CN116555251A - 一种生产苏氨酸的重组微生物及其应用 - Google Patents
一种生产苏氨酸的重组微生物及其应用 Download PDFInfo
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- CN116555251A CN116555251A CN202210107366.3A CN202210107366A CN116555251A CN 116555251 A CN116555251 A CN 116555251A CN 202210107366 A CN202210107366 A CN 202210107366A CN 116555251 A CN116555251 A CN 116555251A
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- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
本发明涉及微生物工程技术领域，具体涉及一种生产苏氨酸的重组微生物及其应用。本发明提供一种重组微生物，其中，磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp片段被替换为强启动子。本发明通过对磷酸烯醇式丙酮酸羧化酶编码基因的启动子的特异优化和基因编码区的突变，显著提高了菌株合成苏氨酸的能力，苏氨酸产量较未经改造的菌株提高25‑40％，上述改造方法可应用于苏氨酸生产菌株的构建和苏氨酸发酵生产中，具有较好的应用价值。
Description
技术领域
本发明涉及微生物工程技术领域，具体涉及一种生产苏氨酸的重组微生物及其应用。
背景技术
L-苏氨酸(L-Threonin，化学名为β-羟基-α-氨基丁酸)的分子式为C4H9NO3，相对分子质量为119.12，是一种必需氨基酸，主要用于医药、化学试剂、食品强化剂、饲料添加剂等领域。
谷氨酸棒状杆菌中，由草酰乙酸生成苏氨酸需要五步催化反应，其催化酶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)。目前利用谷氨酸棒状杆菌生产苏氨酸的报道主要集中在对苏氨酸的合成代谢路径的改造，其中包括：抗反馈抑制的hom基因和lysC基因(Reinscheid D J,Eikmanns B J,Sahm H.Analysis of a Corynebacteriumglutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230；Eikmanns B J,Eggeling L,Sahm H.Molecular aspects of lysine,threonine,and isoleucine biosynthesis inCorynebacterium glutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163.)；以及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(Simic P,Willuhn J,Sahm H,et al.Identification of glyA(Encoding SerineHydroxymethyltransferase)and Its Use Together with the Exporter ThrE ToIncrease l-Threonine Accumulation by Corynebacterium glutamicum[J].Appliedand Environmental Microbiology,2002,68(7):3321-3327.)等。而关于苏氨酸生产的前体供应等方面的研究报道仍然较少。
发明内容
本发明的目的是通过修饰磷酸烯醇式丙酮酸羧化酶编码基因的启动子区域使菌株生产苏氨酸的能力得到提升，从而提供一种生产苏氨酸的重组微生物及其应用。
具体地，本发明提供以下技术方案：
首先，本发明提供磷酸烯醇式丙酮酸羧化酶编码基因(ppc)的强化表达在提高棒状杆菌属细菌的苏氨酸产量或构建生产苏氨酸的棒状杆菌属细菌中的应用，所述强化表达通过将磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp替换为强启动子实现。
以上所述的棒状杆菌属细菌优选为谷氨酸棒状杆菌(Corynebacteriumglutamicum)。
本发明发现，在谷氨酸棒状杆菌中，提高磷酸烯醇式丙酮酸羧化酶的酶活有利于提高苏氨酸合成前体草酰乙酸的供应，进而促进苏氨酸的合成和积累。对于提高磷酸烯醇式丙酮酸羧化酶的酶活性的方式，本发明进行了大量的尝试和筛选，意外地发现，将其编码基因上游非编码区中的特定区域替换为强启动子，能够显著提高磷酸烯醇式丙酮酸羧化酶的表达量。与增加拷贝数的改造方式相比，这种改造方式具有更高的稳定性，而且，与其它替换强启动子的改造方式相比，这种改造方式对于磷酸烯醇式丙酮酸羧化酶的酶活性提升效果明显更优。
优选地，所述强化表达通过将磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp替换为强启动子。
进一步优选地，所述强启动子为Ptuf。所述强启动子Ptuf的核苷酸序列优选如SEQID NO.4所示。
