CN116622596A - 一种修饰的棒状杆菌属微生物及其构建方法与在生产苏氨酸中的应用 - Google Patents
一种修饰的棒状杆菌属微生物及其构建方法与在生产苏氨酸中的应用 Download PDFInfo
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- CN116622596A CN116622596A CN202210125703.1A CN202210125703A CN116622596A CN 116622596 A CN116622596 A CN 116622596A CN 202210125703 A CN202210125703 A CN 202210125703A CN 116622596 A CN116622596 A CN 116622596A
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Abstract
本发明涉及微生物工程技术领域,具体公开了一种修饰的棒状杆菌属微生物及其构建方法与在生产苏氨酸中的应用。本发明的修饰的棒状杆菌属微生物为尿嘧啶磷酸核糖转移酶失活或活力降低的棒状杆菌。进一步相比于未修饰的微生物具有增强的苏氨酸生产能力。本发明的修饰的棒状杆菌属微生物可更好地提升苏氨酸的产量。
Description
技术领域
本发明涉及微生物工程技术领域,具体地说,涉及一种修饰的棒状杆菌属微生物及其构建方法与在生产苏氨酸中的应用。
背景技术
L-苏氨酸(L-Threonin),化学名称为β-羟基-α-氨基丁酸,分子式为C4H9NO3,相对分子质量为119.12。L-苏氨酸是一种必需的氨基酸,苏氨酸主要用于医药、化学试剂、食品强化剂、饲料添加剂等方面。
谷氨酸棒杆菌中,由草酰乙酸生成苏氨酸需要五步催化反应,分别为天冬氨酸激酶(lysC编码)、天冬氨酸半醛脱氢酶(asd编码)、高丝氨酸脱氢酶(hom编码)、高丝氨酸激酶(thrB编码)以及苏氨酸合酶(thrC编码)。Hermann Sahm等人一直致力于高产苏氨酸的谷棒菌株的开发,并取得一定突破,获得了抗反馈抑制的hom基因(Reinscheid D J,Eikmanns BJ,Sahm H.Analysis of a Corynebacterium glutamicum hom gene coding for afeedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230.)、lysC基因(Eikmanns B J,Eggeling L,Sahm H.Molecular aspectsof lysine,threonine,and isoleucine biosynthesis in Corynebacteriumglutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163.)。继Hermann Sahm之后,Lothar Eggling在该领域进行了进一步的探索,弱化苏氨酸利用途径中的编码基因glyA,同时过表达苏氨酸外运蛋白ThrE,使得苏氨酸的产量由49mM提高到67mM(Simic P,Willuhn J,Sahm H,et al.Identification of glyA(Encoding SerineHydroxymethyltransferase)and Its Use Together with the Exporter ThrE ToIncreasel-Threonine Accumulation by Corynebacterium glutamicum[J].Applied andEnvironmental Microbiology,2002,68(7):3321-3327.)。
但目前利用谷氨酸棒状杆菌生产苏氨酸的报道主要集中在其合成路径中,尚未见嘧啶代谢与苏氨酸合成相关的报道。仍有必要对谷氨酸棒状杆菌生产苏氨酸进行进一步研究。
发明内容
本发明的目的是通过失活尿嘧啶磷酸核糖转移酶使菌株生产苏氨酸的能力得到提升,从而提供一种产苏氨酸(L-苏氨酸)菌株及其构建方法与应用。
为了实现本发明目的,第一方面,本发明提供一种修饰的棒状杆菌属微生物,所述微生物相比于未修饰的微生物,其尿嘧啶磷酸核糖转移酶的活性降低或丧失,且所述微生物相比于未修饰的微生物具有增强的苏氨酸生产能力。优选地,尿嘧啶磷酸核糖转移酶在NCBI上的参考序列编号为WP_003858289.1,或与其相似性为90%的氨基酸序列。
进一步地,所述微生物体内尿嘧啶磷酸核糖转移酶的活性降低或丧失是通过降低编码尿嘧啶磷酸核糖转移酶基因的表达或敲除内源的编码尿嘧啶磷酸核糖转移酶的基因来实现的。
可以采用诱变、定点突变或同源重组的方法来降低编码尿嘧啶磷酸核糖转移酶基因的表达或敲除内源的编码尿嘧啶磷酸核糖转移酶的基因。
进一步地,所述微生物与未修饰的微生物相比,其体内苏氨酸合成和/或前体供应途径相关的酶的活性增强;其中,与所述苏氨酸合成和/或前体供应途径相关的酶选自天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶中的至少一种;优选地,它们在NCBI上的参考序列编号分别为WP_003855724.1、WP_011013506.1、WP_003854900.1、WP_011014183.1、WP_011013816.1、WP_011014465.1、WP_011013497.1、WP_011014964.1,或与上述参考序列相似度为90%的氨基酸序列。
