WO2023151411A1 - 一种生产苏氨酸的重组微生物及其构建方法和应用 - Google Patents

一种生产苏氨酸的重组微生物及其构建方法和应用 Download PDF

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WO2023151411A1
WO2023151411A1 PCT/CN2022/143101 CN2022143101W WO2023151411A1 WO 2023151411 A1 WO2023151411 A1 WO 2023151411A1 CN 2022143101 W CN2022143101 W CN 2022143101W WO 2023151411 A1 WO2023151411 A1 WO 2023151411A1
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enhanced
threonine
activity
enzyme
microorganism
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康培
姚嘉琪
宫卫波
何君
李岩
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廊坊梅花生物技术开发有限公司
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Definitions

  • the invention relates to the technical field of microbial engineering, in particular to a threonine-producing recombinant microorganism and its construction method and application.
  • threonine is ⁇ -hydroxy- ⁇ -aminobutyric acid
  • the molecular formula is C 4 H 9 NO 3
  • the relative molecular mass is 119.12. It is an essential amino acid, mainly used in medicine, chemical reagents, food Fortifiers, feed additives, etc.
  • Corynebacterium glutamicum is an important producer of amino acid fermentation.
  • the generation of threonine from oxaloacetate requires five steps of catalytic reactions, which are respectively composed of aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), homoserine dehydrogenase Catalyzed by hydrogenase (encoded by hom), homoserine kinase (encoded by thrB), and threonine synthase (encoded by thrC).
  • the purpose of the present invention is to improve the threonine-producing ability of the bacterial strain by inactivating acetate kinase (ackA), thereby providing a threonine-producing recombinant microorganism and its construction method and application.
  • ackA acetate kinase
  • the metabolic engineering of threonine synthesis by Corynebacterium glutamicum mainly focuses on the synthesis pathway of threonine, mainly the synthesis pathway from oxaloacetate to threonine; and pyruvate is an important intermediate in the microbial metabolic network. Metabolites mainly enter the tricarboxylic acid cycle to provide energy and precursor substances for bacterial growth. However, when the upstream and downstream metabolic pathways are unbalanced, it will cause metabolic overflow of pyruvate, resulting in waste of pyruvate.
  • oxaloacetate can be catalyzed by pyruvate carboxylase to generate pyruvate, and it can also be generated by pyruvate entering the tricarboxylic acid cycle through a series of enzyme-catalyzed reactions.
  • the present invention finds that by reducing the metabolic overflow of pyruvate, the flow of pyruvate to threonine synthesis precursor oxaloacetate can be increased, thereby promoting the synthesis of threonine.
  • the effect of reducing or losing the activity of acetate kinase is significantly better.
  • it can effectively reduce the metabolic overflow of pyruvate and increase the synthesis precursor of threonine. supply, thereby significantly improving the threonine synthesis ability of the strain.
  • the present invention provides a modified microorganism of the genus Corynebacterium, said microorganism has reduced or lost activity of acetate kinase compared to an unmodified microorganism, and said microorganism is compared to Unmodified microorganisms have enhanced threonine production capacity.
  • the reference sequence number of acetate kinase on NCBI is WP_003862874.1, or an amino acid sequence with 90% similarity and equivalent function.
  • Mutagenesis, site-directed mutation or homologous recombination can be used to reduce the expression of the gene encoding acetate kinase or to knock out the endogenous gene encoding acetate kinase.
  • the activity of pyruvate carboxylase is enhanced and/or feedback inhibition is relieved.
  • the reference sequence number of pyruvate carboxylase on NCBI is WP_011013816.1, or an amino acid sequence with 90% similarity and equivalent function.
  • the reference sequence number of malate quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase on NCBI is WP_011014814 .1, NP_600790.1, NP_600669.1, FOB93_04945, or an amino acid sequence having 90% similarity thereto and having equivalent functions.
  • the microorganism is any one of the following 1 ⁇ 6:
  • the enhancement of the activity of the enzymes described above is achieved by being selected from the following 1) to 6), or an optional combination:
  • the enhanced activity of the enzyme is achieved by replacing the original promoter of the gene encoding the enzyme with a strong promoter with stronger activity, and/or, mutating the start codon of the gene to ATG.
  • the strong promoter includes Psod, Ptuf or PcspB.
  • the nucleotide sequences of promoters Psod, Ptuf or PcspB are shown in SEQ ID NO.1, 2 and 3 respectively.
  • pyruvate carboxylase encoding gene malate quinone oxidoreductase encoding gene, glucose-6-phosphate dehydrogenase encoding gene, 6-phosphogluconate dehydrogenase encoding gene, aspartokinase encoding gene 1.
  • the enhanced expression of the threonine synthase coding gene is realized by replacing its original promoter with the Psod promoter;
  • the enhanced expression of the gene encoding homoserine dehydrogenase was achieved by replacing its original promoter with the PcspB promoter.