为增强基因表达，通常会将基因的原始启动子替换为强启动子或者在起始密码子上游直接插入强启动子，在进行启动子替换时，通常会将目的基因与上游基因之间的间隔序列全部替换为强启动子，或者，将目的基因的原始启动子区域替换为强启动子。但本发明发现，与替换磷酸烯醇式丙酮酸羧化酶编码基因的启动子区域相比，将起始密码子上游27bp的DNA片段替换为强启动子(尤其是强启动子Ptuf)，对于提升磷酸烯醇式丙酮酸羧化酶的表达量的作用明显更优。
本发明所述的起始密码子上游27bp片段是指：以起始密码子的上游第一个碱基为第1位碱基，向上游方向延伸至第27位碱基，所获得的长度为27bp的DNA片段。以谷氨酸棒状杆菌野生型菌株ATCC13032为例，磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp片段的序列如SEQ ID NO.3所示。
本发明所述的磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.1或2所示，或者与SEQ ID NO.1或2所示序列相似性至少为90％且具有同等功能的氨基酸序列。
优选磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.1或2所示。
其中，SEQ ID NO.1所示的序列为谷氨酸棒状杆菌野生型磷酸烯醇式丙酮酸羧化酶的氨基酸序列。SEQ ID NO.2所示的序列为在谷氨酸棒状杆菌野生型磷酸烯醇式丙酮酸羧化酶基础上，将第299位D突变为N得到的突变型蛋白(D299N)。该突变可使得磷酸烯醇式丙酮酸解除反馈抑制。在上述启动子区改造的基础上结合磷酸烯醇式丙酮酸羧化酶D299N的突变，可使得菌株的苏氨酸产量显著提升。因此，优选磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.2所示。
进一步地，本发明提供一种重组微生物，所述重组微生物中，磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp片段被替换为强启动子。
优选地，所述重组微生物中，磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp片段被替换为强启动子。
进一步优选地，所述强启动子为Ptuf。
上述磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.1或2所示，或者与SEQID NO.1或2所示序列相似性至少为90％且具有同等功能的氨基酸序列。
优选磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.1或2所示。在上述启动子区改造的基础上结合磷酸烯醇式丙酮酸羧化酶D299N的突变，可使得菌株的苏氨酸产量显著提升。因此，优选重组微生物的磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.2所示。
以上所述的重组微生物优选采用以下方法构建得到：在出发菌株中，将出发菌株的磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp片段替换为强启动子Ptuf。优选所述方法还包括将其磷酸烯醇式丙酮酸羧化酶编码基因突变，使得其编码的磷酸烯醇式丙酮酸羧化酶发生D299N突变的步骤。
其中，出发菌株优选为能够积累苏氨酸的菌株。
优选地，所述重组微生物中，以下(1)～(7)中的任意一个或多个酶的酶活性被增强和/或解除反馈抑制：
(1)天冬氨酸激酶；
(2)天冬氨酸半醛脱氢酶；
(3)高丝氨酸脱氢酶；
(4)苏氨酸合酶；
(5)高丝氨酸激酶；
(6)天冬氨酸转氨酶；
(7)苏氨酸外运蛋白；
优选地，所述苏氨酸外运蛋白为大肠杆菌来源的苏氨酸外运蛋白。
上述天冬氨酸激酶、高丝氨酸脱氢酶、天冬氨酸半醛脱氢酶、天冬氨酸氨基转移酶、高丝氨酸激酶、苏氨酸合酶在NCBI上的参考序列编号分别为WP_003855724.1、WP_003854900.1、WP_011013506.1、WP_011013497.1、WP_011014183.1、WP_011014964.1，或与上述参考序列相似性至少为90％且具有同等功能的氨基酸序列。
大肠杆菌的苏氨酸外运蛋白在NCBI上的参考序列编号为YP_026264.1，或与上述参考序列相似性至少为90％且具有同等功能的氨基酸序列。
优选地，所述微生物为如下①～⑥中的任一种：
①磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶活性增强和/或解除反馈抑制的微生物；
②磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、天冬氨酸激酶和/或天冬氨酸半醛脱氢酶活性增强和/或解除反馈抑制的微生物；
③磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶和/或高丝氨酸脱氢酶活性增强和/或解除反馈抑制的微生物；
④磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、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和/或解除反馈抑制的微生物；