优选地,所述微生物为如下①~⑥中的任一种:
①尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶和/或高丝氨酸激酶活性增强的微生物;
②尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或丙酮酸羧化酶活性增强的微生物;
③尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或磷酸烯醇式丙酮酸羧化酶活性增强的微生物;
④尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或天冬氨酸氨基转移酶活性增强的微生物;
⑤尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或苏氨酸合酶活性增强的微生物;
⑥尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶和/或苏氨酸合酶活性增强的微生物。
所述微生物体内苏氨酸合成途径相关的酶的活性的增强是由选自以下1)~6),或任选的组合实现的:
1)通过导入具有所述酶的编码基因的质粒而增强;
2)通过增加染色体上所述酶的编码基因的拷贝数而增强;
3)通过改变染色体上所述酶的编码基因的启动子序列而增强;
4)通过将强启动子与所述酶的编码基因可操作地连接而增强;
5)通过对酶的氨基酸序列进行改变而增强;
6)通过对编码酶的基因的核苷酸序列进行改变而增强。
优选地,本发明所述棒杆菌为谷氨酸棒状杆菌(Corynebacterium glutamicum),谷氨酸棒状杆菌包括ATCC13032、ATCC13870、ATCC13869、ATCC21799、ATCC21831、ATCC14067、ATCC13287等(参见NCBI Corunebacterium glutamicum进化树https://www.ncbi.nlm.nih.gov/genome/469),更优选谷氨酸棒状杆菌ATCC 13032。
第二方面,本发明提供产苏氨酸菌株的构建方法,所述方法包括:
A、弱化具有氨基酸生产能力的棒杆菌中编码尿嘧啶磷酸核糖转移酶的基因,获得基因弱化菌株;所述弱化包括敲除或降低尿嘧啶磷酸核糖转移酶编码基因的表达;和/或
B、增强步骤A基因弱化菌株中与苏氨酸合成和/或前体供应途径相关的酶,获得酶活增强菌株;
所述增强的途径选自以下1)~6),或任选的组合:
1)通过导入具有所述酶的编码基因的质粒而增强;
2)通过增加染色体上所述酶的编码基因的拷贝数而增强;
3)通过改变染色体上所述酶的编码基因的启动子序列而增强;
4)通过将强启动子与所述酶的编码基因可操作地连接而增强;
5)通过对酶的氨基酸序列进行改变而增强;
6)通过对编码酶的基因的核苷酸序列进行改变而增强;
其中,与所述苏氨酸合成和/或前体供应途径相关的酶选自天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶中的至少一种。
第三方面,本发明提供一种生产苏氨酸的方法,所述方法包括如下步骤:
a)培养上述微生物,以获得所述微生物的培养物;
b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
第四方面,本发明提供编码尿嘧啶磷酸核糖转移酶的基因的敲除或降低表达在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
进一步地,通过失活具有氨基酸生产能力的棒杆菌(Corynebacterium)中的尿嘧啶磷酸核糖转移酶来提高苏氨酸的发酵产量。
优选地,本发明所述棒杆菌为谷氨酸棒状杆菌(Corynebacterium glutamicum),谷氨酸棒状杆菌包括ATCC13032、ATCC13870、ATCC13869、ATCC21799、ATCC21831、ATCC14067、ATCC13287等(参见NCBI Corunebacterium glutamicum进化树https://www.ncbi.nlm.nih.gov/genome/469),更优选谷氨酸棒状杆菌ATCC 13032。
第五方面,本发明提供所述修饰的棒状杆菌属微生物或按照上述方法构建得到的产苏氨酸菌株在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
上述有关菌株的改造方法包括基因的强化和弱化等均为本领域技术人员可知的改造方式,参见满在伟.高产L-精氨酸钝齿棒杆菌的系统途径工程改造[D].江南大学,2016;崔毅.代谢工程改造谷氨酸棒杆菌生产L-亮氨酸[D].天津科技大学;徐国栋.L-异亮氨酸生产菌株的构建及发酵条件优化.天津科技大学,2015。
优选,本发明中,通过将基因组上编码尿嘧啶磷酸核糖转移酶基因的编码区敲除的方式,从而使尿嘧啶磷酸核糖转移酶失活。
