WO2023151406A1 - 苏氨酸生产菌株的构建方法 - Google Patents

苏氨酸生产菌株的构建方法 Download PDF

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WO2023151406A1
WO2023151406A1 PCT/CN2022/142840 CN2022142840W WO2023151406A1 WO 2023151406 A1 WO2023151406 A1 WO 2023151406A1 CN 2022142840 W CN2022142840 W CN 2022142840W WO 2023151406 A1 WO2023151406 A1 WO 2023151406A1
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enzyme
threonine
gene
enhanced
changing
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康培
吴涛
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 belongs to the technical field of microbial engineering, and in particular relates to a method for constructing a threonine-producing bacterial strain.
  • L-threonine (L-Threonin), chemical name is ⁇ -hydroxy- ⁇ -aminobutyric acid, molecular formula C 4 H 9 NO 3 , relative molecular mass 119.12.
  • L-threonine is an essential amino acid, mainly used in medicine, chemical reagents, food fortifiers, feed additives and other fields.
  • threonine from oxaloacetate requires a five-step catalytic reaction, which are aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), and homoserine dehydrogenase. Hydrogenase (hom coded), homoserine kinase (thrB) and threonine synthase (thrC) coded.
  • Hermann Sahm and others have been committed to the development of high-threonine-producing Corynebacterium glutamicum, and have made some breakthroughs, obtaining the hom gene that is resistant to feedback inhibition (Reinscheid D J, Eikmanns B J, Sahm H.
  • Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230), lysC gene (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).
  • the purpose of the present invention is to provide a method for constructing a threonine producing strain.
  • the present invention superposes and modifies multiple enzymes related to threonine synthesis, including aspartate aminotransferase, aspartate kinase, threonine synthase, citrate synthase, isocitrate Dehydrogenase, HTH transcription regulator RamB and malate quinone oxidoreductase, etc., systematically enhance the threonine synthesis pathway, thereby improving the strain's ability to produce threonine.
  • enzymes related to threonine synthesis including aspartate aminotransferase, aspartate kinase, threonine synthase, citrate synthase, isocitrate Dehydrogenase, HTH transcription regulator RamB and malate quinone oxidoreductase, etc.
  • the present invention provides a modified microorganism of the genus Corynebacterium, which has enhanced enzyme activity related to the threonine synthesis pathway and enzyme activity related to the tricarboxylic acid cycle compared with unmodified microorganisms. Alterations are made to increase the supply of precursors in the threonine synthesis pathway, and the microorganism has enhanced threonine production capacity compared to an unmodified microorganism.
  • the enzyme related to the threonine synthesis pathway is selected from at least one of aspartate aminotransferase, aspartate kinase, and threonine synthase;
  • the enzymes related to the tricarboxylic acid cycle are selected from at least one of citrate synthase, isocitrate dehydrogenase, HTH transcription regulator RamB, and malate quinone oxidoreductase.
  • the enzyme activity changes include: enzyme activity enhancement, reduction or loss
  • aspartate aminotransferase Aspartate kinase, threonine synthase, malate quinone oxidoreductase, citrate synthase, isocitrate dehydrogenase, HTH transcription regulator RamB reference on NCBI
  • the serial number is wp_011013497.1, WP_003855724.1, WP_011014964.1, WP_011014814.1, WP_011013914.1, WP_011013800.1, WP_003859703.1, or 90 % of its similarity.
  • Amino acid sequence Amino acid sequence.
  • the enhancement of enzyme activity is achieved by being selected from the following 1) to 6), or an optional combination:
  • the reduction or loss of enzyme activity is achieved by being selected from the following 1)-5), or an optional combination:
  • Mutagenesis, site-directed mutagenesis or homologous recombination can be used to change the activities of enzymes in the threonine synthesis pathway and the tricarboxylic acid cycle.
  • microorganisms Compared with unmodified microorganisms, the microorganisms have enhanced activities of aspartate aminotransferase, aspartate kinase, threonine synthase and malate quinone oxidoreductase, and citrate synthase, isocitrate Hydrogenase and HTH transcriptional regulator RamB activities were reduced or lost.
  • the enhancement of aspartate aminotransferase activity is achieved by inserting the sod promoter upstream of the initiation codon of the aspB gene.
  • the enhancement of aspartokinase activity is achieved by inserting the sod promoter upstream of the start codon of the lysC gene, and at the same time mutating the start codon GTG to ATG, and replacing the 311th amino acid encoded by the lysC gene with threonine acid mutation to isoleucine.
  • the enhancement of threonine synthase activity is achieved by inserting the sod promoter upstream of the start codon of thrC gene, and mutating the start codon GTG to ATG.
  • malate quinone oxidoreductase activity was achieved by inserting the sod promoter upstream of the initiation codon of the mqo gene.
  • citrate synthase activity is achieved by mutating the start codon of the gltA gene from ATG to GTG.
  • the reduction or loss of isocitrate dehydrogenase activity is achieved by mutating the start codon of the icd gene from ATG to TTG.