⑤磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、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和/或解除反馈抑制的微生物；
⑥磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、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、苏氨酸合酶和/或苏氨酸外运蛋白活性增强和/或解除反馈抑制的微生物。
上述酶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)通过对编码酶的核苷酸序列进行改变而增强。
作为本发明的优选方案，所述重组微生物为以下(1)-(6)任一种：
(1)磷酸烯醇式丙酮酸羧化酶基因引入突变位点D299N并将编码基因起始密码子上游27bp片段替换为Ptuf启动子，同时，天冬氨酸转氨酶被强化表达；
(2)磷酸烯醇式丙酮酸羧化酶引入突变位点D299N并将编码基因起始密码子上游27bp片段替换为Ptuf启动子，同时，天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶被强化表达，天冬氨酸激酶解除反馈抑制；
(3)磷酸烯醇式丙酮酸羧化酶引入突变位点D299N并将编码基因起始密码子上游27bp片段替换为Ptuf启动子，同时，天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶被强化表达，天冬氨酸激酶、高丝氨酸脱氢酶解除反馈抑制；
(4)磷酸烯醇式丙酮酸羧化酶引入突变位点D299N并将编码基因起始密码子上游27bp片段替换为Ptuf启动子，同时，天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、、高丝氨酸脱氢酶、高丝氨酸激酶被强化表达，天冬氨酸激酶、高丝氨酸脱氢酶解除反馈抑制；
(5)磷酸烯醇式丙酮酸羧化酶引入突变位点D299N并将编码基因起始密码子上游27bp片段替换为Ptuf启动子，同时，天冬氨酸转氨酶、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、苏氨酸合酶被强化表达，天冬氨酸激酶、高丝氨酸脱氢酶解除反馈抑制；
(6)磷酸烯醇式丙酮酸羧化酶引入突变位点D299N并将编码基因起始密码子ATG上游27bp替换为Ptuf启动子和天冬氨酸转氨酶、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，苏氨酸合酶被强化表达，且表达大肠杆菌来源的苏氨酸外运蛋白，天冬氨酸激酶、高丝氨酸脱氢酶解除反馈抑制。
优选地，以上所述的酶活性的增强或强化表达通过将基因的原始启动子替换为强启动子实现，或者通过将基因的起始密码子突变以使得其编码蛋白的第1位氨基酸由缬氨酸突变为蛋氨酸实现。
强启动子优选为启动子Psod、PcspB或Ptuf。其中，启动子Psod、PcspB的核苷酸序列分别如SEQ ID NO.5和6所示。
优选地，天冬氨酸激酶编码基因、苏氨酸合酶编码基因的强化表达通过将其原始启动子替换为启动子Psod，并将其起始密码子由GTG突变为ATG实现。
天冬氨酸半醛脱氢酶编码基因、天冬氨酸转氨酶编码基因的强化表达通过将其原始启动子替换为启动子Psod实现。
高丝氨酸脱氢酶编码基因、高丝氨酸激酶编码基因的强化表达通过将其原始启动子替换为启动子PcspB实现。
上述大肠杆菌的苏氨酸外运蛋白的表达可通过将大肠杆菌的苏氨酸外运蛋白编码基因的表达盒整合至微生物的染色体中实现。优选的整合位点为cg2009基因下游，其表达盒的启动子为Psod。
上述天冬氨酸激酶的解除反馈抑制优选通过将天冬氨酸激酶编码基因突变，使得其编码的天冬氨酸激酶发生T311I突变实现；高丝氨酸脱氢酶的解除反馈抑制优选通过将高丝氨酸脱氢酶编码基因突变，使得高丝氨酸脱氢酶发生G378E突变实现。
以上所述的重组微生物优选为棒状杆菌属细菌，更优选为谷氨酸棒状杆菌(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ATCC13032。
本发明还提供以上所述的重组微生物的构建方法，包括：将出发菌株的磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp片段替换为强启动子Ptuf。
优选地，所述方法还包括将其磷酸烯醇式丙酮酸羧化酶编码基因突变，使得其编码的磷酸烯醇式丙酮酸羧化酶发生D299N突变。
进一步优选地，所述方法还包括：增强以下(1)～(7)中的任意一个或多个酶的酶活性和/或将其解除反馈抑制：
(1)天冬氨酸激酶；
(2)天冬氨酸半醛脱氢酶；
(3)高丝氨酸脱氢酶；
(4)苏氨酸合酶；
(5)高丝氨酸激酶；
(6)天冬氨酸转氨酶；
(7)苏氨酸外运蛋白；
其中，酶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)通过对编码酶的核苷酸序列进行改变而增强。
上述有关菌株的改造方法包括基因的强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.