  • the release of feedback inhibition as described above is preferably achieved by the following mutation: the release of feedback inhibition of pyruvate carboxylase is achieved by mutating the gene encoding pyruvate carboxylase so that the encoded pyruvate carboxylase undergoes a P458S mutation;
  • the release of feedback inhibition of glucose-6-phosphate dehydrogenase is achieved by mutating the glucose-6-phosphate dehydrogenase gene so that the encoded glucose-6-phosphate dehydrogenase has an A243T mutation;
  • the release of feedback inhibition of homoserine dehydrogenase is achieved by mutating the gene encoding homoserine dehydrogenase, so that the homoserine dehydrogenase undergoes a G378E mutation.
  • the microorganism described in the present invention is Corynebacterium glutamicum.
  • Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc.
  • Corynebacterium acid ATCC 13032.
  • the enzymes related to the threonine synthesis pathway are selected from the group consisting of aspartokinase, homoserine dehydrogenase, threonine at least one of acid synthases;
  • the enhanced pathway is selected from the following 1) to 6), or an optional combination:
  • the present invention provides a method for producing threonine, the method comprising the steps of:
  • step b) collecting the threonine produced from said culture obtained in step a).
  • the present invention provides the application of reducing or losing the enzymatic activity of acetate kinase in the fermentative production of threonine or increasing the fermentative yield of threonine.
  • Glucose-6-phosphate dehydrogenase encoding gene name zwf, NCBI number: cg1778, Cgl1576, NCgl1514.
  • 6-phosphogluconate dehydrogenase encoding gene name gnd, NCBI number: cg1643, Cgl1452, NCgl1396.
  • NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans encoding gene name gapN, NCBI number: FOB93_04945.
  • Embodiment 1 strain genome transformation plasmid construction
  • the upstream homology arm up was obtained by PCR amplification with the P21/P22 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P23/P24 primer pair
  • the Psod was obtained by PCR amplification with the P25/P26 primer pair.
  • lysC a1g-T311I was amplified by PCR with the P27/P28 primer pair to obtain the downstream homology arm dn. Fusion PCR was performed with P21/P24 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-lysC a1g-T311I -dn was obtained by fusion PCR with P21/P28 primer pair and up-Psod, lysC a1g-T311I , dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII.
  • the digested up-Psod-lysC a1g-T311I -dn and pK18mobsacB were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-Psod-lysC a1g-T311I was obtained.
  • the plasmid construction method refers to the above 1, and the primers used are P29, P30, P31, P32, P33, P34, P35, and P36.
  • the plasmid construction method refers to the above 1, and the primers used are P37, P38, P39, P40, P41, and P42.
  • the plasmid construction method refers to the above 1, and the primers used are P13, P14, P15, P16, P17, P18, P19, and P20.
  • the upstream homology arm up was obtained by PCR amplification with the P165/P166 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P167/P168 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR using the P165/P168 primer pair and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII.
  • the digested up-dn and pK18mobsacB were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB- ⁇ ackA was obtained.
  • the plasmid construction method refers to the above 1, and the primers used are P169, P170, P171, P172, P173, and P174.
  • the primers used are P129, P130, P131, P132, P133, P134, P135, and P136.
  • the primers used are P123, P124, P125, P126, P127, and P128.
  • the full-length fragment up-Ptuf-gapN-dn was obtained by fusion PCR with P137/P144 primer pair and up-Ptuf, gapN, dn as template.
  • pK18mobsacB was digested with BamHI/HindIII.
  • the digested up-Ptuf-gapN-dn and pK18mobsacB were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-Ptuf-gapN was obtained.
  • Embodiment 2 Construction of Genome Modification Strain
  • ATCC13032 competent cells were prepared according to the classic method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-Psod-lysC a1g-T311I was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/L kanamycin, in which the target gene was inserted into the chromosome due to homology middle.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm.
  • the strain construction method refers to the above 1, using SMCT106 as the starting bacterium, the plasmid pK18mobsacB-PcspB-hom G378E is introduced into SMCT106, and the transformation of homoserine dehydrogenase expression enhancement is carried out.
  • the obtained modified strain is named SMCT107.
  • the mutation of the hom gene of the strain resulted in the G378E mutation of its encoded protein, and the promoter of the hom gene was replaced by the PcspB promoter derived from ATCC14067.
  • SMCT109 As the starting bacterium, the plasmid pK18mobsacB-Psod-mqo was introduced into SMCT109, and the expression of malate dehydrogenase was enhanced.
  • the modified strain obtained was named SMCT111.
  • the The promoter of the mqo gene of the strain was replaced by the Psod promoter.
  • SMCT108, SMCT109, SMCT111, SMCT112, SMCT113, and SMCT114 as the starting bacteria, respectively.
  • the plasmid pK18mobsacB- ⁇ ackA was introduced into the above starting bacteria, and the transformation of acetate kinase inactivation was carried out.
  • the obtained modified strains were named as SMCT119, SMCT110, SMCT115, SMCT116, SMCT117, SMCT118, compared with their corresponding origin strains, the ackA gene was knocked out.