  • the reduction or loss of the activity of the HTH transcription regulator RamB is achieved by mutating the start codon of the ramB gene from ATG to GTG, or knocking out the coding region of the ramB gene.
  • the present invention starts from an unmodified corynebacterium, such as Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc.
  • Corynebacterium glutamicum which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc.
  • Phylogenetic tree https://www.ncbi.nlm.nih.gov/genome/469
  • Corynebacterium glutamicum ATCC 13032.
  • the present invention provides a method for constructing a threonine-producing strain, the method comprising: using genetic engineering means to enhance the genes related to the threonine synthesis pathway in corynebacteria with amino acid production ability, and transforming the genes related to the three Genes related to the carboxylic acid cycle to increase the supply of precursor substances in the threonine synthesis pathway; preferably, the modification of the genes related to the tricarboxylic acid cycle results in: enhancement, reduction or loss of enzyme activity;
  • the gene related to the threonine synthesis pathway is selected from at least one of aspB, lysC, thrC;
  • the reference sequence numbers of the genes aspB, lysC, thrC, gltA, icd, ramB, and mqo on NCBI are cg0294, cg0306, cg2437, cg0949, cg0766, cg0444, and cg2192, respectively.
  • the enhanced pathway is selected from the following 1) to 6), or an optional combination:
  • the reduction or loss of enzyme activity is achieved by being selected from the following 1)-5), or an optional combination:
  • the present invention starts from an unmodified corynebacterium, such as Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc.
  • Corynebacterium glutamicum which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc.
  • Phylogenetic tree https://www.ncbi.nlm.nih.gov/genome/469
  • Corynebacterium glutamicum ATCC 13032.
  • 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 starts from an unmodified corynebacterium, such as Corynebacterium glutamicum, which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc.
  • Corynebacterium glutamicum which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc.
  • Phylogenetic tree https://www.ncbi.nlm.nih.gov/genome/469
  • Corynebacterium glutamicum ATCC 13032.
  • the present invention provides the use of the modified microorganism of the genus Corynebacterium or the threonine-producing strain constructed according to the above-mentioned method in the fermentative production of threonine or in increasing the fermentative yield of threonine.
  • transformation methods of the above-mentioned related strains are transformation methods known to those skilled in the art.
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention superposes and modifies multiple enzymes related to threonine synthesis in corynebacteria (such as Corynebacterium glutamicum), including aspartate aminotransferase, aspartate kinase, threonine synthase, citrate synthase , isocitrate dehydrogenase, HTH transcription regulator and malate quinone oxidoreductase, etc., have improved the ability of the strain to produce threonine, and its threonine output has increased to 4.9g/L. It provides effective means for large-scale production of threonine and has broad application prospects.
  • corynebacteria such as Corynebacterium glutamicum
  • the invention provides an engineering bacterium for producing threonine, the strain transformation process and effect:
  • the aspartate transaminase was expressed in the starting strain Corynebacterium glutamicum ATCC 13032, and the strain SMCT286 was obtained.
  • the threonine production capacity of the strain was 0.8 g/L.
  • threonine synthase was enhanced to obtain the strain SMCT291, and the threonine production capacity of the strain was increased from 1.8g/L to 2.3g/L.
  • the citrate synthase was weakened to obtain the strain SMCT294, and the threonine production ability of the strain was increased from 2.3g/L to 3.0g/L.
  • the HTH transcription regulator RamB was weakened to obtain the strain SMCT297, and the threonine production ability of the strain was increased from 3.0g/L to 3.5g/L; the HTH transcription regulator RamB was inactivated to obtain the strain SMCT298, The ability of the strain to produce threonine increased from 3.0g/L to 3.6g/L.
  • the isocitrate dehydrogenase was weakened to obtain the strain SMCT299, and the threonine production ability of the strain was increased from 3.6g/L to 4.2g/L.
  • the enhanced malate quinone oxidoreductase was expressed to obtain the strain SMCT300, and the threonine production capacity of the strain increased from 4.2g/L to 4.9g/L.
  • the expression enhancement during the transformation process includes promoter replacement, ribosome binding site change, copy number increase, plasmid overexpression and other means, all of which are known to those skilled in the art.
  • the above means cannot be exhausted by examples, and the specific examples only use promoter enhancement as a representative for illustration.
  • Expression attenuation or inactivation during the transformation process includes removal of the coding region, replacement and change of the start codon, change of the ribosome binding site, and promoter replacement, all of which are known to those skilled in the art.
  • the aspartate aminotransferase is encoded by the aspB gene, and the present invention inserts a sod promoter upstream of the start codon of the aspB gene, thereby realizing enhanced expression of the aspB gene.
  • Aspartokinase is encoded by the lysC gene.
  • the present invention inserts the sod promoter upstream of the start codon of the lysC gene, mutates the start codon GTG to ATG, and mutates the 311th amino acid from threonine to isoleucine Amino acid, so as to realize the overexpression of lysC gene.
  • the threonine synthase is encoded by the thrC gene.