本发明提供以上所述的重组微生物的以下任一种应用：
(1)在发酵生产苏氨酸或其衍生物中的应用；
(2)在作为出发菌株用于构建苏氨酸或其衍生物的生产菌株中的应用；
(3)在提高苏氨酸或其衍生物的产量和/或转化率中的应用。
本发明还提供一种发酵生产苏氨酸或其衍生物的方法，包括培养以上所述的重组微生物并从培养物中分离得到苏氨酸或其衍生物的步骤。
具体地，上述方法包括：将所述重组微生物接种于种子培养基中进行种子培养，得到种子液，将种子液接种于发酵培养基中培养，得到发酵液，将发酵液经分离提取得到苏氨酸或其衍生物。
优选地，所述发酵培养基包含如下组分：玉米浆45-55mL/L，葡萄糖25-35g/L，硫酸铵3-5g/L，MOPS 25-35g/L，磷酸二氢钾8-12g/L，尿素15-25g/L，生物素8-12mg/L，硫酸镁5-7g/L，硫酸亚铁0.5-1.5g/L，VB1·HCl 35-45mg/L，泛酸钙45-55mg/L，烟酰胺35-45mg/L，硫酸锰0.5-1.5g/L，硫酸锌15-25mg/L，硫酸铜15-25mg/L，pH 7.0-7.2。
本发明的有益效果在于：本发明通过对磷酸烯醇式丙酮酸羧化酶编码基因的启动子的特异优化和基因编码区的突变，显著提高了菌株合成苏氨酸的能力，苏氨酸产量较未经上述改造的菌株提高25-40％，上述改造方法可应用于苏氨酸生产菌株的构建和苏氨酸发酵生产中，具有较好的应用价值。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
本发明重点考察了磷酸烯醇式丙酮酸羧化酶的启动子区修饰对苏氨酸生产的影响，经验证，将磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp替换为强启动子可显著促进苏氨酸的生产。
本发明进一步对菌株的苏氨酸合成、转运路径进行强化，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、天冬氨酸转氨酶、高丝氨酸激酶、苏氨酸合酶、苏氨酸外运蛋白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将其磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp替换为强启动子后，其苏氨酸的生产能力有所提升；同时与仅将其磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp替换为强启动子的菌株相比，将磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp替换为强启动子和苏氨酸合成、转运路径中的酶表达强化相结合的菌株在苏氨酸的生产上更有优势。
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，具体实施例中仅以启动子强化作为代表进行说明。
本发明采用如下技术方案：
本发明的技术方案之一，提供一种利用磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶表达强化和/或解调控的菌株生产苏氨酸的方法；
本发明的技术方案之二，提供一种利用磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶至少一个表达强化和/或解调控的菌株生产苏氨酸的方法；
本发明的技术方案之三，提供一种利用磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶至少一个表达强化和/或解调控的菌株生产苏氨酸的方法；
本发明的技术方案之四，提供一种利用磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶至少一个表达强化和/或解调控的菌株生产苏氨酸的方法；
本发明的技术方案之五，提供一种利用磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、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、苏氨酸合酶至少一个表达强化和/或解调控的菌株生产苏氨酸的方法；
本发明的技术方案之六，提供一种利用磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子，且天冬氨酸转氨酶、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、苏氨酸合酶、苏氨酸外运蛋白至少一个表达强化和/或解调控的菌株生产苏氨酸的方法。
上述菌株为棒状杆菌属细菌，优选谷氨酸棒状杆菌，最优选谷氨酸棒状杆菌ATCC13032。
本发明所涉及的酶和基因的详细信息如下：
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：Cgl0252，Cg0307、NCgl0248；
高丝氨酸脱氢酶，编码基因名称hom，NCBI编号：Cg1337、Cgl1183、NCgl1136；
苏氨酸合酶，编码基因名称thrC，NCBI编号：cg2437、Cgl2220、NCgl2139；
高丝氨酸激酶，编码基因thrB，NCBI编号：Cgl1184，Cg1338、NCgl1137；
磷酸烯醇式丙酮酸羧化酶，编码基因ppc，NCBI编号：cg1787、Cgl1585、NCgl1523；
天冬氨酸转氨酶，编码基因名称aspB，NCBI编号：cg0294、cg0294和cg0294；
大肠杆菌来源苏氨酸外运蛋白，编码基因名称rhtC，NCBI编号：948317。
实施例1菌株基因组改造质粒的构建
1、天冬氨酸激酶-天冬氨酸半醛脱氢酶操纵子表达强化质粒pK18mobsacB-Psod-lysCg1a-T311I-asd的构建