通过使编码天冬氨酸激酶的基因lysC突变,从而使其起始密码子由GTG突变为ATG且使其编码蛋白携带T311I突变,并且使lysC基因由Psod启动转录,最终实现天冬氨酸激酶的表达强化及解调控。Psod的核苷酸序列如SEQ ID NO.61所示。
通过使编码天冬氨酸半醛脱氢酶的基因asd由Psod启动转录,最终实现天冬氨酸半醛脱氢酶的表达强化。
通过使编码高丝氨酸脱氢酶的基因hom突变,从而使其编码蛋白携带G378E突变,并且使hom基因由PcspB启动转录,最终实现高丝氨酸脱氢酶的表达强化和解调控。PcspB的核苷酸序列如SEQ ID NO.62所示。
通过使编码高丝氨酸激酶的基因thrB由PcspB启动转录,最终实现高丝氨酸激酶的表达强化。PcspB的核苷酸序列如SEQ ID NO.62所示。
通过使编码丙酮酸羧化酶的基因pyc突变,从而使其编码蛋白携带P458S突变,并且使pyc基因由Psod启动转录,最终实现丙酮酸羧化酶的表达强化。Psod的核苷酸序列如SEQ ID NO.61所示。
通过使编码磷酸烯醇式丙酮酸羧化酶的基因ppc突变,从而使其编码蛋白携带D299N突变,并且使ppc基因由Ptuf启动转录,最终实现磷酸烯醇式丙酮酸羧化酶的表达强化。Ptuf的核苷酸序列如SEQ ID NO.63所示。
通过使编码天冬氨酸氨基转移酶的基因aspB由Psod启动转录,最终实现天冬氨酸氨基转移酶的表达强化。
通过使编码苏氨酸合酶的基因thrC突变,从而使其起始密码子由GTG突变为ATG,并且使thrC基因由Psod启动转录,最终实现苏氨酸合酶的表达强化。
本发明的有益效果至少在于:
本发明将尿嘧啶磷酸核糖转移酶失活菌株应用于苏氨酸生产,其苏氨酸的产量较未改造之前最高可提高10%~17.5%。进一步将其与苏氨酸合成和/或前体供应路径中的天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶中的至少一个表达强化相组合时,其苏氨酸的产量均有15倍~42倍的提升。为提升苏氨酸的生产能力提供了一个新的思路。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。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。
本发明涉及的蛋白及其编码基因如下:
尿嘧啶磷酸核糖转移酶,编码基因upp,NCBI编号:cg0786、Cgl0684、NCgl0654;
天冬氨酸激酶,编码基因名称lysC,NCBI编号:cg0306、Cgl0251、NCgl0247;
天冬氨酸半醛脱氢酶,编码基因名称asd,NCBI编号:cg0307、Cgl0252、NCgl0248;
高丝氨酸脱氢酶,编码基因名称hom,NCBI编号:cg1337、Cgl1183、NCgl1136;
高丝氨酸激酶,编码基因thrB,NCBI编号:cg1338、Cgl1184、NCgl1137;
丙酮酸羧化酶,编码基因pyc,NCBI编号:cg0791、Cgl0689、NCgl0659;
磷酸烯醇式丙酮酸羧化酶,编码基因ppc,NCBI编号:cg1787、Cgl1585、NCgl1523;
天冬氨酸氨基转移酶,编码基因aspB,NCBI编号:cg0294、Cgl0240、NCgl0237;
苏氨酸合酶,编码基因名称thrC,NCBI编号:cg2437、Cgl2220、NCgl2139。
尿嘧啶磷酸核糖转移酶催化尿嘧啶合成尿苷-磷酸(UMP),是嘧啶代谢的一部分。本发明在ATCC13032菌株的基础上失活尿嘧啶磷酸核糖转移酶的编码基因,得到菌株SMCT046,苏氨酸含量为0.2g/L。由此获知,尿嘧啶磷酸核糖转移酶活性丢失有利于苏氨酸的生产。在此基础上本发明进一步验证了尿嘧啶磷酸核糖转移酶活性丢失对苏氨酸生产的影响,对菌株的苏氨酸合成路径进一步强化,主要包括天冬氨酸激酶、高丝氨酸脱氢酶、天冬氨酸半醛脱氢酶、高丝氨酸激酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶中的至少一个表达强化或解调控。从摇瓶结果可以看出所有生产苏氨酸的菌株在尿嘧啶磷酸核糖转移酶活性丢失后,其苏氨酸的生产能力均有所提升;同时与仅失活编码尿嘧啶磷酸核糖转移酶编码基因的菌株相比,尿嘧啶磷酸核糖转移酶活性丢失和苏氨酸合成路径中的酶表达强化相结合的菌株其苏氨酸的生产上更有优势。
改造过程中的失活或弱化包括启动子的替换,核糖体结合位点的改变、点突变、序列的缺失等手段,改造过程中的表达强化包括启动子的替换,核糖体结合位点的改变、拷贝数的增加、质粒过表达等手段,且以上手段均为本领域研究人员公知手段。以上手段无法通过举例而穷尽,因此本发明中的实施例仅用敲除其编码基因作为代表进行失活说明,用启动子强化作为代表进行强化说明。
实施例1菌株基因组改造质粒构建
1)尿嘧啶磷酸核糖转移酶失活方案重组质粒pK18mobsacB-△upp
以ATCC13032基因组为模板,以PW23/PW24引物对进行PCR扩增得到上游同源臂up,以PW25/PW26引物对进行PCR扩增得到下游同源臂dn,以PW23/PW26引物对以up、dn为模板进行融合PCR,获得全长片段△upp。pK18mobsacB用BamHI/HindIII酶切。两者用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-△upp。
2)天冬氨酸激酶表达强化方案重组质粒pK18mobsacB-Psod-lysCg1a-T311I