  • Seed activation medium BHI 3.7%, agar 2%, pH 7.
  • Seed medium peptone 5/L, yeast extract 5g/L, sodium chloride 10g/L, ammonium sulfate 16g/L, urea 8g/L, potassium dihydrogen phosphate 10.4g/L, dipotassium hydrogen phosphate 21.4g /L, biotin 5mg/L, magnesium sulfate 3g/L, glucose 50g/L, pH 7.2.
  • Seed culture pick the slant seeds of strains SMCT108, SMCT109, SMCT110, SMCT111, SMCT112, SMCT113, SMCT114, SMCT115, SMCT116, SMCT117, SMCT118, SMCT119 and connect them to a 500mL Erlenmeyer flask containing 20mL seed medium , 30°C, 220r/min shaking culture for 16h, to obtain seed liquid.
  • Fermentation culture Inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture with shaking at 33° C. and 220 r/min for 24 hours to obtain a fermentation liquid.
  • the production of the protein was further improved, and the increase was significantly higher than that of SMCT108 inactivating acetate kinase, indicating that the combination of the above-mentioned gene modification and the inactivation of acetate kinase was more conducive to the production of threonine.

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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构建方法和应用。本发明通过构建乙酸激酶失活的菌株并将其应用于苏氨酸生产，显著提高了菌株生产苏氨酸的能力，结合苹果酸醌氧化还原酶、葡萄糖-6-磷酸脱氢酶、6-磷酸葡糖酸脱氢酶等的表达强化，苏氨酸的产量进一步提升，为大规模生产苏氨酸提供了新方法，具有较高的应用价值。

Description

一种生产苏氨酸的重组微生物及其构建方法和应用 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构建方法和应用。
背景技术
苏氨酸(Threonin)的化学名称为β-羟基-α-氨基丁酸，分子式为C 4H 9NO 3，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、饲料添加剂等方面。
谷氨酸棒杆菌是氨基酸发酵的重要生产菌。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，分别由天冬氨酸激酶(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)催化。目前利用谷氨酸棒杆菌生产苏氨酸的报道主要集中在其合成路径中，已有抗反馈抑制的hom基因(Reinscheid D J,Eikmanns B J,Sahm H.Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230.)、lysC基因(Eikmanns B J,Eggeling L,Sahm H.Molecular aspects of lysine,threonine,and isoleucine biosynthesis in Corynebacterium glutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163.)的报道。但是，目前极少有关于苏氨酸的前体供应和苏氨酸合成过程中丙酮酸的代谢溢流的代谢工程改造的报道。
发明内容
本发明的目的是通过失活乙酸激酶(ackA)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构建方法和应用。
目前关于利用谷氨酸棒杆菌合成苏氨酸的代谢工程改造主要集中在苏氨酸的合成路径，主要为草酰乙酸到苏氨酸的合成路径；而丙酮酸作为微生物代谢网络中重要的中间代谢产物，主要进入三羧酸循环为菌体生长提供能量和前体物质，然而，当上下游代谢通路不平衡时会造成丙酮酸的代谢溢流，从而造成丙酮酸的浪费。虽然苏氨酸的前体是草酰乙酸，但草酰乙酸可以通过丙酮酸羧化酶催化丙酮酸生成，同时也可由丙酮酸进入三羧酸循环经过一系列的酶催化反应生成。本发明在苏氨酸的代谢工程研究过程中发现，通过减少丙酮酸的代谢溢流， 能够提高丙酮酸向苏氨酸合成前体草酰乙酸的流量，进而促进苏氨酸的合成，而与其它减少丙酮酸代谢溢流的方法相比，降低或丧失乙酸激酶的活性的效果明显更优，通过降低或丧失乙酸激酶的活性能够有效减少丙酮酸代谢溢流，提高苏氨酸合成前体的供应，进而显著提高菌株的苏氨酸合成能力。
为实现本发明的目的，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，其乙酸激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，乙酸激酶在NCBI上的参考序列编号为WP_003862874.1，或与其相似性为90％且具有同等功能的氨基酸序列。
进一步地，所述微生物体内乙酸激酶的活性降低或丧失是通过降低编码乙酸激酶基因的表达或敲除内源的编码乙酸激酶的基因来实现的。