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为模板进行融合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pK18mobsacB和up-Psod-lysCg1a-T311I-dn用无缝克隆试剂盒进行组装，转化Trans1T1感受态细胞，获得重组质粒pK18mobsacB-Psod-lysCg1a-T311I-asd。
2、天冬氨酸转氨酶表达强化质粒pK18mobsacB-Psod-aspB的构建
质粒构建方法参考上述1，所用引物为P103、P104、P105、P106、P107和P108。
3、高丝氨酸脱氢酶-高丝氨酸激酶操纵子表达强化质粒pK18mobsacB-PcspB-homG378E-thrB的构建
质粒构建方法参考上述1，所用引物为P29、P30、P31、P32、P33、P34、P35、P36。
4、苏氨酸合酶表达强化质粒pK18mobsacB-Psod-thrCg1a的构建
质粒构建方法参考上述1，所用引物为P37、P38、P39、P40、P41和P42。
5、苏氨酸外运蛋白表达强化质粒pK18mobsacB-Psod-rhtC的构建
质粒构建方法参考上述1，所用引物为P157、P158、P159、P160、P161、P162、P163和P164。
6、磷酸烯醇式丙酮酸羧化酶表达强化质粒pK18mobsacB-Ptuf-ppcD299N的构建
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为模板进行融合PCR，获得片段up-Ptuf。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pK18mobsacB和up-Ptuf-ppcD299N-dn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。
以上质粒构建过程中所使用的引物如表1所示。
表1引物序列
实施例2基因组改造菌株的构建
1、天冬氨酸转氨酶强化表达菌株的构建
按照谷氨酸棒状杆菌经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC13032感受态细胞。重组质粒pK18mobsacB-Psod-aspB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落的基因组中不携带插入的载体序列。通过PCR扩增目的片段并进行核苷酸测序分析，获得目的突变菌株命名为SMCT061，该菌株与菌株ATCC13032相比，aspB基因的启动子被替换为Psod启动子。
2、天冬氨酸激酶-天冬氨酸半醛脱氢酶操纵子强化表达菌株的构建
菌株构建方法参考上述1，以SMCT061为出发菌，将pK18mobsacB-Psod-lysCg1a-T311I-asd质粒导入SMCT061中，进行天冬氨酸激酶-天冬氨酸半醛脱氢酶操纵子强化的改造，获得的菌株命名为SMCT062，该菌株与菌株SMCT061相比，lysC基因发生突变导致其起始密码子由GTG突变为ATG，编码氨基酸序列的第311位由苏氨酸突变为异亮氨酸，同时lysC-asd操纵子的启动子被替换为Psod启动子。
3、高丝氨酸脱氢酶-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SMCT062为出发菌，将pK18mobsacB-PcspB-homG378E-thrB质粒导入SMCT062中，进行高丝氨酸脱氢酶-高丝氨酸激酶表达强化的改造，获得的改造菌株命名为SMCT063，该菌株与菌株SMCT062相比，hom基因发生突变导致其编码蛋白产生G378E的突变，同时hom-thrB操纵子的启动子被替换为PcspB启动子。
4、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SMCT063为出发菌，将pK18mobsacB-Psod-thrCg1a质粒导入SMCT063中，进行苏氨酸合酶表达强化的改造，获得的改造菌株命名为SMCT064，该菌株与菌株SMCT063相比，thrC基因的起始密码子突变为ATG，且thrC基因的启动子被替换为Psod。
5、苏氨酸外运蛋白表达强化菌株的构建
菌株构建方法参考上述1，以SMCT064为出发菌，将pK18mobsacB-Psod-rhtC质粒导入SMCT064中，进行苏氨酸外运蛋白表达强化的改造，获得的改造菌株命名为SMCT065，该菌株与SMCT064相比，在cg2009基因下游插入了来源于大肠杆菌的苏氨酸外运蛋白基因rhtC。
6、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，分别以SMCT061、SMCT062、SMCT063、SMCT064、SMCT065为出发菌，将pK18mobsacB-Ptuf-ppcD299N质粒分别导入上述出发菌中，进行磷酸烯醇式丙酮酸羧化酶表达强化的改造，获得的改造菌株分别命名为SMCT066、SMCT067、SMCT068、SMCT079、SMCT070，这些改造菌株与其对应的出发菌相比，磷酸烯醇式丙酮酸羧化酶基因ppc突变使得其编码蛋白产生D299N突变，且ppc基因起始密码子上游27bp片段被替换为Ptuf启动子。
上述通过基因改造获得的菌株的基因型如表2所示。
表2菌株基因型信息
| 菌株 | 基因型 |
| SMCT061 | ATCC13032,Psod-aspB |
| SMCT062 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd |
| SMCT063 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,PcspB-homG378E-thrB |
