、pdxS、pdxT、GDH1、GLT1、IDP1、AGC1,gltX、hemL、pgr7、hemA和rhtA相关酶活性,同时抑制了SDH5、LSC1、LSC2、YOR131C、GDH2酶活性,强化KGD2活性和弱化HEM2活性,以及携带蛋白支架序列GBD1SH32PDZ2。
2.本发明的酿酒酵母菌株在优化培养基中可生产3.53g/L的5-氨基乙酰丙酸,是当前酵母中合成5-ALA最高产量。
3.本发明的酿酒酵母5-氨基乙酰丙酸生产菌株在构建过程中不需要添加抗生素,保证菌株食品安全,减少发酵成本。
附图说明
图1为CRISPR/Cas9操作系统的pCas9质粒的结构图。
图2为生物反应器发酵BY12-MPCP菌株5-ALA产量示意图。
具体实施方式
本发明通过下述实施例进一步阐明,但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。除非另有说明,所有技术和科学术语都具有本领域所知的常见含义。所有专利、专利申请、公开出版物、序列、以及其他公开材料均援引加入本文,除非另有说明。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下面结合实施例对本发明做进一步说明,下述实施例是为了使本领域的技术人员能够更好地理解本发明,但对本发明不作任何限制。
本发明所用到的原始菌株BY4741为通用菌株(ATCC编号4040002,基因型为MATa;his3Δ1;leu2Δ0;met15Δ0;ura3Δ0)。
原始质粒pCAS9来源于(Zhang,Y.,J.Wang,Z.Wang,et al.Nat Commun,2019,10(1):1053.)。
所用限制性内切酶、DNA聚合酶等分子生物学试剂购买自上海生工生物技术有限公司。
所用PCR仪,电转仪和分光光度计购自Bio-Rad。
本发明所涉及的基因,其中来自大肠杆菌E.coli的aceA和aceB基因克隆于大肠杆菌,来源于Yarrowia lipolytica的ACH1基因克隆于解脂耶氏酵母基因组,来源于Bacillussubtilis的pdxS和pdxT基因克隆于枯草芽孢杆菌,PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3基因克隆于酿酒酵母,来源于Pichia pastoris的HEM1基因,来自Homo sapiens的AGXT基因,来自Hansenula anomala的AOX1基因,来自大肠杆菌E.coli的gltX、hemL、hemA和rhtA基因经密码子优化后并合成(金维智,苏州,中国),支架蛋白结合域序列GBD1SH32PDZ2由苏州金维智合成。
YPD固体培养基(蛋白胨20g/L,酵母抽提物10g/L,葡萄糖20g/L,琼脂2%)余量是水。
YPD液体培养基(蛋白胨20g/L,酵母抽提物10g/L,葡萄糖20g/L)余量是水。
OPYPD液体培养基:蛋白胨20g/L,酵母抽提物10g/L,葡萄糖20g/L,甘氨酸5g/L、硫酸亚铁1.87mM,乙酰丙酸34mM,硫酸镁0.05mM,硫酸铵3.93g/L,5g/L谷氨酸、半乳糖糖2%,pH(5-6);
SC-dropout液体培养基(YNB 6.7g/L,dropout powder 1.3g/L,葡萄糖20g/L)余量是水。
发酵液中5-氨基乙酰丙酸的测定方法:采用Modified Ehrlich’s Reagent方法(Feng LL,et al.(2016).Biotechnol Bioeng 113(6):1284-1293.)检测。
胞内5-ALA检测的测定方法:取5mL发酵培养液,在10000×g 4℃条件下离心5min去除上清,保留沉淀菌体,沉淀菌体在真空冷冻干燥机中去除多余水分,然后用1mL蒸馏水重悬沉淀菌体;往上述悬浮液中分别加入2mL 1M乙酸盐缓冲液(pH 4.6)和0.4mL乙酰乙酸乙酯,然后置于100℃条件下水浴12min;上述混合液降至室温后,加入4mL乙酸乙酯,震荡混匀,然后静止至分层。取适量乙酸乙酯层加入等体积Ehrlich’s reagent进行显色反应检测胞内5-ALA含量。