可以采用诱变、定点突变或同源重组的方法来降低编码乙酸激酶基因的表达或敲除内源的编码乙酸激酶的基因。
进一步地，所述微生物与未修饰的微生物相比，丙酮酸羧化酶的活性增强和/或解除反馈抑制。
优选地，丙酮酸羧化酶在NCBI上的参考序列编号为WP_011013816.1，或与其相似性为90％且具有同等功能的氨基酸序列。
进一步地，所述微生物与未修饰的微生物相比，以下(1)～(4)中的任意一个或多个酶的活性增强和/或解除反馈抑制：
(1)苹果酸醌氧化还原酶；
(2)葡萄糖-6-磷酸脱氢酶；
(3)6-磷酸葡糖酸脱氢酶；
(4)NADP依赖的甘油醛-3-磷酸脱氢酶。
优选地，苹果酸醌氧化还原酶、葡萄糖-6-磷酸脱氢酶、6-磷酸葡糖酸脱氢酶、NADP依赖的甘油醛-3-磷酸脱氢酶在NCBI上的参考序列编号为WP_011014814.1、NP_600790.1、NP_600669.1、FOB93_04945，或与其相似性为90％且具有同等功能的氨基酸序列。
优选地，所述微生物与未修饰的微生物相比，其体内与苏氨酸合成途径相关的酶的活性增强和/或解除反馈抑制；其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶中的至少一种。
优选地，天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶在NCBI上的参考序列编号为WP_003855724.1、WP_003854900.1、WP_011014964.1，或与其相似性为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乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶和/或苏氨酸合酶活性增强和/或解除反馈抑制的微生物；
②乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶和/或丙酮酸羧化酶活性增强和/或解除反馈抑制的微生物；
③乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或苹果酸醌氧化还原酶活性增强和/或解除反馈抑制的微生物；
④乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或葡萄糖-6-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物；
⑤乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或6-磷酸葡糖酸脱氢酶活性增强和/或解除反馈抑制的微生物；
⑥乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物。
以上所述的酶的活性增强是由选自以下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。
优选地，所述的酶的活性增强通过将酶的编码基因的原始启动子替换为活性更强的强启动子，和/或，将基因的起始密码子突变为ATG实现。
其中，所述强启动子包括Psod、Ptuf或PcspB。
启动子Psod、Ptuf或PcspB的核苷酸序列分别如SEQ ID NO.1、2和3所示。
优选地，丙酮酸羧化酶编码基因、苹果酸醌氧化还原酶编码基因、葡萄糖-6-磷酸脱氢酶编码基因、6-磷酸葡糖酸脱氢酶编码基因、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Psod启动子实现；
变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶编码基因的强化表达通过将由Ptuf启动子启动转录的变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶编码基因整合至菌株 的染色体上实现；
高丝氨酸脱氢酶编码基因的强化表达通过将其原始启动子替换为PcspB启动子实现。
以上所述的解除反馈抑制优选通过以下突变实现：丙酮酸羧化酶的解除反馈抑制通过将丙酮酸羧化酶编码基因突变，使得其编码的丙酮酸羧化酶发生P458S突变实现；
葡萄糖-6-磷酸脱氢酶的解除反馈抑制通过将葡萄糖-6-磷酸脱氢酶基因突变，使得其编码的葡萄糖-6-磷酸脱氢酶发生A243T突变实现；
天冬氨酸激酶的解除反馈抑制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。
优选地，本发明所述微生物为谷氨酸棒状杆菌(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状杆菌中编码乙酸激酶的基因，获得基因弱化菌株；所述弱化包括敲除或降低乙酸激酶编码基因的表达；和/或
B、增强丙酮酸羧化酶的活性和/或将其解除反馈抑制；
C、增强以下(1)～(4)中的任意一个或多个酶的活性和/或将其解除反馈抑制：
(1)苹果酸醌氧化还原酶；
(2)葡萄糖-6-磷酸脱氢酶；
(3)6-磷酸葡糖酸脱氢酶；
(4)NADP依赖的甘油醛-3-磷酸脱氢酶；
和/或
D、增强与苏氨酸合成途径相关的酶的活性和/或将其解除反馈抑制，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶中的至少一种；
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)通过对编码酶的核苷酸序列进行改变而增强。
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)培养所述微生物，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，本发明提供乙酸激酶的酶活性降低或丧失在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
优选地，所述微生物体内乙酸激酶的活性降低或丧失是通过降低编码乙酸激酶基因的表达或敲除内源的编码乙酸激酶的基因来实现的。
进一步地，通过失活具有氨基酸生产能力的棒状杆菌(Corynebacterium)中的乙酸激酶来提高苏氨酸的发酵产量。
优选地，本发明所述棒状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.