| SMCT064 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,PcspB-homG378E-thrB,Psod-thrCg1a |
| SMCT065 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,PcspB-homG378E-thrB,Psod-thrCg1a,Psod-rhtC |
| SMCT066 | ATCC13032,Psod-aspB,Ptuf-ppcD299N |
| SMCT067 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,Ptuf-ppcD299N |
| SMCT068 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,PcspB-homG378E-thrB,Ptuf-ppcD299N |
| SMCT069 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,PcspB-homG378E-thrB,Psod-thrCg1a,Ptuf-ppcD299N |
| SMCT070 | ATCC13032,Psod-aspB,Psod-lysCg1a-T311I-asd,PcspB-homG378E-thrB,Psod-thrCg1a,Psod-rhtC,Ptuf-ppcD299N |
实施例3菌株的摇瓶发酵验证
对实施例2构建的各改造菌株进行摇瓶发酵验证，具体如下：
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SMCT061、SMCT062、SMCT063、SMCT064、SMCT065、SMCT066、SMCT067、SMCT068、SMCT069和SMCT070斜面种子1环接至装有20mL种子培养基的500mL三角瓶中，于30℃、220r/min振荡培养16h，得到种子液。
(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，于33℃、220r/min振荡培养24h，得到发酵液。
(3)取1mL发酵液离心(12000rpm，2min)，收集上清液，采用HPLC检测工程菌与对照菌发酵液中的L-苏氨酸。
苏氨酸的摇瓶发酵结果如表3所示。
表3发酵检测结果
结果表明，在苏氨酸生产出发菌SMCT061、SMCT062、SMCT063、SMCT064、SMCT065中优化磷酸烯醇式丙酮酸羧化酶编码基因的启动子并将磷酸烯醇式丙酮酸羧化酶进行D299N突变，苏氨酸的产量进一步提升，菌株SMCT066、SMCT067、SMCT068、SMCT069、SMCT070的苏氨酸产量分别提升25％、33％、35％、38％和40％，表明通过将磷酸烯醇式丙酮酸羧化酶编码基因起始密码子上游20～30bp片段替换为强启动子并将磷酸烯醇式丙酮酸羧化酶进行D299N突变可增加苏氨酸合成前体草酰乙酸的供应，进而促进苏氨酸的合成。此外，将上述磷酸烯醇式丙酮酸羧化酶的改造与苏氨酸合成路径中的不同酶以及苏氨酸外运蛋白的强化表达相结合，苏氨酸的产量进一步提升，表明上述改造相结合更有利于提升菌株的苏氨酸生产能力。
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> 一种生产苏氨酸的重组微生物及其应用
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<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gtaaaacgac ggccagtgcc aagcttagcc tggtaagagg aaacgt 46
<210> 15
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aattcgagct cggtacccgg ggatccctgc gggcagatcc ttttga 46
<210> 16
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atttctttat aaacgcaggt catatctacc aaaactacgc 40
<210> 17
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gcgtagtttt ggtagatatg acctgcgttt ataaagaaat 40
<210> 18
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gtatatctcc ttctgcagga ataggtatcg aaagacgaaa 40
<210> 19
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tttcgtcttt cgatacctat tcctgcagaa ggagatatac 40
<210> 20
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tagccaattc agccaaaacc cccacgcgat cttccacatc c 41
<210> 21
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ggatgtggaa gatcgcgtgg gggttttggc tgaattggct a 41
<210> 22
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gtaaaacgac ggccagtgcc aagcttgctg gctcttgccg tcgata 46