  • the present invention inserts the sod promoter upstream of the start codon of the thrC gene, and mutates the start codon GTG to ATG, thereby realizing the overexpression of the thrC gene.
  • the citrate synthase is encoded by the gltA gene.
  • the invention mutates the start codon of the gltA gene from ATG to GTG, thereby realizing the weakening of the gltA gene.
  • the HTH transcription regulator is encoded by the ramB gene.
  • the start codon of the ramB gene is mutated from ATG to GTG, thereby realizing the weakening of the ramB gene; or, the ramB gene coding region is knocked out and mutated to inactivate the ramB gene .
  • the isocitrate dehydrogenase is encoded by the icd gene.
  • the invention mutates the start codon of the icd gene from ATG to TTG, thereby realizing the weakening of the icd gene.
  • the malate quinone oxidoreductase is encoded by the mqo gene, and the present invention inserts a sod promoter upstream of the start codon of the mqo gene, so as to enhance the expression of the mqo gene.
  • Corynebacterium preferred Corynebacterium glutamicum, most preferably Corynebacterium glutamicum ATCC 13032.
  • Aspartate aminotransferase encoding gene name aspB, NCBI number: cg0294, cg0294 and cg0294.
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Threonine synthase encoding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139.
  • Citrate synthase encoding gene name gltA, NCBI number: cg0949, Cgl0829, NCgl0795.
  • Isocitrate dehydrogenase encoding gene name icd, NCBI number: cg0766, Cgl0664, NCgl0634.
  • HTH transcription regulator RamB encoding gene name ramB, NCBI number: cg0444, Cgl0369, NCgl0358.
  • genes lysC, thrC, gltA, ramB, and icd are from Corynebacterium glutamicum, and the nucleotide sequences of the wild-type genes lysC, thrC, gltA, ramB, and icd are respectively shown in SEQ ID NO: 1-5.
  • the PCR amplification system is as follows:
  • the PCR amplification procedure is as follows:
  • Transformation method refer to the instructions of Trans1-T1 Phage Resistant Chemically Competent Cell.
  • the upstream homology arm up was obtained by PCR amplification with the P103/P104 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P105/P106 primer pair
  • the P107/P108 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • Fusion PCR was carried out with P103/P106 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR using the P103/P108 primer pair and up-Psod and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -aspB.
  • 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 P25/P26 primer The downstream homology arm dn-1 was obtained by PCR amplification
  • the downstream homology arm dn-2 was obtained by PCR amplification with the P27/P28 primer pair. Fusion PCR was performed with P21/P24 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the fragment dn was obtained.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR using the P21/P28 primer pair and up-Psod and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I .
  • g1a means that the first base of the start codon of the lysC gene (see SEQ ID NO: 1 for the wild-type gene sequence of lysC) is mutated from g to a
  • T311I means that the 311th base of the aspartokinase encoded by the lysC gene is The amino acid is mutated from T to I.
  • the upstream homology arm up was obtained by PCR amplification with the P37/P38 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P39/P40 primer pair
  • the P41/P42 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • Fusion PCR was performed with P37/P40 primer pair and up and Psod as a template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR using the P37/P42 primer pair and up-Psod and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -thrC g1a .
  • g1a means that the first base of the start codon of the thrC gene (see SEQ ID NO: 2 for the wild-type gene sequence of thrC) is mutated from g to a.
  • the upstream homology arm up was obtained by PCR amplification with the P153/P154 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P155/P156 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR with primer pair P153/P156 and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-gltA a1g .
  • a1g means that the first base of the start codon of the gltA gene (see SEQ ID NO: 3 for the gltA wild-type gene sequence) is mutated from a to g.
  • the upstream homology arm up was obtained by PCR amplification with the P115/P116 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P117/P118 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR using the P115/P118 primer pair and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-ramB a1g .
  • a1g means that the first base of the start codon of the ramB gene (see SEQ ID NO: 4 for the ramB wild-type gene sequence) is mutated from a to g.
  • the upstream homology arm up was obtained by PCR amplification with the P119/P120 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P121/P122 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR with primer pair P119/P122 and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and obtained the recombinant plasmid pK18mobsacB- ⁇ ramB.
  • the upstream homology arm up was obtained by PCR amplification with the P149/P150 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P151/P152 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR using the P149/P152 primer pair and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-icd a1t .
  • a1t means that the first base of the initiation codon of the icd gene (see SEQ ID NO: 5 for the sequence of the icd wild-type gene) is mutated from a to t. 8. Construction of expression-enhanced plasmid pK18mobsacB-Psod-mqo for malate quinone oxidoreductase
  • the upstream homology arm up was obtained by PCR amplification with the P169/P170 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P171/P172 primer pair
  • the P173/P174 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • Fusion PCR was carried out with P169/P172 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR using the P169/P174 primer pair and up-Psod and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with the seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -mqo.
  • ATCC 13032 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -aspB was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT286.
  • Competent cells were prepared using the SMCT286 obtained above as the starting bacteria.
  • the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I was used to transform the competent cells by electroporation, and the transformant was screened on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was eliminated due to homology. inserted into the chromosome.