以ATCC13032基因组为模板,以P21/P22引物对进行PCR扩增得到上游同源臂up,以P23/P24引物对进行PCR扩增得到启动子片段Psod,以P25/P26引物对进行PCR扩增得到lysCg1a-T311I,以P27/P28引物对进行PCR扩增得到下游同源臂dn。以P21/P24引物对以up、Psod为模版进行融合PCR,获得片段up-Psod。以P21/P28引物对以up-Psod、lysCg1a-T311I、dn为模板进行融合PCR获得全长片段up-Psod-lysCg1a-T311I-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Psod-lysCg1a-T311I-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-Psod-lysCg1a-T311I。
3)天冬氨酸半醛脱氢酶表达强化方案重组质粒pK18mobsacB-Psod-asd
以ATCC13032基因组为模板,以P1/P2引物对进行PCR扩增得到上游同源臂up,以P3/P4引物对进行PCR扩增得到启动子片段Psod,以P5/P6引物对进行PCR扩增得到下游同源臂dn,以P1/P6引物对以up、Psod、dn为模板进行融合PCR,获得全长片段up-Psod-dn。pK18mobsacB用BamHI/HindIII酶切。两者用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-Psod-asd。
4)高丝氨酸脱氢酶表达强化方案重组质粒pK18mobsacB-PcspB-homG378E
以ATCC13032基因组为模板,以P29/P30引物对进行PCR扩增得到上游同源臂up,以ATCC14067基因组为模板以P31/P32引物对进行PCR扩增得到启动子片段PcspB,以ATCC13032基因组为模板以P33/P34引物对进行PCR扩增得到homG378E,以P35/P36引物对进行PCR扩增得到下游同源臂dn。以P29/P32引物对以up、PcspB为模版进行融合PCR,获得片段up-PcspB。以P29/P36引物对以up-PcspB、homG378E、dn为模板进行融合PCR获得全长片段up-PcspB-homG378E-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-PcspB-homG378E-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-PcspB-homG378E。
5)高丝氨酸激酶表达强化方案重组质粒pK18mobsacB-PcspB-thrB
以ATCC13032基因组为模板,以P7/P8引物对进行PCR扩增得到上游同源臂up,以ATCC14067基因组为模板以P9/P10引物对进行PCR扩增得到启动子片段PcspB,以ATCC13032基因组为模板以P11/P12引物对进行PCR扩增得到下游同源臂dn。以P7/P12引物对以up、PcspB、dn为模板进行融合PCR获得全长片段up-PcspB-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-PcspB-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-PcspB-thrB。
6)丙酮酸羧化酶表达强化方案重组质粒pK18mobsacB-Psod-pycP458S
以ATCC13032基因组为模板,以P13/P14引物对进行PCR扩增得到上游同源臂up,以P15/P16引物对进行PCR扩增得到启动子片段Psod,以P17/P18引物对进行PCR扩增得到pycP458S,以P19/P20引物对进行PCR扩增得到下游同源臂dn。以P13/P16引物对以up、Psod为模版进行融合PCR,获得片段up-Psod。以P13/P20引物对以up-Psod、pycP458S、dn为模板进行融合PCR获得全长片段up-Psod-pycP458S-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Psod-pycP458S-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-Psod pycP458S。
7)磷酸烯醇式丙酮酸羧化酶表达强化方案重组质粒pK18mobsacB-Ptuf-ppcD299N
以ATCC13032基因组为模板,以P53/P54引物对进行PCR扩增得到上游同源臂up,以P55/P56引物对进行PCR扩增得到启动子片段Ptuf,以P57/P58引物对进行PCR扩增得到ppcD299N,以P59/P60引物对进行PCR扩增得到下游同源臂dn。以P53/P56引物对以up、Ptuf为模版进行融合PCR,获得片段up-Pstuf。以P53/P60引物对以up-Ptuf、ppcD299N、dn为模板进行融合PCR获得全长片段up-Ptuf-ppcD299N-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Ptuf-ppcD299N-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-Ptuf-ppcD299N。