<210> 23
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
attcgagctc ggtacccggg gatccgccgt tgatcattgt tcttca 46
<210> 24
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
cccggaataa ttggcagcta ggatataacc ctatcccaag 40
<210> 25
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
cttgggatag ggttatatcc tagctgccaa ttattccggg 40
<210> 26
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
acgcgtcgaa atgtagtcca tgggtaaaaa atcctttcgt a 41
<210> 27
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
tacgaaagga ttttttaccc atggactaca tttcgacgcg t 41
<210> 28
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
gtaaaacgac ggccagtgcc aagcttgaat acgcggattc cctcgc 46
<210> 29
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
aattcgagct cggtacccgg ggatcctacg tcgtcgagca gacccg 46
<210> 30
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
cattcgcagg gtaacggcca agggtgttgg cgtgcatgag 40
<210> 31
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ctcatgcacg ccaacaccct tggccgttac cctgcgaatg 40
<210> 32
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tcgcgtaaaa aatcagtcat tgtatgtcct cctggacttc 40
<210> 33
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gaagtccagg aggacataca atgactgatt ttttacgcga 40
<210> 34
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gtgaccttat tcatgcggtt cgacaggctg agctcatgct 40
<210> 35
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
agcatgagct cagcctgtcg aaccgcatga ataaggtcac 40
<210> 36
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gtaaaacgac ggccagtgcc aagcttggtg acttgggcgc gttcga 46
<210> 37
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gagctcggta cccggggatc cgcagggtat tgcagggact ca 42
<210> 38
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
caagcccgga ataattggca gctaaactgc gtacctccgc atgtggtgg 49
<210> 39
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tagctgccaa ttattccggg cttgt 25
<210> 40
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gggtaaaaaa tcctttcgta ggttt 25
<210> 41
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ggaaacctac gaaaggattt tttacccatg agttcagttt cgctgcagga ttt 53
<210> 42
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
acgacggcca gtgccaagct tacaccggaa caacccacat g 41
<210> 43
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
tacgaattcg agctcggtac ccggggatcc agttaactcc accgaccggg tactgc 56
<210> 44
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
aagcccggaa taattggcag ctatgtcttc gctggaccaa gag 43
<210> 45