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT289.
  • Competent cells were prepared using the SMCT289 obtained above as the starting strain.
  • the recombinant plasmid pK18mobsacB-P sod -thrC g1a was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the in the chromosome.
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT291.
  • Competent cells were prepared using the SMCT291 obtained above as the starting strain.
  • the recombinant plasmid pK18mobsacB-gltA a1g was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology.
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT294.
  • Competent cells were prepared using the SMCT294 obtained above as the starting strain.
  • the recombinant plasmid pK18mobsacB-ramB a1g was electroporated to transform the competent cells, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology.
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT297.
  • Competent cells were prepared using the SMCT294 obtained above as the starting strain.
  • the recombinant plasmid pK18mobsacB- ⁇ ramB 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 gene of interest was inserted into the chromosome due to homology.
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT298.
  • Competent cells were prepared using the SMCT298 obtained above as the starting strain.
  • the recombinant plasmid pK18mobsacB-icd a1t was electroporated to transform the competent cells, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology.
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT299.
  • Competent cells were prepared using the SMCT299 obtained above as the starting strain.
  • the recombinant plasmid pK18mobsacB-Psod-mqo was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology .
  • 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. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT300.
  • Embodiment 3 constructs bacterial strain shaking flask verification
  • Seed activation medium BHI 3.7%, agar 2%, pH7.
  • 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.
  • Fermentation medium corn steep liquor 50mL/L, glucose 30g/L, ammonium sulfate 4g/L, MOPS 30g/L, potassium dihydrogen phosphate 10g/L, urea 20g/L, biotin 10mg/L, magnesium sulfate 6g/L , ferrous sulfate 1g/L, VB1 ⁇ HCl 40mg/L, calcium pantothenate 50mg/L, nicotinamide 40mg/L, manganese sulfate 1g/L, zinc sulfate 20mg/L, copper sulfate 20mg/L, pH 7.2.
  • Seed culture Pick 1 loop of engineering bacteria slant seeds and transfer them to a 500 mL Erlenmeyer flask containing 20 mL of seed medium, and shake and culture at 30° C. and 220 r/min for 16 hours.
  • Fermentation culture inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture at 33° C. and 220 r/min for 24 hours with shaking.

Abstract

提供了一种苏氨酸生产菌株的构建方法，通过对苏氨酸合成相关的多个酶进行叠加修饰，包括天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶、柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子和苹果酸醌氧化还原酶等，提高了棒杆菌（如谷氨酸棒状杆菌）生产苏氨酸的能力，其苏氨酸的产量提高到4.9g/L，为大规模生产苏氨酸提供有效手段。

Description

苏氨酸生产菌株的构建方法 技术领域
本发明属于微生物工程技术领域，具体地说，涉及一种苏氨酸生产菌株的构建方法。
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C 4H 9NO 3，相对分子质量119.12。L-苏氨酸是一种必需氨基酸，主要用于医药、化学试剂、食品强化剂、饲料添加剂等领域。