实施例1
工程菌株构建过程中用到的基因的制备:
根据NCBI上提供的来自大肠杆菌的异柠檬酸裂合酶aceA(GeneID:948517)和苹果酸合酶aceB(GeneID:948512)的核苷酸序列,经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后得到aceA和aceB基因序列。aceA的核苷酸序列如SEQ ID NO:1所示,aceB的核苷酸序列如SEQ ID NO:2所示。
根据NCBI上提供的来自Yarrowia lipolytica的ACH1基因(NC_013741.1)的核苷酸序列经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后的乙酰辅酶A水解酶ACH1。ACH1的核苷酸序列如SEQ ID NO:3所示。
根据NCBI上提供的来自Homo sapiens的丙氨酸-乙醛酸转氨酶AGXT核苷酸序列经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后的丙氨酸-乙醛酸转氨酶AGXT。AGXT的核苷酸序列如SEQ ID NO:4所示。
根据NCBI上提供的来自Hansenula anomala的烟酰胺腺嘌呤二核苷酸替代氧化酶AOX1的核苷酸序列经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后的烟酰胺腺嘌呤二核苷酸替代氧化酶AOX1的核苷酸序列如SEQ ID NO:5所示。
根据NCBI上提供的来自Bacillus subtilis的吡哆醛生物合成裂解酶pdxS和谷氨酰胺氨基转移酶pdxT来源于的核苷酸序列经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后的吡哆醛生物合成裂解酶pdxS的核苷酸序列如SEQ ID NO:6所示,谷氨酰胺氨基转移酶pdxT的核苷酸序列如SEQ ID NO:7所示。
根据NCBI上提供的来自Pichia pastoris的5-氨基乙酰丙酸合酶编码基因HEM1基因经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后的5-氨基乙酰丙酸合酶编码基因HEM1基因的核苷酸序列如SEQ ID NO:8所示。
根据NCBI上提供的来自大肠杆菌的L-苏氨酸/高丝氨酸转运蛋白RhtA(GeneID:947045)的核苷酸序列,经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后得到rhtA基因序列。rhtA的核苷酸序列如SEQ ID NO:9所示。
根据NCBI上提供的来自大肠杆菌的谷氨酸连接酶gltX(GeneID:946906)的核苷酸序列,经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后得到gltX基因序列。gltX的核苷酸序列如SEQ ID NO:10所示。
根据NCBI上提供的来自大肠杆菌的谷氨酸转运核糖核酸还原酶hemA(GeneID:945777)的核苷酸序列,经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后得到hemA基因序列。hemA的核苷酸序列如SEQ ID NO:11所示。
根据NCBI上提供的来自大肠杆菌的谷氨酸L-氨基醛氨基转移酶的编码基因hemL(GeneID:946892)的核苷酸序列,经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后得到hemL基因序列。hemL的核苷酸序列如SEQ ID NO:12所示。
根据NCBI上提供的来自Arabidopsis thaliana的谷氨酸转运核糖核酸还原酶激活蛋白的编码基因PGR7(NC_003074.8)的核苷酸序列,经过密码子优化后,委托苏州金唯智生物科技有限公司合成优化后得到PGR7基因序列。PGR7的核苷酸序列如SEQ ID NO:13所示。