本发明的有益效果在于：本发明通过失活乙酸激酶，降低丙酮酸的代谢溢流，减少溢流代谢物的产生，减少丙酮酸的浪费，使更多的丙酮酸流向苏氨酸合成前体草酰乙酸，从而显著提高了菌株生产苏氨酸的能力，菌株的苏氨酸产量较未经改造的菌株显著提高。结合苹果酸醌氧化还原酶、葡萄糖-6-磷酸脱氢酶、6-磷酸葡糖酸脱氢酶等的表达强化，苏氨酸的产量进一步提升。上述改造可用于苏氨酸的发酵生产中，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。
本发明所涉及的蛋白及其编码基因的信息如下：
乙酸激酶，编码基因名称ackA，NCBI编号：cg3047、Cgl2752、NCgl2656。
天冬氨酸激酶，编码基因名称lysC，NCBI编号：cg0306、Cgl0251、NCgl0247。
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。
丙酮酸羧化酶，编码基因名称pyc，NCBI编号：cg0791、Cgl0689、NCgl0659。
苹果酸醌氧化还原酶，编码基因名称mqo，NCBI编号：cg2192、Cgl2001、NCgl1926。
葡萄糖-6-磷酸脱氢酶，编码基因名称zwf，NCBI编号：cg1778、Cgl1576、NCgl1514。
6-磷酸葡糖酸脱氢酶，编码基因名称gnd，NCBI编号：cg1643、Cgl1452、NCgl1396。
变异链球菌来源NADP依赖的甘油醛-3-磷酸脱氢酶，编码基因名称gapN，NCBI编号：FOB93_04945。
实施例1 菌株基因组改造质粒构建
1、天冬氨酸激酶表达强化质粒pK18mobsacB-Psod-lysC a1g-T311I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lysC a1g-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、lysC a1g-T311I、dn为模板进行融合PCR获得全长片段up-Psod-lysC a1g-T311I-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Psod-lysC a1g-T311I-dn和pK18mobsacB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-lysC a1g-T311I。
2、高丝氨酸脱氢酶表达强化质粒pK18mobsacB-PcspB-hom G378E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。
3、苏氨酸合酶表达强化质粒pK18mobsacB-Psod-thrC a1g的构建
质粒构建方法参考上述1，所用引物为P37、P38、P39、P40、P41、P42。
4、丙酮酸羧化酶表达强化质粒pK18mobsacB-Psod-pyc P458S的构建
质粒构建方法参考上述1，所用引物为P13、P14、P15、P16、P17、P18、P19、P20。
5、乙酸激酶失活质粒pK18mobsacB-ΔackA的构建
以ATCC13032基因组为模板，以P165/P166引物对进行PCR扩增得到上游同源臂up，以P167/P168引物对进行PCR扩增得到下游同源臂dn。以P165/P168引物对以up、dn为模板进行融合PCR获得全长片段up-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-dn和pK18mobsacB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-ΔackA。
6、苹果酸醌氧化还原酶表达强化质粒pK18mobsacB-Psod-mqo的构建
质粒构建方法参考上述1，所用引物为P169、P170、P171、P172、P173、P174。
7、葡萄糖-6-磷酸脱氢酶表达强化质粒pK18mobsacB-Psod-zwf A243T的构建
质粒构建方法参考上述1，所用引物为P129、P130、P131、P132、P133、P134、P135、P136。
8、6-磷酸葡糖酸脱氢酶表达强化质粒pK18mobsacB-Psod-gnd的构建
质粒构建方法参考上述1，所用引物为P123、P124、P125、P126、P127、P128。
9、变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶表达强化质粒pK18mobsacB-Ptuf-gapN的构建
以ATCC13032基因组为模板，以P137/P138引物对进行PCR扩增得到上游同源臂up，以P139/P140引物对进行PCR扩增得到启动子片段Ptuf，以合成的变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶的编码基因为模板以P141/P142引物对进行PCR扩增得到gapN，以ATCC13032基因组为模板以P143/P144引物对进行PCR扩增得到下游同源臂dn。以P137/P140引物对以up、Ptuf为模版进行融合PCR，获得片段up-Ptuf。以P137/P144引物对以up-Ptuf、gapN、dn为模板进行融合PCR获得全长片段up-Ptuf-gapN-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-Ptuf-gapN-dn和pK18mobsacB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-Ptuf-gapN。
以上质粒构建过程中所用的引物如表1所示。
表1 引物序列
名称 序列(5’-3’)(依次为SEQ ID No:4-65)
P21 AATTCGAGCTCGGTACCCGGGGATCCAGCGACAGGACAAGCACTGG
P22 CCCGGAATAATTGGCAGCTATGTGCACCTTTCGATCTACG
P23 CGTAGATCGAAAGGTGCACATAGCTGCCAATTATTCCGGG
P24 TTTCTGTACGACCAGGGCCATGGGTAAAAAATCCTTTCGTA
P25 TACGAAAGGATTTTTTACCCATGGCCCTGGTCGTACAGAAA
P26 TCGGAACGAGGGCAGGTGAAGGTGATGTCGGTGGTGCCGTCT
P27 AGACGGCACCACCGACATCACCTTCACCTGCCCTCGTTCCGA
P28 GTAAAACGACGGCCAGTGCCAAGCTTAGCCTGGTAAGAGGAAACGT
P29 AATTCGAGCTCGGTACCCGGGGATCCCTGCGGGCAGATCCTTTTGA
P30 ATTTCTTTATAAACGCAGGTCATATCTACCAAAACTACGC
P31 GCGTAGTTTTGGTAGATATGACCTGCGTTTATAAAGAAAT
P32 GTATATCTCCTTCTGCAGGAATAGGTATCGAAAGACGAAA
