CN113481233B - 构建依克多因生产菌的方法 - Google Patents
构建依克多因生产菌的方法 Download PDFInfo
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- CN113481233B CN113481233B CN202110750324.7A CN202110750324A CN113481233B CN 113481233 B CN113481233 B CN 113481233B CN 202110750324 A CN202110750324 A CN 202110750324A CN 113481233 B CN113481233 B CN 113481233B
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Abstract
本发明公开了一种构建依克多因生产菌的方法,构建的基因工程菌CGMCC No.22733能够以葡萄糖为碳源,通过发酵直接生产依克多因,5L发酵罐发酵48小时,可产依克多因42.7g/L,具有开发和应用潜力。
Description
技术领域
本发明属于代谢工程和基因工程领域,具体地说,涉及一种构建依克多因生产菌的方法、构建得到的基因工程菌及其应用。
背景技术
依克多因(Ectoin),化学名为2-甲基-1,4,5,6,-四氢嘧啶-4-羧酸,又名四氢甲基嘧啶羧酸,易溶于水、甘油、丙二醇、乙醇等。分子为C6H10O2N2,结构式如下所示:
1985年,Galinski教授在埃及沙漠发现嗜盐菌在高温、干燥、强UV照射、高盐度环境下,会在细胞外层生成一种天然保护成分-依克多因,能使嗜盐菌免受伤害,从而进行自我修护;研究表明,依克多因同样对皮肤有很好的修复保护作用。他是欧莱雅、雅诗兰黛等高档化妆品采用的生物工程制剂之一,同时还可用于医疗保健,比如应激性皮炎,肺炎,过敏性鼻炎和老年痴呆症等。有文献报道,依克多因的市场售价达1000美元/公斤(Genes2018,9(4),177.),未来具有较大市场潜力。
目前,Ectoin主要是以嗜盐菌发酵来制备,高盐发酵不仅影响发酵容器的寿命,对后续发酵废液处理也是很大的挑战。1999年,Ono等报道了高嗜盐菌(Halomonas elongata)中依克多因的合成途径并鉴定了相关酶(J Bacteriol.1999,181:91-9.),早期发酵生产主要是使用这个菌株,同时还生成氢化依克多因。随着基因工程技术的发展,研究人员尝试用大肠杆菌和谷氨酸棒杆菌等常规发酵宿主,通过基因工程改造使其产Ectoin(参见MicrobCell Fact.2021,20:76.),大肠杆菌基因工程菌株可产25g/L依克多因,转化率达到0.11g/g葡萄糖(Metabolic Engineering.2016,36:10-18.);Becker等以谷氨酸棒杆菌为宿主菌构建了高产赖氨酸的基因工程菌株,发酵浓度可达120g/L(Metabolic Engineering.2011,13:159-168.);随后,他们又在谷氨酸棒杆菌LYS-1中表达了来源于Pseudomonas stutzeri的ectABC基因,并敲除lysE基因以便防止赖氨酸的外排,在低盐浓度下可产依克多因4.5g/L(Microbial Cell Factories.2013,12:110.);Giesselmann等以Corynebacteriumglutamicum lysCfbr作为出发菌株,通过不同的启动子组合优化ectA、ectB和ectC的表达,平衡了相关代谢流,最终获得菌株发酵56h可产65g/L依克多因(Biotechnol.J.2019,14,1800417),转化率达到0.19g/g葡萄糖。
发明内容
发明人对已报道的谷氨酸棒杆菌中依克多因的生物合成路线进行了研究,开发出一种新的代谢路径,以产赖氨酸的谷氨酸棒杆菌为出发菌株,通过弱化hom基因,敲除pck基因,并用sod启动子增强lysC和tkt-tal-zwf的表达,然后在ddh基因位点和噬菌体转座酶IS30(IS30-like element,ISCg2family transposase)基因位点整合ectABC基因,构建出依克多因生产菌。因此,本发明包括如下技术方案。
一种构建依克多因生产菌的方法,其包括以下步骤:
A.以产赖氨酸的谷氨酸棒杆菌为出发菌株,敲减或弱化基因组中编码高丝氨酸激酶的hom基因,得到下调高丝氨酸激酶表达的菌株A;其中,hom基因的敲减包括、但不限于敲除hom基因、下调hom基因的表达、或者弱化hom的高丝氨酸激酶的酶活力,例如可以使hom基因发生突变从而弱化高丝氨酸激酶的功能;
B.敲除菌株A基因组中编码磷酸烯醇式丙酮酸羧激酶的pck基因,得到缺失磷酸烯醇式丙酮酸羧激酶的菌株B;
C.增强菌株B的基因组中编码天冬氨酸激酶的基因lysC的表达,得到过表达天冬氨酸激酶的菌株C;
D.增强菌株C的基因组中编码转酮醇酶基因tkt、编码酪氨酸解氨酶基因tal和编码6-磷酸葡萄糖酸脱氢酶基因zwf的表达,得到氧化磷酸化途径增强的菌株D;
E.在菌株D基因组中二氨基丙酸脱氢酶的编码基因ddh位点整合来源于施氏假单胞菌Pseudomonas stutzeri的ectABC基因(即ectA、ectB和ectC,简写ECT),得到依克多因合成途径增强的菌株E;
F.在菌株E基因组中噬菌体转座酶IS30基因位点整合来源于施氏假单胞菌Pseudomonas stutzeri的ectABC基因,得到依克多因合成途径进一步增强的菌株F,筛选阳性克隆,得到依克多因生产菌。
优选地,上述的方法还包括在基因组中叠加整合ectABC基因的下述步骤:
G.在步骤F所得菌株F的基因组中进一步整合来源于施氏假单胞菌Pseudomonasstutzeri的ectABC基因,筛选依克多因合成途径增强的阳性克隆。
在一种实施方式中,步骤A中所述出发菌株是Corynebacterium glutamicumATCC13032LysCfbr。该菌株可按照文献Biotechnol.J.2019,14,1800417.报道的方法构建得到。
上述步骤A中hom基因的敲减可以通过使hom基因发生T176C突变而实现,高丝氨酸激酶发生T176C突变后酶活力会发生降低。
例如,hom基因发生T176C突变的步骤为:将核苷酸序列为SEQ ID NO:4的质粒pK18mobSacB-homT176C导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
上述步骤B中pck基因的敲除可以包括下述步骤:将核苷酸序列为SEQ ID NO:2的质粒pK18mobSacB-KOpck导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
上述步骤C中的基因lysC表达的增强可以通过将lysC基因置于sod启动子下游而实现。例如,可以包括下述步骤:将核苷酸序列为SEQ ID NO:1的质粒pK18mobSacB-Psod-lysC导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
上述步骤D中的基因tkt、tal和zwf表达的增强可以通过用sod启动子替换其天然启动子而实现。例如,可以包括下述步骤:将核苷酸序列为SEQ ID NO:3的质粒pK18mobSacB-Psod-tkt-tal-zwf导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
上述步骤E中ectABC基因在ddh基因位点的整合可以包括下述步骤:将核苷酸序列为SEQ ID NO:5的质粒pK18mobSacB-ddh-ECT导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
上述步骤F中ectABC基因在IS30基因位点的整合可以包括下述步骤:将核苷酸序列为SEQ ID NO:6的质粒pK18mobSacB-IS30-ECT导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
上述步骤中质粒的导入可以是氯化钙转化法或电转化,优选电转化。
本发明的第二个目的在于提供一种依克多因生产菌,其通过上述的方法构建和筛选得到。
优选地,上述依克多因生产菌可以是谷氨酸棒杆菌(Corynebacteriumglutamicum),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo.22733。
本发明的第三个目的在于提供上述依克多因生产菌比如CGMCC No.22733用于生产依克多因的用途。
例如,可以通过上述依克多因生产菌比如CGMCC No.22733的发酵来生产依克多因。
在发酵时,需要将葡萄糖作为碳源。