根据NCBI上提供的Saccharomyces cerevisiae中丙酮酸羧化酶基因PYC2(GeneID:852519),丙酮酸脱氢酶(乙酰基转移)亚基基因PDA1(GeneID:856925),乌头酸酶同工酶基因ACO2(GeneID:853230),烟酰胺腺嘌呤二核苷酸磷酸-泛氨基酮氧化还原酶基因NDI1(GeneID:854919),苏氨酸醛酶基因GLY1(GeneID:856665),线粒体甘氨酸转运蛋白基因HEM25(GeneID:851439),线粒体内膜甘氨酸转运蛋白基因YMC1(GeneID:856171),磷酸葡萄糖酸脱氢酶基因GND1(GeneID:856589),吡啶磷酸氧化酶PDX3(GeneID:852323),假定的卤酸脱卤酶样水解酶基因YOR131C(GeneID:854299),线粒体载体的线粒体蛋白基因MTM1(GeneID:853173),非蛋白水解三磷酸腺苷酶基因MCX1(GeneID:852528)核苷酸序列,线粒体烟酰胺腺嘌呤二核苷酸磷酸特异性异位酸脱氢酶编码基因IDP1(GeneID:851493),烟酰胺腺嘌呤二核苷酸磷酸依赖性谷氨酸合酶编码基因GLT1(GeneID:851383),烟酰胺腺嘌呤二核苷酸磷酸依赖性谷氨酸脱氢酶编码基因GDH1(GeneID:854557),线粒体谷氨酸转运蛋白编码基因AGC1(GeneID:856132),α-酮戊二酸脱氢酶编码基因KGD2(GeneID:851726),卟啉原合酶编码基因HEM2(GeneID:852842),线粒体定位肽Cox4(GeneID:852688)核苷酸序列,根据引物表1中的引物从Saccharomyces cerevisiae基因组中PCR获得。
表1引物序列
实施例2
本实施例用到的CRISRPR/Cas9操作系统质粒的结构见图1。
本实施例所使用的基因敲除或敲入系统是根据已有的CRISRPR/Cas9操作系统。针对不同的sgRNA进行的质粒构建都是基于原始质粒pCAS9,来源为来源于(Zhang,Y.,J.Wang,Z.Wang,et al.Nat Commun,2019,10(1):1053.)。该质粒上有一个酶切位点BsaI,该酶切位点上游含有启动子SNR52用于启动sgRNA的表达,将sgRNA的20bp序列插入该位点即获得了sgRNA质粒。插入HEM1所用的sgRNA序列为5‘-CAACATTCACCCATTTCTCA-3’。插入基因PYC2和PDA1所用的sgRNA序列为5‘-TGCTTCTCGCTATACCAGAG-3’。插入基因CIT2所用的sgRNA为5‘-CTGCATGGTCGTGCGGTCGT-3’。敲入基因aceA和aceB所用的sgRNA为5‘-GACATTTTTGTTCCATGCGG-3’。敲除基因SDH5所用的sgRNA为5‘-CTGCATGGTCGTGCGGTCGT-3’。敲除基因LSC1/2并插入ACH1用的sgRNA1为5‘-TTAGTTGGGCCCAACTGCCC-3’,sgRNA2为5‘-TGGCGGCTTCTACCAGCCCC-3’。插入基因AOX1和NDI1的sgRNA为5‘-AATACAGGTTTATATGTATC-3’。插入基因AGXT1和GLY1所用的sgRNA为5‘-AGCGTGCGTCTGATTTCTAC-3’。插入基因YMC1和HEM25所用的sgRNA为5‘-ccacaggtacatgcagagac-3’。敲除基因YOR131C并插入基因pdsT/X所用的sgRNA为5‘-TACTAGAAATGTCGGAGCCC-3’。插入基因PDX3和GND1所用的sgRNA为5‘-AATACAGGTTTATATGTATC-3’。插入基因MCX1和MTM1所用的sgRNA为5‘-TCTTTCCTTGCAAAGTTCAT-3’。插入COX4-GDH1和COX4-GLT1所用的sgRNA序列为5‘-GACATTTTTGTTCCATGCGG-3’。插入基因COX4-GLT1所用的sgRNA序列为5‘-AGTTCTTCAAAATAAATAAA-3’。插入基因CIT2所用的sgRNA为5‘-CTGCATGGTCGTGCGGTCGT-3’。