P33 TTTCGTCTTTCGATACCTATTCCTGCAGAAGGAGATATAC
P34 TAGCCAATTCAGCCAAAACCCCCACGCGATCTTCCACATCC
P35 GGATGTGGAAGATCGCGTGGGGGTTTTGGCTGAATTGGCTA
P36 GTAAAACGACGGCCAGTGCCAAGCTTGCTGGCTCTTGCCGTCGATA
P37 ATTCGAGCTCGGTACCCGGGGATCCGCCGTTGATCATTGTTCTTCA
P38 CCCGGAATAATTGGCAGCTAGGATATAACCCTATCCCAAG
P39 CTTGGGATAGGGTTATATCCTAGCTGCCAATTATTCCGGG
P40 ACGCGTCGAAATGTAGTCCATGGGTAAAAAATCCTTTCGTA
P41 TACGAAAGGATTTTTTACCCATGGACTACATTTCGACGCGT
P42 GTAAAACGACGGCCAGTGCCAAGCTTGAATACGCGGATTCCCTCGC
P13 AATTCGAGCTCGGTACCCGGGGATCCTGACAGTTGCTGATCTGGCT
P14 CCCGGAATAATTGGCAGCTATAGAGTAATTATTCCTTTCA
P15 TGAAAGGAATAATTACTCTATAGCTGCCAATTATTCCGGG
P16 GAAGATGTGTGAGTCGACACGGGTAAAAAATCCTTTCGTA
P17 TACGAAAGGATTTTTTACCCGTGTCGACTCACACATCTTC
P18 GGTGGAGCCTGAAGGAGGTGCGAGTGATCGGCAATGAATCCGG
P19 CCGGATTCATTGCCGATCACTCGCACCTCCTTCAGGCTCCACC
P20 GTAAAACGACGGCCAGTGCCAAGCTTCGCGGCAGACGGAGTCTGGG
P165 CGAGCTCGGTACCCGGGGATCCACCCGGGTGTGGCGCGCAAGAAGATGCCAG
P166 TAAATGTTGTACGCGGACCAGAACAAGATTCCGCCGTGGACCACGC
P167 GGCGGAATCTTGTTCTGGTCCGCGTACAACATTTACATACACC
P168 GTAAAACGACGGCCAGTGCCAAGCTTAGCAAGGTGTTAGAGCAAATTTTCG
P169 AATTCGAGCTCGGTACCCGGGGATCCTCCGTATGCTCCCAAACCTC
P170 CCCGGAATAATTGGCAGCTAGTTCAACTTCCTTTTATCTC
P171 GAGATAAAAGGAAGTTGAACTAGCTGCCAATTATTCCGGG
P172 TTCTTCGGGGAATCTGACATGGGTAAAAAATCCTTTCGTA
P173 TACGAAAGGATTTTTTACCCATGTCAGATTCCCCGAAGAA
P174 GTAAAACGACGGCCAGTGCCAAGCTTCCTTCAAGAACTTAGGGGTC
P129 CATGATTACGAATTCGAGCTCGGTACCCGGGGATCCGATGAGGCTTTGGCTCTGCG
P130 AGCCCGGAATAATTGGCAGCTAGATGGTAGTGTCACGATCCT
P131 AGGATCGTGACACTACCATCTAGCTGCCAATTATTCCGGGCT
P132 GGGTCGTGTTTGTGCTCATGGGTAAAAAATCCTTTCGTA
P133 TACGAAAGGATTTTTTACCCATGAGCACAAACACGACCCCCT
P134 CACCCAAGCCAATATCTTCAGTCATGGTGATCTGGACGTGGTCA
P135 TGACCACGTCCAGATCACCATGACTGAAGATATTGGCTTGGGTG
P136 TCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTTCGAATCACGATGGCGTTT
P123 ACGAATTCGAGCTCGGTACCCGGGGATCCCGATGTGGGTGACACATGGGGTGCCGTCA
P124 GGAAACCTACGAAAGGATTTTTTACCCATGACTAATGGAGATAATCTCGCACAG
P125 CTGTGCGAGATTATCTCCATTAGTCATGGGTAAAAAATCCTTTCGTAGGTTTCC
P126 GTAAAATCGCCACTACCCCCAAATGGTTAGCTGCCAATTATTCCGGGCTTGTGA
P127 TCACAAGCCCGGAATAATTGGCAGCTAACCATTTGGGGGTAGTGGCGATTTTAC
P128 GTTGTAAAACGACGGCCAGTGCCAAGCTTCATGGTGCGCAGTGTGGTTCGTGCGACG
P137 ACGAATTCGAGCTCGGTACCCGGGGATCCTGTTTACCTGACACTCAAGCCCCGTGCAC
P138 GCCGATTTCAAGATATCTAACAAGCCGCTTAGTCTGAGATAATCTGGGTCAGTGGT
P139 ACCACTGACCCAGATTATCTCAGACTAAGCGGCTTGTTAGATATCTTGAAATCGGC
P140 TTGACATAATTTTTATATTGTTTTGTCATTTACTGAATCCTAAGGGCAACGGCGTTGA
P141 TCAACGCCGTTGCCCTTAGGATTCAGTAAATGACAAAACAATATAAAAATTATGTCAA
P142 AGATGAAGTAGGTGGGTGAATATAGCTGTTATTTGATATCAAATACGACGGATTTA
P143 TAAATCCGTCGTATTTGATATCAAATAACAGCTATATTCACCCACCTACTTCATCT
P144 TTGTAAAACGACGGCCAGTGCCAAGCTTGATTGGAATCGGCATGGGTGTTCTGCGT
实施例2 基因组改造菌株的构建
1、天冬氨酸激酶强化表达菌株的构建
按照谷氨酸棒杆菌经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC13032感受态细胞。重组质粒pK18mobsacB-Psod-lysC a1g-T311I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SMCT106，该菌株中，该菌株与ATCC13032菌株相比，lysC基因起始密码子由GTG突变为ATG，其编码的氨基酸第311为由苏氨酸变为异亮氨酸，同时lysC基因的启动子被替换为Psod启动子。