在一种实施方式中,摇瓶发酵种子培养基的组成可以为:蛋白胨10g/L,牛肉浸膏5g/L,酵母粉5g/L,氯化钠2.5g/L,尿素2g/L,葡萄糖10g/L。使用时115℃灭菌15min。
摇瓶发酵培养基可以为:
发酵罐培养基可以为:
发酵温度优选是30℃左右。
本发明开发出了一种新的依克多因代谢路线,使得谷氨酸棒杆菌能够通过发酵直接产生依克多因,值得进一步开发利用。
本发明构建的依克多因高产基因工程菌的拉丁学名是Corynebacteriumglutamicum,中文名称是谷氨酸棒杆菌或谷氨酸棒状杆菌,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期是2021年6月18日,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC No.22733。
附图说明
图1为本发明构建的谷氨酸棒杆菌中依克多因的代谢路线图。
图2是本发明构建的质粒pK18mobsacB的结构示意图。
图3是本发明构建的质粒pK18mobSacB-Psod-lysC的结构示意图。其核苷酸序列为SEQ ID NO:1。
图4是本发明构建的质粒pK18mobSacB-KOpck的结构示意图。其核苷酸序列为SEQID NO:2。
图5是本发明构建的质粒pK18mobSacB-Psod-tkt-tal-zwf的结构示意图。其核苷酸序列为SEQ ID NO:3。
图6是本发明构建的质粒pK18mobSacB-homT176C的结构示意图。其核苷酸序列为SEQ ID NO:4。
图7是本发明构建的质粒pK18mobSacB-ddh-ECT的结构示意图。其核苷酸序列为SEQ ID NO:5。
图8是本发明构建的质粒pK18mobSacB-IS30-ECT的结构示意图。其核苷酸序列为SEQ ID NO:6。
图9是本发明构建的基因工程菌ATCC13032-ECT-01的发酵曲线图。其中横坐标为发酵取样时间,纵坐标为发酵液中依克多因浓度。
具体实施方式
本发明对产赖氨酸的谷氨酸棒杆菌内的依克多因合成的代谢途径进行了改变,如图1所示,可以从葡萄糖出发,通过酶系催化实现依克多因的生物合成。
为了创建新的依克多因代谢路线,需要将待敲除、弱化、整合、过表达、强化表达的各种基因分别克隆在质粒载体上。然后分别地、先后地或者同时地转入谷氨酸棒杆菌(C.glutamicum)感受态细胞中;也可以将两个以上基因克隆在一个质粒上,然后分别地、先后地或者同时地转入谷氨酸棒杆菌感受态细胞中。
其中,可用的质粒载体包括pK18mobsacB等,但不限于此。
应理解,在构建本发明的基因工程菌的具体操作中,步骤A、步骤B、步骤C、步骤D、步骤E、步骤F、步骤G的排序并非完全根据英文字母顺序由前到后地固定不变,它们可以交叉、颠倒地操作,只要每个步骤能实现各自的功能、完成宿主细胞基因型的定向改变即可。
在本文中,为了描述简便,有时会将某种酶比如高丝氨酸激酶(hom)与其编码基因(DNA)名称混用,本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如,对于hom,用于描述高丝氨酸激酶的功能或类别时,指的是蛋白质;在作为一种基因描述时,指的是编码该酶的基因。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
实施例中的引物和基因合成、测序由江苏金唯智生物技术有限公司和安徽通生物技术有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等,主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB液体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠。
LB固体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,20g/L琼脂粉。
BHIS培养基:37g/L BHI,91g/L山梨醇。
BHIS-suc培养基:37g/LBHI,91g/L山梨醇,200g/L蔗糖,10g/L葡萄糖。
BHI培养基:37g BHI粉末,加入到1L纯水中,115℃灭菌15min。
LB-SUC100:1L LB固体培养基中加入100g蔗糖。
LB-SUC100-K25:1L LB固体培养基中加入100g蔗糖,25mg卡那霉素。