8)天冬氨酸氨基转移酶表达强化方案重组质粒pK18mobsacB-Psod-aspB
以ATCC13032基因组为模板,以P103/P104引物对进行PCR扩增得到上游同源臂up,以P105/P106引物对进行PCR扩增得到启动子片段Psod,以P107/P108引物对进行PCR扩增得到下游同源臂dn。以P103/P108引物对以up、Psod、dn为模板进行融合PCR获得全长片段up-Psod-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Psod-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-Psod-aspB。
9)苏氨酸合酶表达强化质粒pK18mobsacB-Psod-thrCg1a
以ATCC13032基因组为模板,以P37/P38引物对进行PCR扩增得到上游同源臂up,以P39/P40引物对进行PCR扩增得到启动子片段Psod-thrCg1a,以P41/P42引物对进行PCR扩增得到下游同源臂dn。以P37/P42引物对以up、Psod-thrCg1a、dn为模板进行融合PCR获得全长片段up-Psod-thrCg1a-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Psod-thrCg1a-dn与pK18mobsacB用无缝克隆试剂盒进行组装,转化Trans1 T1感受态细胞,获得重组质粒pK18mobsacB-Psod-thrCg1a。
质粒构建过程中所用的引物如下表1所示:
表1
注:表1中加粗字体及下划线为引入相应点突变的引物。
实施例2基因组改造菌株的构建
1)尿嘧啶磷酸核糖转移酶失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC13032感受态细胞。重组质粒pK18mobsacB-△upp以电穿孔方法转化该感受态细胞,并在含有15mg/L卡那霉素的选择培养基上筛选转化子,其中目的基因由于同源性被插入到染色体中。将筛得的转化子过夜培养于普通液体脑心浸液培养基中,培养温度为30℃,回转摇床220rpm振荡培养。此培养过程中,转化子发生第二次重组,通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4),稀释液涂布在含有10%蔗糖的普通固体脑心浸液培养基上,33℃静置培养48h。蔗糖培养基上长出的菌株在其基因组中不携带插入的载体序列。通过PCR扩增目的序列,核苷酸测序分析,获得目的突变菌株分别命名为SMCT046。
2)天冬氨酸激酶强化表达及解调控菌株的构建
菌株构建方法参考上述1),以ATCC13032为出发菌,进行天冬氨酸激酶强化及解调控的改造(将pK18mobsacB-Psod-lysCg1a-T311I导入ATCC13032),获得的菌株命名为SMCT047。该菌株中,lysC基因被突变,起始密码子由GTG突变为ATG且第311位的氨基酸由苏氨酸突变为异亮氨酸,且lysC基因的启动子被替换为强启动子Psod。
3)天冬氨酸半醛脱氢酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT047为出发菌,进行天冬氨酸半醛脱氢酶表达强化的改造(将pK18mobsacB-Psod-asd导入SMCT047),获得的改造菌株命名为SMCT048。该菌株中,asd基因的启动子被替换为强启动子Psod。
4)高丝氨酸脱氢酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT048为出发菌,进行高丝氨酸脱氢酶表达强化的改造(将pK18mobsacB-PcspB-homG378E导入SMCT048),获得的改造菌株命名为SMCT049。该菌株中,hom基因进一步被突变,对应的氨基酸突变位点为G378E,且hom基因的启动子被替换为强启动子PcspB。
5)高丝氨酸激酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT049为出发菌,进行高丝氨酸激酶表达强化菌株的改造(将pK18mobsacB-PcspB-thrB导入SMCT049),获得的改造菌株命名为SMCT050。该菌株中,thrB基因的启动子被替换为强启动子PcspB。
6)丙酮酸羧化酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT050为出发菌,进行丙酮酸羧化酶表达强化的改造(将pK18mobsacB-Psod-pycP458S导入SMCT050),获得的改造菌株命名为SMCT051。该菌株中,pyc基因进一步被突变,对应的氨基酸突变位点为P458S,且pyc基因的启动子被替换为强启动子Psod。
7)磷酸烯醇式丙酮酸羧化酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT050为出发菌,进行磷酸烯醇式丙酮酸羧化酶表达强化的改造(将pK18mobsacB-Ptuf-ppcD299N导入SMCT050),获得的改造菌株命名为SMCT052。该菌株中,ppc基因进一步被突变,对应的氨基酸突变位点为D299N,且ppc基因的启动子被替换为强启动子Ptuf。