2、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SMCT106为出发菌，将质粒pK18mobsacB-PcspB-hom G378E导入SMCT106中，进行高丝氨酸脱氢酶表达强化的改造，获得的改造菌株命名为SMCT107，与菌株SMCT106相比，该菌株的hom基因发生突变导致其编码蛋白产生G378E的突变，同时hom基因的启动子被替换为ATCC14067来源的PcspB启动子。
3、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SMCT107为出发菌，将质粒pK18mobsacB-Psod-thrC a1g导入SMCT107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SMCT108，与菌株SMCT107相比，该菌株的thrC基因发生突变导致其起始密码子由GTG突变为ATG，同时thrC基因的启动子被替换为Psod启动子。
4、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SMCT108为出发菌，将质粒pK18mobsacB-Psod-pyc P458S导入SMCT108中，进行丙酮酸羧化酶表达强化的改造，获得的改造菌株命名为SMCT109，与菌株SMCT108相比，该菌株的pyc基因发生突变导致其编码蛋白产生P458S的突变，同时pyc基因的启动子被替换为Psod启动子。
5、苹果酸醌氧化还原酶表达强化菌株的构建
菌株构建方法参考上述1，以SMCT109为出发菌，将质粒pK18mobsacB-Psod-mqo导入SMCT109中，进行苹果酸脱氢酶表达强化的改造，获得的改造菌株命名为SMCT111，与菌株SMCT109相比，该菌株的mqo基因的启动子被替换为Psod启动子。
6、葡萄糖-6-磷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SMCT109为出发菌，将质粒pK18mobsacB-Psod-zwf A243T导入SMCT109中，进行葡萄糖-6-磷酸脱氢酶表达强化的改造，获得的改造菌株命名为SMCT112，与菌株SMCT109相比，该菌株的zwf基因发生突变导致其编码蛋白产生A243T的突变，同时zwf基因的启动子被替换为Psod启动子。
7、6-磷酸葡糖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SMCT109为出发菌，将质粒pK18mobsacB-Psod-gnd导入SMCT109中，进行6-磷酸葡糖酸脱氢酶表达强化的改造，获得的改造菌株命名为SMCT113， 与菌株SMCT109相比，该菌株的gnd基因的启动子被替换为Psod启动子。
8、变异链球菌来源的NADP依赖的甘油醛-3-磷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SMCT109为出发菌，进行NADP依赖的甘油醛-3-磷酸脱氢酶表达强化的改造，将质粒pK18mobsacB-Ptuf-gapN导入SMCT109中，获得的改造菌株命名为SMCT114，与菌株SMCT109相比，该菌株在染色体的Cgl1705后插入由Ptuf启动转录的gapN基因。
9、乙酸激酶失活菌株的构建
菌株构建方法参考上述1，分别以SMCT108、SMCT109、SMCT111、SMCT112、SMCT113、SMCT114为出发菌，将质粒pK18mobsacB-ΔackA分别导入上述出发菌中，进行乙酸激酶失活的改造，获得的改造菌株命名为SMCT119、SMCT110、SMCT115、SMCT116、SMCT117、SMCT118，这些菌株与其对应的出发菌相比，ackA基因被敲除。
以上获得的菌株的基因型信息如表2所示。
表2 菌株基因型信息
菌株名称 基因型
SMCT106 ATCC13032,P sod-lysC a1g-T311I
SMCT107 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E
SMCT108 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g
SMCT109 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S
SMCT110 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,ΔackA
SMCT111 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P sod-mqo
SMCT112 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P sod-zwf A243T
SMCT113 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P sod-gnd
SMCT114 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,Ps od-pyc P458S,P tuf-gapN
SMCT115 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P sod-mqo,ΔackA
SMCT116 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P sod-zwf A243T,ΔackA
SMCT117 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P sod-gnd,ΔackA
SMCT118 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,P sod-pyc P458S,P tuf-gapN,ΔackA
SMCT119 ATCC13032,P sod-lysC a1g-T311I,P cspB-hom G378E,P sod-thrC a1g,ΔackA
实施例3 菌株的摇瓶发酵验证
对实施例2构建的各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，pH 7.2。
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：挑取菌株SMCT108、SMCT109、SMCT110、SMCT111、SMCT112、SMCT113、SMCT114、SMCT115、SMCT116、SMCT117、SMCT118、SMCT119的斜面种子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，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，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，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