20X电转母液:80g/L甘氨酸,2%吐温80。
以下实施例中,当使用含卡那霉素的培养基时,所述卡那霉素在培养基中的终浓度为50μg/ml。
依克多因的HPLC检测方法:安捷伦1260高效液相色谱仪;色谱柱为安捷伦AQ-C18,检测器VWD检测器,检测波长210nm,流动相为10Mm磷酸二氢钾(pH3.5):乙腈=99:1,流速0.7ml/min,柱温箱20℃。依克多因的RT为5.4min。
实施例中所使用的出发菌株为谷氨酸棒杆菌ATCC13032LysCfbr,由浙江华睿生物技术有限公司按照参考文献Biotechnol.J.2019,14,1800417.报道的方法构建。
实施例中所使用的质粒pK18mobsacB、pK18mobSacB-Psod-lysC、pK18mobSacB-KOpck、pK18mobSacB-Psod-tkt-tal-zwf、pK18mobSacB-homT176C、pK18mobSacB-ddh-ECT和pK18mobSacB-IS30-ECT由浙江华睿生物技术有限公司构建,任何单位和个人都可以获得这些质粒用于验证本发明,但未经允许不得用作其他用途,包括开发利用、科学研究和教学。
实施例中使用的引物序列信息如表1所示。
表1、引物序列
注:表中引物名称后缀F代表正向引物,R代表反向引物。
实施例1:基因改造靶点质粒构建
1.1构建质粒pK18-Psod-lysC
以谷氨酸棒杆菌ATCC13032LysCfbr基因组为模板,以Psod-lysC-up-F/Psod-lysC-up-R和Psod-lysC-down-F/Psod-lysC-down-R引物对分别进行PCR扩增。PCR扩增条件:95℃5min;92℃30s,58℃30s,65℃35s,30个循环;65℃10min。扩增得到Psod-lysC-up、Psod-lysC-down片段,长度均为1k,切胶回收。
以ATCC1303LysCfbr基因组为模板,以Psod-lysC-F,Psod-lysC-R为引物进行扩增,得到Psod-lysC片段,长度为200bp,切胶回收。以Psod-lysC-up-F,Psod-lysC-down-R为引物,将Psod-lysC-up,Psod-lysC-down,Psod-lysC三个片段进行overlap PCR,产物切胶回收,成为Psod-lysC片段。以pK18mobsacB质粒为模板,以Psod-lysC-Z-F,Psod-lysC-Z-R为引物进行扩增,得到Psod-lysC-Z片段,长度为5.7kb,切胶回收。将Psod-lysC-Z与Psod-lysC片段进行组装(Transgen,Seamless Cloning and Assembly Kit),转化DH5a感受态细胞,复苏,涂布kan平板,以M13F,M13R为鉴定引物进行菌落PCR,阳性克隆应有2.2kb的条带,挑选阳性克隆接种试管,抽提质粒备用。
获得的质粒pK18-Psod-lysC的结构见图3,其核苷酸序列为SEQ ID NO:1。
1.2构建质粒pK18-KOpck
以ATCC13032LysCfbr基因组为模板,分别以KOpck-up-F/KOpck-up-R和KOpck-down-F/KOpck-down-R引物对分别进行扩增。PCR扩增条件:95℃5min;92℃30s,58℃30s,65℃35s,30个循环;65℃10min。扩增获得KOpck-up,KOpck-down片段,长度均为1kb,切胶回收。以pK18mobsacB质粒为模板进行扩增,获得KOpck-Z片段,长度为5.7kb,切胶回收。将KOpck-up,KOpck-down以及KOpck-Z进行Gibson组装,转化DH5a宿主,复苏,涂布kan平板,以M13F,M13R为鉴定引物进行鉴定,阳性克隆应有2kb的条带,接种菌落PCR阳性克隆,抽提质粒备用。
获得的质粒pK18-KOpck的结构见图4,其核苷酸序列为SEQ ID NO:2。
1.3构建质粒pK18-Psod-tkt-tal-zwf