插入基因COX4-gltx和COX4-hemL所用的sgRNA为5‘-TACCCAAAGACCAGTCTACT-3’。插入基因COX4-hemA和COX4-PGR7所用的sgRNA为5‘-TGTCCCCTCTATCTCCGCTC-3’。插入来源于(Dueber,J.E.,B.J.Yeh,K.Chak,et al.Science,2003,301(5641):1904-1908.)支架蛋白结合域序列GBD1SH32PDZ2所用的sgRNA为5‘-TGAACGCACATTGCGCCCCT-3’。插入基因AGC1所用的sgRNA为5‘-AAGGGTGACTGGAACGGTGC-3’。插入基因rhtA所用的sgRNA为5‘-TTGTCACAGTGTCACATCAG-3’。替换KGD2基因的启动子所用的sgRNA为5‘-ACAGCCGTCCTAGGCTTGCA-3’。替换HEM2基因的启动子所用的sgRNA为5‘-CAAAAATTCAGCTGTATGCA-3’。插入基因MATα所用的sgRNA为5‘-GTTCTAAAAATGCCCGTGCT-3’。
实施例3
构建高产5-ALA酿酒酵母工程菌株BY12-MPCP。
基于已有的CRISPR/Cas9操作系统,将含有HEM1的基因表达盒片段和对应的基因编辑质粒pCAS9导入S.cerevisiae BY4741中,在基因组的delta位点处进行整合。
具体方法如下:
(1)取一环酵母菌接种于5mL YPD液体培养基中,220rpm,30℃培养12-16h。然后按初始OD600=0.2转接到50mL YPD液体培养基中,在30℃220rpm条件下培养4-5h至OD=1.3-1.4。
(2)上述所有酵母细胞在3000rpm 4℃离心3min收集沉淀菌体,然后用20mL预冷的1M山梨醇溶液重悬,并在相同的离心条件下离心收集沉淀细胞。
(3)沉淀细胞用16mL 1M山梨醇溶液,2mL 10×TE缓冲液和2mL 1M醋酸锂溶液重悬然后在30℃孵育30min。
(4)在孵育的菌液中加200uL 1M DTT溶液在30℃继续孵育15min,然后在3000rpm4℃离心收集菌体;
(5)沉淀细胞再用20mL预冷1M山梨醇溶液洗两次,去上清,沉淀细胞重悬在0.4mL1M山梨醇溶液中,每管分装100-200μL保存在-80℃备用;
(6)在100μL感受态细胞中加入16.5μg donors片段和0.5μg质粒或者10μL GoldenGate反应液。
(7)电转后的感受态细胞立即加2mL的YPD和1mL 1M山梨醇溶液洗在30℃220rpm恢复培养5h,然后转到SC-dropout液体培养基中在30℃220rpm继续培养24h,然后涂布至SC-dropout板;
(8)酵母电穿孔转化电击条件:电压:1500V;电阻:400Ω;电容:25μF;脉冲时间:10ms;一次电击。
(9)随机挑选转化子,利用引物F:5’-GCAAAGCAGGACCAACAAC-3’和R:5‘-CGTTAGAGCTGATATAGAGGCG-3’验证。(10)根据胞内外的检测方法分别检测转化子5-ALA水平。
为了进一步提高菌株5-ALA素产量,本实施例基于已有的CRISPR/Cas9操作系统,在上述菌株中过表达PYC2、PDA1、CIT1和ACO2基因。由于酿酒酵母ICL1基因的表达会被葡萄糖抑制,因此进一步引入大肠杆菌来源的aceA以及aceB基因。在酿酒酵母中,琥珀酸在琥珀酸脱氢酶(由SDH5基因编码)的催化下降解为富马酸;而且琥珀酰辅酶a也会被琥珀酰辅酶a连接酶(由LCS1/2基因编码)降解为琥珀酸。因此,进一步敲除SDH5、LCS1和LCS2基因,以及在这两个位点同时过表达ACH1基因。为了提高胞内辅酶水平,从而提高5-氨基乙酰丙酸的合成,进一步过表达AOX1和NDI1基因。此外在染色体XI-3.3位点过表达AGXT、GLY1、HEM25和YMC1基因,敲除YOR131C基因阻断了PLP降解途径,进一步过表达了来源枯草芽孢杆菌的BSpdxS/T基因以及酿酒酵母自身的GND1和PDX3基因。由于PLP同样需要有细胞质转运至线粒体细胞器,因此,我们进一步过表达了PLP转运蛋白MTM1基因和激活PLP和5-ALA合酶结合的激活蛋白的编码基因MCX1。然后敲除GDH2基因,过表达了IDP1和AGC1基因,并将GDH1、GLT1和C5途径基因gltX、hemL、pgr7、hemA定位至线粒体和细胞质中,进一步引入5-ALA转运蛋白RhtA,用CIT2p替换KGD2p启动子、用KEX2p调控HEM2基因表达,用MATa替换MATα基因表达盒片,将不同配型细胞融合获得重组菌株BY12-MPCP。