菌株编号 OD562 L-苏氨酸(g/L) 菌株编号 OD562 L-苏氨酸(g/L)
SMCT108 23 3.0 SMCT119 23 3.3
SMCT109 23 3.6 SMCT110 23 4.2
SMCT111 23 4.2 SMCT115 23 4.8
SMCT112 22 4.3 SMCT116 22 5.4
SMCT113 22 4.3 SMCT117 22 5.4
SMCT114 22 4.5 SMCT118 23 5.9
由表3结果可以看出，乙酸激酶失活后，不同的苏氨酸生产菌的产量提升在10％～30％之间。同时以SMCT108为出发菌，进一步强化表达丙丙酮酸羧化酶、苹果酸醌氧化还原酶、葡萄糖-6-磷酸脱氢酶、6-磷酸葡糖酸脱氢酶、变异链球菌NADP依赖的甘油醛-3-磷酸脱氢酶的改造菌的苏氨酸产量均有进一步提升，说明上述位点的改造有利于苏氨酸的生产，同时在这些改造的基础上失活乙酸激酶，苏氨酸的产量进一步提升，且提升幅度较SMCT108失活乙酸激酶的提升幅度明显更高，说明上述基因的改造与乙酸激酶的失活的改造组合更有利于苏氨酸的生产。
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。
序列说明
SEQ ID No:1人工序列(Artificial Sequence)Psod
Figure PCTCN2022143101-appb-000001
Figure PCTCN2022143101-appb-000002
SEQ ID No:2人工序列(Artificial Sequence)Ptuf
Figure PCTCN2022143101-appb-000003
SEQ ID No:3人工序列(Artificial Sequence)PcspB
Figure PCTCN2022143101-appb-000004

Claims (11)

  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，其乙酸激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，所述微生物体内乙酸激酶的活性降低或丧失是通过降低编码乙酸激酶基因的表达或敲除内源的编码乙酸激酶的基因来实现的。
  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乙酸激酶基因的表达或敲除内源的编码乙酸激酶的基因。
  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，丙酮酸羧化酶的活性增强和/或解除反馈抑制；
    优选地，所述微生物与未修饰的微生物相比，以下(1)～(4)中的任意一个或多个酶的活性增强和/或解除反馈抑制：
    (1)苹果酸醌氧化还原酶；
    (2)葡萄糖-6-磷酸脱氢酶；
    (3)6-磷酸葡糖酸脱氢酶；
    (4)NADP依赖的甘油醛-3-磷酸脱氢酶。
  5. 根据权利要求1～4任一项所述的微生物，其特征在于，所述微生物与未修饰的微生物相比，其体内与苏氨酸合成途径相关的酶的活性增强和/或解除反馈抑制；
    其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶中的至少一种。
  6. 根据权利要求5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乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶和/或苏氨酸合酶活性增强和/或解除反馈抑制的微生物；
    ②乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶和/或丙酮酸羧化酶活性增强和/或解除反馈抑制的微生物；
    ③乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或苹果酸醌氧化还原酶活性增强和/或解除反馈抑制的微生物；
    ④乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或葡萄糖-6-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物；
    ⑤乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或6-磷酸葡糖酸脱氢酶活性增强和/或解除反馈抑制的微生物；
    ⑥乙酸激酶活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物。
  7. 根据权利要求4～6任一项所述的微生物，其特征在于，所述酶的活性增强是由选自以下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1-7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)。
  9. 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状杆菌中编码乙酸激酶的基因，获得基因弱化菌株；所述弱化包括敲除或降低乙酸激酶编码基因的表达；
    和可选的：
    B、增强丙酮酸羧化酶的活性和/或将其解除反馈抑制；
    C、增强以下(1)～(4)中的任意一个或多个酶的活性和/或将其解除反馈抑制：
    (1)苹果酸醌氧化还原酶；
    (2)葡萄糖-6-磷酸脱氢酶；
    (3)6-磷酸葡糖酸脱氢酶；
    (4)NADP依赖的甘油醛-3-磷酸脱氢酶；
    和/或
    D、增强与苏氨酸合成途径相关的酶的活性和/或将其解除反馈抑制，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶中的至少一种；
    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)通过对编码酶的核苷酸序列进行改变而增强。
  10. 根据权利要求9所述的方法，其特征在于，所述棒杆菌为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  11. 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1-8任一项所述的微生物或用权利要求9或10所述的方法构建的产苏氨酸菌株，以获得所述微生物或产苏氨酸菌株的培养物；
    b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
PCT/CN2022/143101 2022-02-08 2022-12-29 一种生产苏氨酸的重组微生物及其构建方法和应用 WO2023151411A1 (zh)

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