谷氨酸棒杆菌中，由草酰乙酸生成苏氨酸需五步催化反应，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)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氨酸棒状杆菌的开发，并取得一定突破，获得了抗反馈抑制的hom基因(Reinscheid D J,Eikmanns 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)。继Hermann Sahm之后，Lothar Eggling通过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 Serine Hydroxymethyltransferase)and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2002,68(7):3321-3327)。
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途径中，关于前体供应等方面的报道较少。且现有报道仅对苏氨酸合成途径做了初步研究，并未形成系统。
发明内容
本发明的目的是提供一种苏氨酸生产菌株的构建方法。
为了实现本发明目的，本发明通过对苏氨酸合成相关的多个酶进行叠加修饰，包括天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶、柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子RamB和苹果酸醌氧化还原酶等，系统地增强苏氨酸合成途径，从而实现提高菌株生产苏氨酸的能力。
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，其与苏氨酸合成途径相关的酶活性增强，且与三羧酸循环相关的酶活性发生改变以提高苏氨酸合成途径中前体物质的供应，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。
其中，与苏氨酸合成途径相关的酶选自天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶中的至少一种；
与三羧酸循环相关的酶选自柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子RamB、苹果酸醌氧化还原酶中的至少一种。
优选地，所述酶活性发生改变包括：酶活性增强、降低或丧失
优选地，天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶、苹果酸醌氧化还原酶、柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子RamB在NCBI上的参考序列编号分别为WP_011013497.1、WP_003855724.1、WP_011014964.1、WP_011014814.1、WP_011013914.1、WP_011013800.1、WP_003859703.1，或与其相似性为90％的氨基酸序列。
优选地，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)通过改变编码上述酶的核苷酸序列而增强。
酶活性降低或丧失是由选自以下1)-5)，或任选的组合实现的：
1)通过改变所述酶的编码基因的启动子序列而降低或丧失；
2)通过改变所述酶的编码基因的核糖体结合位点而降低或丧失；
3)通过改变所述酶的氨基酸序列而降低或丧失；
4)通过改变编码所述酶的核苷酸序列而降低或丧失；
5)通过敲除所述酶的编码序列而丧失。
可以采用诱变、定点突变或同源重组的方法来改变苏氨酸合成途径和三羧酸循环中酶的活性。
所述微生物与未修饰的微生物相比，其体内天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶和苹果酸醌氧化还原酶活性增强，且柠檬酸合酶、异柠檬酸脱氢酶和HTH转录调节因子RamB活性降低或丧失。
进一步地，天冬氨酸氨基转移酶活性的增强是通过在aspB基因起始密码子上游插入sod启动子来实现的。
进一步地，天冬氨酸激酶活性的增强是通过在lysC基因起始密码子上游插入sod启动子，同时将起始密码子GTG突变为ATG，并将lysC基因编码的第311位氨基酸由苏氨酸突变为异亮氨酸来实现的。
进一步地，苏氨酸合酶活性的增强是通过在thrC基因起始密码子上游插入sod启动子，并将起始密码子GTG突变为ATG来实现的。
进一步地，苹果酸醌氧化还原酶活性的增强是通过在mqo基因起始密码子上游插入sod启动子来实现的。
进一步地，柠檬酸合酶活性降低或丧失是通过将gltA基因的起始密码子由ATG突变为GTG来实现的。
进一步地，异柠檬酸脱氢酶活性降低或丧失是通过将icd基因的起始密码子由ATG突变为TTG来实现的。
进一步地，HTH转录调节因子RamB活性降低或丧失是通过将ramB基因的起始密码子由ATG突变为GTG，或者对ramB基因编码区进行敲除来实现的。
优选地，本发明从未修饰的棒杆菌出发，例如为谷氨酸棒状杆菌(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，本发明提供苏氨酸生产菌株的构建方法，所述方法包括：利用基因工程手段，增强具有氨基酸生产能力的棒杆菌中的与苏氨酸合成途径相关的基因，并改造与三羧酸循环相关的基因以提高苏氨酸合成途径中前体物质的供应；优选地，所述改造与三羧酸循环相关的基因导致：酶活性的增强、降低或丧失；
其中，与苏氨酸合成途径相关的基因选自aspB、lysC、thrC中的至少一种；
与三羧酸循环途径相关的及基因选自gltA、icd、ramB、mqo中的至少一种；
优选地，基因aspB、lysC、thrC、gltA、icd、ramB、mqo在NCBI上的参考序列编号分别为cg0294、cg0306、cg2437、cg0949、cg0766、cg0444、cg2192。
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)通过改变编码上述酶的核苷酸序列而增强。
酶活性降低或丧失是由选自以下1)-5)，或任选的组合实现的：
1)通过改变所述酶的编码基因的启动子序列而降低或丧失
2)通过改变所述酶的编码基因的核糖体结合位点而降低或丧失
3)通过改变所述酶的氨基酸序列而降低或丧失
4)通过改变编码所述酶的核苷酸序列而降低或丧失
5)通过敲除所述酶的编码序列而丧失
酶活改变的方法可选自诱变、定点突变、同源重组等中的至少一种。
优选地，本发明从未修饰的棒杆菌出发，例如为谷氨酸棒状杆菌(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)培养所述修饰的棒状杆菌属微生物，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。
优选地，本发明从未修饰的棒杆菌出发，例如为谷氨酸棒状杆菌(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。
第四方面，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苏氨酸生产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。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明通过对棒杆菌(如谷氨酸棒状杆菌)苏氨酸合成相关的多个酶进行叠加修饰，包括天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶、柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子和苹果酸醌氧化还原酶等，提高了菌株生产苏氨酸的能力，其苏氨酸的产量提高到4.9g/L。为大规模生产苏氨酸提供有效手段，应用前景广阔。