详细步骤如下:(1)取相同生物量(OD600)的两种交配型的新鲜酵母细胞,重悬于4mL YPD液体培养基中,继续培养4-6h;期间每隔1-2h用显微镜观察细胞的形态,直到菌株出现哑铃状结构停止培养;(2)将上述混合的菌液涂布于YPD固体培养基上,在30℃下培养1~2d;(3)从上述YPD固体培养基上挑取少量菌体,划线到另一新鲜的YPD固体培养基上,多次划线以得到单菌落;(4).待平板中长出单菌落后,通过PCR验证交配型。验证引物为MAT-F5’AGTCACATCAAGATCGTTTATGG3’;MAT-a5’ACTCCACTTCAAGTAAGAGTTTG3’;MAT-alpha5’GCACGGAATATGGGACTACTTCG3’。在PCR反应体系中加入上述三条引物,a型酵母细胞的PCR产物为544bp,α型酵母细胞的PCR产物为404bp,a/α型酵母细胞的PCR产物为两条分别是404bp和544bp。
实施例4
一种高产5-ALA酿酒酵母工程菌株BY12-MPCP的发酵培养基:
菌株的种子培养基中(YPD),包括葡萄糖1~3%;蛋白胨1~3%;酵母提取物0.5~2%;
菌株的发酵优化培养基中(OPYPD),包括甘氨酸(5-10g/L)、硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L)、5-10g/L谷氨酸、葡萄糖(2~6%);蛋白胨(2~6%);酵母提取物(1~4%)、pH(5-6)。
葡萄糖阶段的补料液:甘氨酸(5-10g/L)、硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L)、葡萄糖(60-80%)。
半乳糖阶段的补料液:谷氨酸(5-10g/L)、硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L)、半乳糖(60-80%)。
实施例5
一种高产5-ALA酿酒酵母工程菌株BY12-MPCP的发酵方法,包括如下述步骤:
(1)活化菌株:将高产5-ALA酿酒酵母工程菌株BY12-MPCP在YPD固体培养基中划线,30℃培养20-26h,使菌株复壮;
(2)种子液的培养:将步骤(1)获得的活化菌接种到YPD液体培养基中,30℃,220rpm培养12h,然后将所得菌液按照体积比为10%的接种量接种到一种实施例4制备的用于高产5-ALA酿酒酵母工程菌株的发酵培养基中,30℃,220rpm培养20-26h,得到种子液;
(3)发酵罐发酵:将步骤(2)所获得的种子液按照体积比为1%的比例接种到装有2L实施例4制备的一种用于高产5-ALA酿酒酵母工程菌株的发酵培养基(OPYPD)的5L发酵罐中,在30℃,pH=5.5,0-12h期间搅拌转速300rpm,12h以后搅拌转速为300-800rpm的条件下发酵,采用连续补料的方式补充实施例4制备的一种用于高产5-ALA酿酒酵母工程菌株的葡萄糖阶段的补料培养基,维持葡萄浓度在1-2g/L,pH在5-6、温度在30℃、通气量为1-2VVM,溶氧为35-45%;
(4)当菌株生长至OD600为38-40时,开始补加半乳糖阶段的补料液:硫酸亚铁(1.44-1.87mM)、乙酰丙酸(30-34mM)、硫酸镁(0.01-0.05mM)、硫酸铵(3-4g/L)、谷氨酸(5-10g/L),维持半乳糖浓度在1-2g/L、pH在5-6、温度在30℃、通气量为1-2VVM,溶氧设置为35-45%,培养至5-ALA的产量不增加时停止发酵。经过76h发酵,5-ALA产量达到3.53g/L(图2)。