8)天冬氨酸氨基转移酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT050为出发菌,进行天冬氨酸氨基转移酶表达强化的改造(将pK18mobsacB-Psod-aspB导入SMCT050),获得的改造菌株命名为SMCT053。该菌株中,aspB基因的启动子被替换为强启动子Psod。
9)苏氨酸合酶表达强化菌株的构建
菌株构建方法参考上述1),以SMCT050为出发菌,进行苏氨酸合酶表达强化菌株的改造(将pK18mobsacB-Psod-thrCg1a导入SMCT050),获得的改造菌株命名为SMCT054。该菌株中,thrC基因进一步被突变,起始密码子由GTG突变为ATG,且thrC基因的启动子被替换为强启动子Psod。
10)上述位点叠加菌株的构建
以SMCT051为出发菌,进行磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶表达强化的改造(将pK18mobsacB-Ptuf-ppcD299N、pK18mobsacB-Psod-aspB、pK18mobsacB-Psod-thrCg1a导入SMCT051),获得的改造菌命名为SMCT385。
11)尿嘧啶磷酸核糖转移酶失活菌株的构建
菌株构建方法参考上述1),以SMCT050、SMCT051、SMCT052、SMCT053、SMCT054、SMCT385为出发菌,进行尿嘧啶磷酸核糖转移酶失活菌株的构建(将pK18mobsacB-△upp分别导入上述菌株),获得的改造菌命名为SMCT055、SMCT056、SMCT057、SMCT058、SMCT059、SMCT060。
获得的菌株列表如下表2。
表2
实施例3构建菌株摇瓶验证
1.培养基
种子活化培养基:BHI 3.7%,琼脂2%,pH 7。
种子培养基:蛋白胨5/L,酵母抽提物5g/L,氯化钠10g/L,硫酸铵16g/L,尿素8g/L,磷酸二氢钾10.4g/L,磷酸氢二钾21.4g/L,生物素5mg/L,硫酸镁3g/L。葡萄糖50g/L,pH7.2。
发酵培养基:玉米浆50mL/L,葡萄糖30g/L,硫酸铵4g/L,MOPS 30g/L,磷酸二氢钾10g/L,尿素20g/L,生物素10mg/L,硫酸镁6g/L,硫酸亚铁1g/L,VB1·HCl 40mg/L,泛酸钙50mg/L,烟酰胺40mg/L,硫酸锰1g/L,硫酸锌20mg/L,硫酸铜20mg/L,pH 7.2。
2.工程菌摇瓶发酵生产L-苏氨酸
(1)种子培养:挑取ATCC13032、SMCT046、SMCT047、SMCT048、SMCT049、SMCT050、SMCT051、SMCT052、SMCT053、SMCT054、SMCT385、SMCT055、SMCT056、SMCT057、SMCT058、SMCT059、SMCT060斜面种子1环接至装有20mL种子培养基的500mL三角瓶中,30℃、220r/min振荡培养16h。
(2)发酵培养:将2mL种子液接种至装有20mL发酵培养基的500mL三角瓶中,33℃、220r/min振荡培养24h。
(3)取1mL发酵液离心(12000rpm,2min),收集上清液,用HPLC检测工程菌与对照菌发酵液中的L-苏氨酸,其浓度如下表3-表4所示。
(4)苏氨酸摇瓶发酵结果如下表3-表4所示。
表3
菌株编号 | OD562 | 苏氨酸(g/L) |
ATCC13032 | 25 | — |
SMCT046 | 23 | 0.2 |
SMCT047 | 23 | 1.2 |
SMCT048 | 24 | 1.5 |
SMCT049 | 24 | 2.1 |
SMCT050 | 23 | 2.8 |
由上表可以看出,随着苏氨酸合成路径基因的组合强化,苏氨酸产量也在逐步提升。
表4
菌株编号 | OD562 | 苏氨酸(g/L) | 菌株编号 | OD562 | 苏氨酸(g/L) |
ATCC13032 | 25 | — | SMCT046 | 23 | 0.2 |
SMCT050 | 23 | 2.8 | SMCT055 | 25 | 3.1 |
SMCT051 | 23 | 3.2 | SMCT056 | 23 | 3.7 |
SMCT052 | 24 | 3.9 | SMCT057 | 23 | 4.5 |
SMCT053 | 24 | 4.0 | SMCT058 | 24 | 4.7 |
SMCT054 | 23 | 3.8 | SMCT059 | 24 | 4.4 |
SMCT385 | 23 | 7.2 | SMCT060 | 23 | 8.4 |
由上表可以看出,尿嘧啶磷酸核糖转移酶失活后的菌株的苏氨酸产量较失活前有所提高,其产量提高在10%~17.5%之间,不同的尿嘧啶磷酸核糖转移酶失活菌株之间的苏氨酸产量有所差异,在0.1g/L~5.3g/L之间,说明尿嘧啶磷酸核糖转移酶的失活与不同位点的组合有不同的效果。尿嘧啶磷酸核糖转移酶失活与苏氨酸合成途径基因强化整合菌株SMCT060苏氨酸产量最高,为8.4g/L。当尿嘧啶磷酸核糖转移酶失活与苏氨酸合成路径中的天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶中的至少一个表达强化相组合时,其苏氨酸的产量均有提升,且其提升幅度比单独进行苏氨酸合成路径的改造及尿嘧啶磷酸核糖转移酶两者的加和要高,说明其叠加不仅仅是单纯的产量放大,两者的叠加有进一步放大的作用。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 一种修饰的棒状杆菌属微生物及其构建方法与在生产苏氨酸中的应用