具体实施方式
本发明提供一种生产苏氨酸的工程菌，菌株改造过程及效果：
首先，在出发菌株谷氨酸棒状杆菌ATCC 13032中强化表达天冬氨酸转氨酶，获得菌株SMCT286，菌株生产苏氨酸的能力为0.8g/L。
在改造菌SMCT286的基础上，强化表达天冬氨酸激酶，获得菌株SMCT289，菌株生产苏氨酸的能力由0.8g/L提高至1.8g/L。
在改造菌SMCT289的基础上，强化表达苏氨酸合酶，获得菌株SMCT291，菌株生产苏氨酸的能力由1.8g/L提高至2.3g/L。
在改造菌SMCT291的基础上，弱化柠檬酸合酶，获得菌株SMCT294，菌株生产苏氨酸的能力由2.3g/L提高至3.0g/L。
在改造菌SMCT294的基础上，弱化HTH转录调节因子RamB，获得菌株SMCT297，菌株生产苏氨酸的能力由3.0g/L提高至3.5g/L；失活HTH转录调节因子RamB，获得菌株SMCT298，菌株生产苏氨酸的能力由3.0g/L提高至3.6g/L。
在改造菌SMCT298的基础上，弱化异柠檬酸脱氢酶，获得菌株SMCT299，菌株生产苏氨酸的能力由3.6g/L提高至4.2g/L。
在改造菌SMCT299的基础上，表达强化苹果酸醌氧化还原酶，得到菌株SMCT300，菌株生产苏氨酸的能力由4.2g/L提高至4.9g/L。
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技术人员公知手段。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。
改造过程中的表达弱化或失活包括编码区的去除、起始密码子的替换及改变、核糖体结合位点的改变和启动子替换等手段，且以上手段均为本领域技术人员公知手段。
具体地，
天冬氨酸转氨酶，由aspB基因编码，本发明在aspB基因起始密码子上游插入sod启动子，从而实现对aspB基因的表达增强。
天冬氨酸激酶，由lysC基因编码，本发明在lysC基因起始密码子上游插入sod启动子，将起始密码子GTG突变为ATG，并将第311位氨基酸由苏氨酸突变为异亮氨酸，从而实现对lysC基因的过表达。
苏氨酸合酶，由thrC基因编码，本发明在thrC基因起始密码子上游插入sod启动子，并将起始密码子GTG突变为ATG，从而实现对thrC基因的过表达。
柠檬酸合酶，由gltA基因编码，本发明将gltA基因的起始密码子由ATG突变为GTG，从而实现对gltA基因的弱化。
HTH转录调节因子，由ramB基因编码，本发明将ramB基因的起始密码子由ATG突变为GTG，从而实现对 ramB基因的弱化；或者，对ramB基因编码区进行敲除突变，失活ramB基因。
异柠檬酸脱氢酶，由icd基因编码，本发明将icd基因的起始密码子由ATG突变为TTG，从而实现对icd基因的弱化。
苹果酸醌氧化还原酶，由mqo基因编码，本发明在mqo基因起始密码子上游插入sod启动子，从而实现对mqo基因的表达增强。
上述菌株为棒杆菌，优选谷氨酸棒状杆菌，最优选谷氨酸棒状杆菌ATCC 13032。
进一步地，将上述菌株用于苏氨酸发酵生产。
本发明涉及的蛋白及其编码基因如下：
天冬氨酸转氨酶，编码基因名称aspB，NCBI编号：cg0294、cg0294和cg0294。
天冬氨酸激酶，编码基因名称lysC，NCBI编号：cg0306、Cgl0251、NCgl0247。
苏氨酸合酶，编码基因名称thrC，NCBI编号：cg2437、Cgl2220、NCgl2139。
柠檬酸合酶，编码基因名称gltA，NCBI编号：cg0949、Cgl0829、NCgl0795。
异柠檬酸脱氢酶，编码基因名称icd，NCBI编号：cg0766、Cgl0664、NCgl0634。
HTH转录调节因子RamB，编码基因名称ramB，NCBI编号：cg0444、Cgl0369、NCgl0358。
苹果酸醌氧化还原酶，编码基因名称mqo，NCBI编号：cg2192、Cgl2001、NCgl1926。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中基因lysC、thrC、gltA、ramB、icd来自谷氨酸棒状杆菌，野生型基因lysC、thrC、gltA、ramB、icd的核苷酸序列分别如SEQ ID NO:1-5所示。
以下实施例中使用的实验材料如下：
Figure PCTCN2022142840-appb-000001
Figure PCTCN2022142840-appb-000002
以下实施例中涉及的实验方法如下：
PCR扩增体系如下：
成分 体积(微升)
灭菌的去离子水 29
5×pfu buffer 10
2.5mM dNTP 5
10μM上游引物 2
10μM下游引物 2
Pfu 1
模板 1(融合PCR模板最大加到2微升)
共计 50
PCR扩增程序如下：
Figure PCTCN2022142840-appb-000003
菌株改造方法：
1、无缝组装反应程序：参照ClonExpress MultiS One Step Cloning Kit说明书。
2、转化方法：参照Trans1-T1 Phage Resistant Chemically Competent Cell说明书。
3、感受态细胞的制备：参照C.glutamicum Handbook,Charpter 23。
以下实施例中涉及到的引物见表1。
表1
Figure PCTCN2022142840-appb-000004
Figure PCTCN2022142840-appb-000005
Figure PCTCN2022142840-appb-000006
注：加粗字体及下划线为引入相应点突变的引物。
实施例1菌株基因组改造质粒的构建
1、天冬氨酸转氨酶表达强化质粒pK18mobsacB-P sod-aspB的构建
以谷氨酸棒状杆菌ATCC 13032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/P106引物对以up、Psod为模版进行融合PCR，获得片段up-Psod。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。两者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-P sod-aspB。
2、天冬氨酸激酶表达强化质粒pK18mobsacB-P sod-lysC g1a-T311I的构建
以谷氨酸棒状杆菌ATCC 13032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扩增得到下游同源臂dn-1，以P27/P28引物对进行PCR扩增得到下游同源臂dn-2。以P21/P24引物对以up、Psod为模版进行融合PCR，获得片段up-Psod。以P25/P28引物对以dn-1、dn-2为模版进行融合PCR，获得片段dn。以P21/P28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Trans1T1感受态细胞，获得重组质粒pK18mobsacB-P sod-lysC g1a-T311I。
其中，g1a表示lysC基因(lysC野生型基因序列见SEQ ID NO:1)起始密码子的第1位碱基由g突变为a，T311I表示lysC基因编码的天冬氨酸激酶的第311为氨基酸由T突变为I。
3、苏氨酸合酶表达强化质粒pK18mobsacB-P sod-thrC g1a的构建