以ATCC13032LysCfbr基因组为模板,分别以Psod-ttz-uP-F/Psod-ttz-up-R和Psod-ttz-down-F/Psod-ttz-down-R物对分别进行扩增。PCR扩增条件:95℃5min;92℃30s,58℃30s,65℃35s,30个循环;65℃10min。扩增得到的三个片段大小分别为1kb,1kb,200bp,获得ttz-up,ttz-down,Psod-ttz片段,切胶回收。以Psod-ttz-uP-F,Psod-ttz-down-R为引物,以ttz-up,ttz-down,Psod-ttz三个片段为模板,进行overlap PCR,长度2.2kb,切胶回收,命名为Psod-ttz-OE片段。Psod-ttz-Z-F/Psod-ttz-Z-R引物对以pK18mobsacB质粒为模板进行扩增,获得Psod-ttz-Z片段,长度为5.7kb,切胶回收。将Psod-ttz-OE,Psod-ttz-Z两个片段进行Gibson组装,转化DH5a宿主,复苏,涂布kan平板,以M13F,M13R为鉴定引物进行鉴定,阳性克隆应有2.2kb的条带,接种菌落PCR阳性克隆抽提质粒备用。
获得的质粒pK18-Psod-tkt-tal-zwf的结构见图5,其核苷酸序列为SEQ ID NO:3。
1.4构建质粒pK18-homT176C
以ATCC13032LysCfbr基因组为模板,分别以homT176C-up-F/homT176C-up-R以及homT176C-down-F/homT176C-down-R为引物对分别进行扩增。PCR扩增条件:95℃5min;92℃30s,58℃30s,65℃35s,30个循环;65℃10min。扩增得到homT176C-up,homT176C-down片段,长度均为1kb,切胶回收。以pK18mobsacB质粒为模板,以homT176C-Z-F,homT176C-Z-R为引物进行扩增,得到homT176C-Z片段,长度为5.7kb,切胶回收。将homT176C-up,homT176C-down,homT176C-Z进行Gibson组装,转化DH5a,复苏,涂布kan平板,以M13F,M13R为鉴定引物进行菌落PCR,阳性克隆条带长2kb,将阳性克隆接种试管抽提质粒备用。
获得的质粒pK18-homT176C的结构见图6,其核苷酸序列为SEQ ID NO:4。
1.5构建质粒pK18mobsacB-ddh-ECT
以ATCC13032LysCfbr基因组为模板,分别以ddh-up-F/Ddh1-up-R和Ddh1-dn-F/Ddh1-dn-R为引物对进行PCR扩增。PCR扩增条件:95℃5min;92℃30s,58℃30s,65℃35s,30个循环;65℃10min。扩增得到的两片段大小都为1kb,琼脂糖凝胶纯化回收。以上述获得的两个纯化回收片段为模板,以ddh-up-F/Ddh1-dn-R为引物对进行overlap PCR,获得2kb大小片段,琼脂糖凝胶纯化回收。
pK18mobsacB质粒用HindIII/EcoRI酶切,纯化回收后与上述overlap PCR回收片段进行Gibson拼接,转化DH5α感受态,涂布卡纳霉素抗性平板。
以M13F,M13R为鉴定引物进行菌落PCR,阳性克隆条带长2kb,将阳性克隆接种试管抽提质粒。质粒用PstI/EcoRI酶切,出现一条单一条带,琼脂糖凝胶纯化回收线性化载体A。
pEKEX2-ECT质粒(苏州金唯智合成)用PstI/EcoRI酶切,出现3.7kb/8.1kb两个条带,回收3.7kb片段;将回收线性化载体片段与上述步骤中的回收线性化载体A用T4连接酶连接转化,涂布卡纳霉素抗性平板。转化子以M13F,M13R为鉴定引物进行菌落PCR,阳性转化子出现5.7kb大小片段。阳性转化子接抗性试管,过夜培养,抽质粒备用。
获得的质粒pK18mobsacB-ddh-ECT的结构见图7,其核苷酸序列为SEQ ID NO:5。
1.6构建质粒pK18mobsacB-IS30-ECT
以ATCC13032LysCfbr基因组为模板,分别用引物对IS30-up-F/IS30-up-R和IS30-dn-F/IS30-dn-R进行PCR扩增。PCR扩增条件:95℃5min;92℃30s,58℃30s,65℃35s,30个循环;65℃10min。扩增得到的两片段大小都为0.6kb,琼脂糖凝胶纯化回收。
以上述获得两纯化回收片段为模板,以IS30-up-F/IS30-dn-R引物对进行overlapPCR,获得1.2kb大小片段,琼脂糖凝胶纯化回收。
pK18mobsacB质粒用HindIII/EcoRI酶切,纯化回收后与上述overlap PCR回收片段进行Gibson拼接,转化DH5α感受态,涂布卡纳霉素抗性平板。
以M13F,M13R为鉴定引物进行菌落PCR,阳性克隆条带长1.2kb,将阳性克隆接种试管抽提质粒;质粒用PstI/EcoRI酶切,出现一条单一条带,琼脂糖凝胶纯化回收线性化载体B。