本发明公开和提出的技术方案,本领域技术人员可通过借鉴本文内容,适当改变条件路线等环节实现,尽管本发明的方法和制备技术已通过较佳实施例子进行了描述,相关技术人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和技术路线进行改动或重新组合,来实现最终的制备技术。特别需要指出的是,所有相类似的替换和改动对本领域技术人员来说是显而易见的,他们都被视为包括在本发明精神、范围和内容中。本发明未尽事宜属于公知技术。
Claims (5)
1.一种5-ALA高产工程菌,其特征在于,以BY4741为出发菌株,经过基因修饰后具有增强的ALAS、PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、aceA、aceB、AGXT、AOX1、ACH1、pdxS、pdxT、GDH1、GLT1、IDP1、AGC1,gltX、hemL、pgr7、hemA和rhtA相关酶活性,同时抑制了SDH5、LSC1、LSC2、YOR131C、GDH2酶活性,强化KGD2活性和弱化HEM2活性,以及携带蛋白支架序列GBD1SH32PDZ2,通过酵母融合从而获得酿酒酵母菌株,简称BY12-MPCP。
2.如权利要求1所述一种5-ALA高产工程菌,其特征在于,所述5-ALA合酶ALAS来源于Pichia pastoris;
所述PDA1、PYC2、CIT2、ACO2、GLY1、HEM25、YMC1、MTM1、MCX1、NDI1、GND1、PDX3、GDH1、GLT1、IDP1、AGC1 SDH5、LSC1、LSC2、YOR131、KGD2、HEM2、GDH2为酿酒酵母内源酶;
所述异柠檬酸裂合酶aceA和苹果酸合酶aceB来自E.coli;
所述丙氨酸-乙醛酸转氨酶AGXT来自Homo sapiens;
所述烟酰胺腺嘌呤二核苷酸替代氧化酶AOX1来自Hansenula anomala;
所述乙酰辅酶A水解酶ACH1来源于Yarrowia lipolytica;
所述吡哆醛生物合成裂解酶pdxS和谷氨酰胺氨基转移酶pdxT来源于Bacillussubtilis;
所述L-苏氨酸/高丝氨酸转运蛋白RhtA、谷氨酸转运核糖核酸连接酶gltX、谷氨酸转运核糖核酸还原酶HemA、谷氨酸L-氨基醛氨基转移酶HemL来自E.coli;
所述谷氨酸转运核糖核酸还原酶激活蛋白PGR7来自Arabidopsis thaliana。
3.权利要求1所述5-ALA高产工程菌应用酿酒酵母工程菌。
4.权利要求3所述5-ALA高产工程菌应用酿酒酵母工程菌的发酵方法,其特征在于;包括如下步骤过程:
(1)将BY12-MPCP菌株接种于种子培养基,培养基包括葡萄糖1-3%、蛋白胨1-3%、酵母提取物0.5-2%,接入菌株后在30℃、220rpm条件下摇瓶培养获得种子液培养;
(2)将上述种子培养液按照体积比为1%的比例转接至发酵优化培养基中,发酵优化培养基包括5-10g/L甘氨酸、1.44-1.87mM硫酸亚铁、30-34mM乙酰丙酸、0.01-0.05mM硫酸镁、3-4g/L硫酸铵、5-10g/L谷氨酸、2-4%葡萄糖、2-6%蛋白胨、1-4%酵母提取物;接入种子培养液后在pH 5-6、在30℃、300rpm条件培养;
(3)待培养基中的葡萄糖消耗完时,补加葡萄糖阶段的补料液:补料液包括5-10g/L甘氨酸、1.44-1.87mM硫酸亚铁、30-34mM乙酰丙酸、0.01-0.05mM硫酸镁、3-4g/L硫酸铵,维持葡萄浓度在1-2g/L,pH在5-6在30℃、通气量为1-2VVM,溶氧为35-45%;
(4)当菌株生长至OD600为38-40,开始补加半乳糖阶段的补料液,补料液包括1.44-1.87mM硫酸亚铁、30-34mM乙酰丙酸、0.01-0.05mM硫酸镁、3-4g/L硫酸铵、5-10g/L谷氨酸,维持半乳糖浓度在1-2g/L、pH在5-6、温度在30℃、通气量为1-2VVM、溶氧为35-45%、培养至5-ALA的产量不增加时停止发酵。
5.权利要求1所述5-ALA高产工程菌应用在食品、饲料、医药、农业及化妆品工业领域。
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