<130> KHP211124935.8
<160> 63
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<212> DNA
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<212> DNA
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<212> DNA
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<212> DNA
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atttctttat aaacgcaggt catatctacc aaaactacgc 40
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<212> DNA
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cccggaataa ttggcagcta ggatataacc ctatcccaag 40
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gtaaaacgac ggccagtgcc aagcttgaat acgcggattc cctcgc 46
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
<400> 50
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<213> 人工序列(Artificial Sequence)
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gggtaaaaaa tcctttcgta ggttt 25
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<212> DNA
<213> 人工序列(Artificial Sequence)
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acgacggcca gtgccaagct tacaccggaa caacccacat g 41
<210> 57
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
catgattacg aattcgagct cggtacccgg ggatccaaag ggtggtgggg ttggcg 56
<210> 58
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
cgtaatgccc ttagaaactt agcttcacat gttaaatcat 40
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
atgatttaac atgtgaagct aagtttctaa gggcattacg 40
<210> 60
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
tcacgacgtt gtaaaacgac ggccagtgcc aagcttgagg atgaagtcca aagctg 56
<210> 61
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
tagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60
ttcgttgcaa tatcaacaaa aaggcctatc attgggaggt gtcgcaccaa gtacttttgc 120
gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180
gattttttac cc 192
<210> 62
<211> 260
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
acctgcgttt ataaagaaat gtaaacgtga tcggatcgat ataaaagaaa cagtttgtac 60
tcaggtttga agcattttct ccaattcgcc tggcaaaaat ctcaattgtc gcttacagtt 120
tttctcaacg acaggctgct aagctgctag ttcggtggcc tagtgagtgg cgtttacttg 180