pEKEX2-ECT质粒(苏州金唯智合成)用PstI/EcoRI酶切,出现3.7kb/8.1kb两个条带,回收3.7kb片段;将回收线性化载体片段与上述步骤中的回收线性化载体B段用T4连接酶连接转化,涂布卡纳霉素抗性平板。转化子以M13F,M13R为鉴定引物进行菌落PCR,阳性转化子出现4.9kb大小片段;阳性转化子接抗性试管,过夜培养,抽质粒备用。
获得的质粒pK18mobsacB-IS30-ECT的结构见图8,其核苷酸序列为SEQ ID NO:6。
实施例2:基因工程菌株构建
2.1将pK18-Psod-lysC质粒吸取3μl(500ng以上)到谷氨酸棒杆菌ATCC13032LysCfbr感受态细胞中,混匀后转移至2μm电转杯中,在2.5kV的条件下进行电击,电击时间为5.3ms,电击后立即转入800μl 46℃预热的BHIS液体培养基中,并在46℃水浴锅中水浴6min,然后置于恒温摇床中30℃,220rpm培养1h,使菌体复苏。复苏之后取100μl菌体涂布在含卡那霉素的BHIS平板上,将平板倒置在30℃恒温培养箱中培养48小时。挑取含卡那霉素的BHIS平板上的转化子,接种到无抗性的BHIS试管培养基中,在恒温摇床中30℃,220rpm培养24h,使其发生双交换。将菌体稀释1000倍后涂布在含20%蔗糖的BHIS-suc平板上,倒置在30℃恒温培养箱中培养48小时。挑取BHIS-suc平板转化子,分别点板BHIS平板和含卡那霉素的BHIS平板,倒置在30℃恒温培养箱中培养24小时。在BHIS平板上能生长而在含卡那霉素的BHIS平板上不能生长的转化子使用引物对Psod-lysC-F/Psod-lysC-down-R进行PCR扩增,阳性转化子PCR扩增条带约为1.2Kb,阴性转化子PCR扩增无条带,测序鉴定启动子整合阳性菌株。挑取阳性转化子到4ml的BHIS试管培养基中,在恒温摇床上30℃,220rpm培养16小时,使用20%甘油保菌,得到了基因型为ATCC13032PsodlysCfbr菌株。
2.2参照上述方法,将pK18-KOpck质粒用电转化方法转化步骤2.1中所得ATCC13032PsodlysCfbr菌株,然后按照sacB反筛的方法进行操作,得到ATCC13032PsodlysCfbrΔpck菌株。
2.3同样地,参照上述方法,分别将实施例1中构建的pK18-Psod-tkt-tal-zwf质粒、pK18-homT176C质粒、pK18mobsacB-ddh-ECT和pK18mobsacB-IS30-ECT质粒一次进行叠加,最终获得ATCC13032-ECT-01菌株。
2.4重复一次pK18mobsacB-IS30-ECT质粒在ATCC13032-ECT-01菌株中的整合步骤,获得ATCC13032-ECT-02菌株。
实施例3:工程菌的发酵
3.1摇瓶发酵:基因工程菌株在BHIS平板划线,30℃培养箱2天左右,挑2-3个单克隆到BHIS试管;30℃220rpm振荡培养14h左右,按照5v/v%比例转接种子摇瓶,30℃220rpm振荡培养10h左右;然后10v/v%接种量接发酵摇瓶,30℃220rpm振荡培养20h左右,取样检测依克多因的含量,发酵结果见表2。
表2、菌株摇瓶发酵生产依克多因的实验结果
菌株名称 | 摇瓶发酵产量g/L | 转化率g/g葡萄糖 |
ATCC13032lysCfbr | 0 | 0 |
ATCC13032-ECT-01 | 1.89 | 0.189 |
ATCC13032-ECT-02 | 2.23 | 0.223 |
由表2可知,本发明构建的基因工程菌株ATCC13032-ECT-01和ATCC13032-ECT-02改变了出发菌株ATCC13032lysCfbr内的代谢路径,从而能够将葡萄糖转化为依克多因。ectABC基因的过表达对于提高依克多因的生物合成是有利的。
3.2发酵罐发酵:从BHIS平板上挑取ATCC13032-ECT-02单克隆到BHI摇瓶,30℃220rpm振荡培养14h左右;5v/v%接种量接5L发酵罐,发酵温度30℃,溶氧维持30%,25%氨水维持pH 7.0,发酵64h终止发酵,定时用HPLC检测发酵液的依克多因含量,发酵结果见图9。
上述实施例表明,采用本发明方法构建的菌株ATCC13032-ECT-02能够实现发酵液中依克多因的积累,5L发酵罐发酵64小时,可产依克多因42.7g/L,有潜力进一步开发应用于工业化生产,已对其进行菌种保藏,保藏编号为CGMCC No.22733。