以谷氨酸棒状杆菌ATCC 13032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，以P41/P42引物对进行PCR扩增得到下游同源臂dn。以P37/P40引物对以up、Psod为模版进行融合PCR，获得片段up-Psod。以P37/P42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Trans1T1感受态细胞，获得重组质粒pK18mobsacB-P sod-thrC g1a。
其中，g1a表示thrC基因(thrC野生型基因序列见SEQ ID NO:2)起始密码子的第1位碱基由g突变为a。
4、柠檬酸合酶表达弱化质粒pK18mobsacB-gltA a1g的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P153/P154引物对进行PCR扩增得到上游同源臂up，以P155/P156引物对进行PCR扩增得到下游同源臂dn。以P153/P156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。两者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-gltA a1g。
其中，a1g表示gltA基因(gltA野生型基因序列见SEQ ID NO:3)起始密码子的第1位碱基由a突变为g。
5、HTH转录调节因子表达弱化质粒pK18mobsacB-ramB a1g的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P115/P116引物对进行PCR扩增得到上游同源臂up，以P117/P118引物对进行PCR扩增得到下游同源臂dn。以P115/P118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。两者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-ramB a1g。
其中，a1g表示ramB基因(ramB野生型基因序列见SEQ ID NO:4)起始密码子的第1位碱基由a突变为g。
6、HTH转录调节因子失活质粒pK18mobsacB-△ramB的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P119/P120引物对进行PCR扩增得到上游同源臂up，以P121/P122引物对进行PCR扩增得到下游同源臂dn。以P119/P122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。两者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-△ramB。
7、异柠檬酸脱氢酶表达弱化质粒pK18mobsacB-icd a1t的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P149/P150引物对进行PCR扩增得到上游同源臂up，以P151/P152引物对进行PCR扩增得到下游同源臂dn。以P149/P152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。两者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-icd a1t。
其中，a1t表示icd基因(icd野生型基因序列见SEQ ID NO:5)起始密码子的第1位碱基由a突变为t。8、苹果酸醌氧化还原酶表达强化质粒pK18mobsacB-Psod-mqo的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P169/P170引物对进行PCR扩增得到上游同源臂up，以P171/P172引物对进行PCR扩增得到启动子片段Psod，以P173/P174引物对进行PCR扩增得到下游同源臂dn。以P169/P172引物对以up、Psod为模版进行融合PCR，获得片段up-Psod。以P169/P174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Trans1T1感受态细胞，获得重组质粒pK18mobsacB-P sod-mqo。
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
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC 13032感受态细胞。重组质粒pK18mobsacB-P sod-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，其中感兴趣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，获得目的突变菌株命名为SMCT286。
2、天冬氨酸激酶表达强化菌株的构建
以上述获得的SMCT286为出发菌，制备感受态细胞。重组质粒pK18mobsacB-P sod-lysC g1a-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，其中感兴趣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，获得目的突变菌株命名为SMCT289。
3、苏氨酸合酶表达强化菌株的构建
以上述获得的SMCT289为出发菌株，制备感受态细胞。重组质粒pK18mobsacB-P sod-thrC g1a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，其中感兴趣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，获得目的突变菌株命名为SMCT291。
4、柠檬酸合酶表达弱化菌株的构建
以上述获得的SMCT291为出发菌株，制备感受态细胞。重组质粒pK18mobsacB-gltA a1g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，其中感兴趣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，获得目的突变菌株命名为SMCT294。
5、HTH转录调节因子表达弱化菌株的构建
以上述获得的SMCT294为出发菌株，制备感受态细胞。重组质粒pK18mobsacB-ramB a1g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，其中感兴趣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，获得目的突变菌株命名为SMCT297。
6、HTH转录调节因子敲除菌株的构建
以上述获得的SMCT294为出发菌株，制备感受态细胞。重组质粒pK18mobsacB-△ramB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，其中感兴趣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，获得目的突变菌株命名为SMCT298。
7、异柠檬酸脱氢酶弱化菌株的构建
以上述获得的SMCT298为出发菌株，制备感受态细胞。重组质粒pK18mobsacB-icd a1t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，其中感兴趣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，获得目的突变菌株命名为SMCT299。