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ctcttggtcc agcgaagaca tagctgccaa ttattccggg ctt 43
<210> 46
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gacggtgaga aataacatca acatgggtaa aaaatccttt cgta 44
<210> 47
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
tacgaaagga ttttttaccc atgttgatgt tatttctcac cgtc 44
<210> 48
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
tgcctctttt agccttttca gagggtcacc gcgaaataat caaatgaa 48
<210> 49
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
ttcatttgat tatttcgcgg tgaccctctg aaaaggctaa aagaggca 48
<210> 50
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gttgtaaaac gacggccagt gccaagctta aaaggcagtc cagtacaccc t 51
Claims (10)
1.磷酸烯醇式丙酮酸羧化酶编码基因的强化表达在提高棒状杆菌属细菌的苏氨酸产量或构建生产苏氨酸的棒状杆菌属细菌中的应用；
所述强化表达通过将磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp片段替换为强启动子实现。
2.根据权利要求1所述的应用，其特征在于，所述强化表达通过将磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp片段替换为强启动子实现；
优选地，所述强启动子为Ptuf。
3.根据权利要求1或2所述的应用，其特征在于，所述磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.1或2所示。
4.一种重组微生物，其特征在于，所述重组微生物中，磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游20～30bp片段被替换为强启动子；
优选地，所述重组微生物中，磷酸烯醇式丙酮酸羧化酶编码基因的起始密码子上游27bp片段被替换为强启动子；
更优选地，所述强启动子为Ptuf。
5.根据权利要求4所述的重组微生物，其特征在于，所述重组微生物的磷酸烯醇式丙酮酸羧化酶的氨基酸序列如SEQ ID NO.1或2所示。
6.根据权利要求4或5所述的重组微生物，其特征在于，所述重组微生物中，以下(1)～(7)中的任意一个或多个酶的酶活性被增强和/或解除反馈抑制：
(1)天冬氨酸激酶；
(2)天冬氨酸半醛脱氢酶；
(3)高丝氨酸脱氢酶；
(4)苏氨酸合酶；
(5)高丝氨酸激酶；
(6)天冬氨酸转氨酶；
(7)苏氨酸外运蛋白；
优选地，所述苏氨酸外运蛋白为大肠杆菌来源的苏氨酸外运蛋白。
7.根据权利要求6所述的重组微生物，其特征在于，所述酶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)通过对编码酶的核苷酸序列进行改变而增强。
8.根据权利要求4-7任一项所述的重组微生物，其特征在于，所述微生物为棒杆状菌属细菌，优选为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
9.权利要求4～8任一项所述的重组微生物的以下任一种应用：
(1)在发酵生产苏氨酸或其衍生物中的应用；
(2)在作为出发菌株用于构建苏氨酸或其衍生物的生产菌株中的应用；
(3)在提高苏氨酸或其衍生物的产量和/或转化率中的应用。
10.一种发酵生产苏氨酸或其衍生物的方法，其特征在于，包括培养权利要求4～8任一项所述的重组微生物并从培养物中分离得到苏氨酸或其衍生物的步骤。
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| CN202210107366.3A CN116555251A (zh) | 2022-01-28 | 2022-01-28 | 一种生产苏氨酸的重组微生物及其应用 |
| PCT/CN2022/143106 WO2023142862A1 (zh) | 2022-01-28 | 2022-12-29 | 一种生产苏氨酸的重组微生物及其应用 |
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| KR100858913B1 (ko) * | 2007-03-09 | 2008-09-17 | 한국과학기술원 | 부위특이적 변이에 의해 제조된 고수율의 ｌ－쓰레오닌생산균주 및 이를 이용한 ｌ－쓰레오닌의 제조방법 |
| CN106867952B (zh) * | 2017-01-09 | 2019-10-18 | 天津科技大学 | 一株大肠杆菌基因工程菌及利用其生产l-苏氨酸的方法 |
| CN107012161A (zh) * | 2017-04-03 | 2017-08-04 | 天津大学 | 利用秸秆水解液高产琥珀酸的谷氨酸棒杆菌及构建及应用 |
| CN107012160A (zh) * | 2017-05-15 | 2017-08-04 | 天津大学 | 高产琥珀酸的谷氨酸棒杆菌菌株及构建方法及应用 |
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