应理解,这些实施例仅用于举例说明目的,而不是对本发明的限制。本领域技术人员在阅读了本发明的构思之后,对其做出的各种改变或调整,均应落入本发明的保护范围内,这些等价形式同样属于本申请所附权利要求书限定的范围。
Claims (11)
1.一种构建依克多因生产菌的方法,其包括以下步骤:
A.以产赖氨酸的谷氨酸棒杆菌Corynebacterium glutamicum ATCC13032 LysCfbr为出发菌株,敲减基因组中编码高丝氨酸激酶的hom基因,得到下调高丝氨酸激酶表达的菌株A;
B.敲除菌株A基因组中编码磷酸烯醇式丙酮酸羧激酶的pck基因,得到缺失磷酸烯醇式丙酮酸羧激酶的菌株B;
C.增强菌株B的基因组中编码天冬氨酸激酶的基因lysC的表达,得到过表达天冬氨酸激酶的菌株C;
D.增强菌株C的基因组中编码转酮醇酶tkt的基因tkt、编码酪氨酸解氨酶tal的基因tal和编码6-磷酸葡萄糖酸脱氢酶zwf的基因zwf的表达,得到氧化磷酸化途径增强的菌株D;
E.在菌株D基因组中二氨基丙酸脱氢酶的编码基因ddh位点整合来源于施氏假单胞菌Pseudomonas stutzeri的ectABC基因,得到依克多因合成途径增强的菌株E,所述整合包括下述步骤:将核苷酸序列为SEQ ID NO:5的质粒pK18mobSacB-ddh-ECT导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆;
F.在菌株E基因组中噬菌体转座酶IS30基因位点整合来源于施氏假单胞菌Pseudomonas stutzeri的ectABC基因,得到依克多因合成途径进一步增强的菌株F,筛选阳性克隆,得到依克多因生产菌,所述整合包括下述步骤:将核苷酸序列为SEQ ID NO:6的质粒pK18mobSacB-IS30-ECT导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
2.如权利要求1所述的方法,其特征在于,还包括下述步骤:
G.在步骤F所得菌株F的基因组中噬菌体转座酶IS30基因位点进一步整合来源于施氏假单胞菌Pseudomonas stutzeri的ectABC基因,筛选依克多因合成途径增强的阳性克隆。
3.如权利要求1所述的方法,其特征在于,步骤A中hom基因的敲减通过使hom基因发生T176C突变而实现。
4.如权利要求3所述的方法,其特征在于,步骤A中hom基因发生T176C突变的步骤为:将核苷酸序列为SEQ ID NO:4的质粒pK18mobSacB-homT176C导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
5.如权利要求1所述的方法,其特征在于,步骤B中pck基因的敲除包括下述步骤:将核苷酸序列为SEQ ID NO:2的质粒pK18mobSacB-KOpck导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆;
步骤C中的基因lysC表达的增强通过将lysC基因置于sod启动子下游而实现;
步骤D中的基因tkt、tal和zwf表达的增强通过用sod启动子替换其天然启动子而实现。
6.如权利要求5所述的方法,其特征在于,步骤C包括下述步骤:将核苷酸序列为SEQ IDNO:1的质粒pK18mobSacB-Psod-lysC导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
7.如权利要求1所述的方法,其特征在于,步骤D包括下述步骤:将核苷酸序列为SEQ IDNO:3的质粒pK18mobSacB-Psod-tkt-tal-zwf导入宿主细胞中;进行SacB蔗糖反筛,筛选阳性克隆。
8.一种依克多因生产菌,其特征在于,通过如权利要求1-7中任一项所述的方法构建得到。
9.如权利要求8所述的依克多因生产菌用于生产依克多因的用途。
10.一种依克多因生产菌,其为谷氨酸棒杆菌(Corynebacterium glutamicum),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.22733。
11.如权利要求10所述依克多因生产菌用于生产依克多因的用途。
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