8、苹果酸醌氧化还原酶表达强化菌株的构建
以上述获得的SMCT299为出发菌株，制备感受态细胞。重组质粒pK18mobsacB-Psod-mqo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，其中感兴趣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，获得目的突变菌株命名为SMCT300。
获得的菌株如表2所示：
表2
菌株名称 基因型
SMCT286 ATCC13032,P sod-aspB
SMCT289 SMCT286,P sod-lysC g1a-T311I
SMCT291 SMCT289,P sod-thrC g1a
SMCT294 SMCT291,gltA a1g
SMCT297 SMCT294,ramB a1g
SMCT298 SMCT294,ΔramB
SMCT299 SMCT298,icd a1t
SMCT300 SMCT299,P sod-mqo
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％，pH7。
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：挑取工程菌斜面种子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(表3)。
表3谷氨酸棒状杆菌生产苏氨酸的能力
Figure PCTCN2022142840-appb-000007
Figure PCTCN2022142840-appb-000008
由表3可以看出，天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶、柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子和苹果酸醌氧化还原酶等的叠加改造，对苏氨酸产量增加有正效果，苏氨酸产量提高到4.9g/L，表明本发明的叠加组合有效，可显著提高苏氨酸的产量。
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，可以对之做一些修改或改进，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。
部分序列
Figure PCTCN2022142840-appb-000009
Figure PCTCN2022142840-appb-000010
Figure PCTCN2022142840-appb-000011

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，其与苏氨酸合成途径相关的酶活性增强，且与三羧酸循环相关的酶活性发生改变以提高苏氨酸合成途径中前体物质的供应，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；
    其中，与苏氨酸合成途径相关的酶选自天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶中的至少一种；
    与三羧酸循环相关的酶选自柠檬酸合酶、异柠檬酸脱氢酶、HTH转录调节因子RamB、苹果酸醌氧化还原酶中的至少一种，
    其中，所述酶活性发生改变包括：酶活性增强、降低或丧失。
  2. 根据权利要求1所述的微生物，其特征在于，酶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)通过改变编码上述酶的核苷酸序列而增强；
    酶活性降低或丧失是由选自以下1)-5)，或任选的组合实现的：
    1)通过改变所述酶的编码基因的启动子序列而降低或丧失；
    2)通过改变所述酶的编码基因的核糖体结合位点而降低或丧失；
    3)通过改变所述酶的氨基酸序列而降低或丧失；
    4)通过改变编码所述酶的核苷酸序列而降低或丧失；
    5)通过敲除所述酶的编码序列而丧失。
  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改变苏氨酸合成途径和三羧酸循环中酶的活性。
  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，其体内天冬氨酸转氨酶、天冬氨酸激酶、苏氨酸合酶和苹果酸醌氧化还原酶活性增强，且柠檬酸合酶、异柠檬酸脱氢酶和HTH转录调节因子RamB活性降低或丧失。
  5. 根据权利要求4所述的微生物，其特征在于，天冬氨酸氨基转移酶活性的增强是通过在aspB基因起始密码子上游插入sod启动子来实现的；和/或
    天冬氨酸激酶活性的增强是通过在lysC基因起始密码子上游插入sod启动子，同时将起始密码子GTG突变为ATG，并将lysC基因编码的第311位氨基酸由苏氨酸突变为异亮氨酸来实现的；和/或
    苏氨酸合酶活性的增强是通过在thrC基因起始密码子上游插入sod启动子，并将起始密码子GTG突变为ATG来实现的；和/或
    苹果酸醌氧化还原酶活性的增强是通过在mqo基因起始密码子上游插入sod启动子来实现的；和/或
    柠檬酸合酶活性降低或丧失是通过将gltA基因的起始密码子由ATG突变为GTG来实现的；和/或
    异柠檬酸脱氢酶活性降低或丧失是通过将icd基因的起始密码子由ATG突变为TTG来实现的；和/或
    HTH转录调节因子RamB活性降低或丧失是通过将ramB基因的起始密码子由ATG突变为GTG，或者对ramB基因编码区进行敲除来实现的。
  6. 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)。
  7. 苏氨酸生产菌株的构建方法，其特征在于，所述方法包括：利用基因工程手段，增强具有氨基酸生产能力的棒杆菌中的与苏氨酸合成途径相关的基因，并改造与三羧酸循环相关的基因以提高苏氨酸合成途径中前体物质的供应；
    其中，所述与苏氨酸合成途径和三羧酸循环相关的基因选自aspB、lysC、thrC、mqo、gltA、icd、ramB中的至少一种；
    其中，所述改造与三羧酸循环相关的基因导致：酶活性的增强、降低或丧失；
    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)通过改变编码上述酶的核苷酸序列而增强；
    酶活性降低或丧失是由选自以下1)-5)，或任选的组合实现的：
    1)通过改变所述酶的编码基因的启动子序列而降低或丧失；
    2)通过改变所述酶的编码基因的核糖体结合位点而降低或丧失；
    3)通过改变所述酶的氨基酸序列而降低或丧失；
    4)通过改变编码所述酶的核苷酸序列而降低或丧失；
    5)通过敲除所述酶的编码序列而丧失。
  8. 如权利要求7所述的构建方法，其特征在于，所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  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1-6任一项所述的微生物或权利要求7或8所述方法构建的苏氨酸生产菌株，以获得所述微生物或菌株的培养物；
    b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
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