gataaaagta atcccatgtc gtgatcagcc attttgggtt gtttccatag catccaaagg 240
tttcgtcttt cgatacctat 260
<210> 63
<211> 200
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
tggccgttac cctgcgaatg tccacagggt agctggtagt ttgaaaatca acgccgttgc 60
ccttaggatt cagtaactgg cacattttgt aatgcgctag atctgtgtgc tcagtcttcc 120
aggctgctta tcacagtgaa agcaaaacca attcgtggct gcgaaagtcg tagccaccac 180
gaagtccagg aggacataca 200
Claims (9)
1.一种修饰的棒状杆菌属微生物,其特征在于,所述微生物相比于未修饰的微生物,其尿嘧啶磷酸核糖转移酶的活性降低或丧失,且所述微生物相比于未修饰的微生物具有增强的苏氨酸生产能力。
2.根据权利要求1所述的微生物,其特征在于,所述微生物体内尿嘧啶磷酸核糖转移酶的活性降低或丧失是通过降低编码尿嘧啶磷酸核糖转移酶基因的表达或敲除内源的编码尿嘧啶磷酸核糖转移酶的基因来实现的。
3.根据权利要求2所述的微生物,其特征在于,采用诱变、定点突变或同源重组的方法来降低编码尿嘧啶磷酸核糖转移酶基因的表达或敲除内源的编码尿嘧啶磷酸核糖转移酶的基因。
4.根据权利要求1所述的微生物,其特征在于,所述微生物与未修饰的微生物相比,其体内苏氨酸合成和/或前体供应途径相关的酶的活性增强;
其中,与所述苏氨酸合成和/或前体供应途径相关的酶选自天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶中的至少一种。
5.根据权利要求4所述的微生物,其特征在于,所述微生物为如下①~⑥中的任一种:
①尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶和/或高丝氨酸激酶活性增强的微生物;
②尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或丙酮酸羧化酶活性增强的微生物;
③尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或磷酸烯醇式丙酮酸羧化酶活性增强的微生物;
④尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或天冬氨酸氨基转移酶活性增强的微生物;
⑤尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶和/或苏氨酸合酶活性增强的微生物;
⑥尿嘧啶磷酸核糖转移酶活性降低或丧失且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶和/或苏氨酸合酶活性增强的微生物。
6.根据权利要求4所述的微生物,其特征在于,所述微生物体内苏氨酸合成途径相关的酶的活性的增强是由选自以下1)~6),或任选的组合实现的:
1)通过导入具有所述酶的编码基因的质粒而增强;
2)通过增加染色体上所述酶的编码基因的拷贝数而增强;
3)通过改变染色体上所述酶的编码基因的启动子序列而增强;
4)通过将强启动子与所述酶的编码基因可操作地连接而增强;
5)通过对酶的氨基酸序列进行改变而增强;
6)通过对编码酶的基因的核苷酸序列进行改变而增强。
7.根据权利要求1-5任一项所述的微生物,其特征在于,所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
8.产苏氨酸菌株的构建方法,其特征在于,所述方法包括:
A、弱化具有氨基酸生产能力的棒杆菌中编码尿嘧啶磷酸核糖转移酶的基因,获得基因弱化菌株;所述弱化包括敲除或降低尿嘧啶磷酸核糖转移酶编码基因的表达;和/或
B、增强步骤A基因弱化菌株中与苏氨酸合成和/或前体供应途径相关的酶,获得酶活增强菌株;
所述增强的途径选自以下1)~6),或任选的组合:
1)通过导入具有所述酶的编码基因的质粒而增强;
2)通过增加染色体上所述酶的编码基因的拷贝数而增强;
3)通过改变染色体上所述酶的编码基因的启动子序列而增强;
4)通过将强启动子与所述酶的编码基因可操作地连接而增强;
5)通过对酶的氨基酸序列进行改变而增强;
6)通过对编码酶的基因的核苷酸序列进行改变而增强;
其中,与所述苏氨酸合成和/或前体供应途径相关的酶选自天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、天冬氨酸氨基转移酶、苏氨酸合酶中的至少一种。
9.一种生产苏氨酸的方法,其特征在于,所述方法包括如下步骤:
a)培养权利要求1-7任一项所述的微生物,以获得所述微生物的培养物;
b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
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