WO2023151409A1 - 高产苏氨酸工程菌的构建方法 - Google Patents

高产苏氨酸工程菌的构建方法 Download PDF

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WO2023151409A1
WO2023151409A1 PCT/CN2022/142849 CN2022142849W WO2023151409A1 WO 2023151409 A1 WO2023151409 A1 WO 2023151409A1 CN 2022142849 W CN2022142849 W CN 2022142849W WO 2023151409 A1 WO2023151409 A1 WO 2023151409A1
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enhanced
gene
threonine
enzyme
microorganism
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康培
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 high-threonine-producing engineering bacteria.
  • L-threonine (L-Threonin), chemical name ⁇ -hydroxy- ⁇ -aminobutyric acid, molecular formula C4H9NO3, relative molecular mass is 119.12.
  • L-threonine is an essential amino acid, mainly used in medicine, chemical reagents, food fortifiers, feed additives and many other aspects.
  • 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 encoded by hom
  • homoserine kinase encoded by thrB
  • threonine synthase encoded by thrC
  • 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 high-threonine-producing engineered bacteria.
  • the present invention enhances the ability of the bacterial strain to produce L-threonine by strengthening the enzymes in the threonine synthesis pathway and simultaneously strengthening the supply of precursors.
  • the present invention provides a modified Corynebacterium microorganism whose aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine
  • the activities of acid synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, and malate/quinone oxidoreductase are enhanced, and the microorganism has enhanced threonine production capacity compared to an unmodified microorganism.
  • aspartate aminotransferase Aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase and malate/quinone redox
  • the reference sequence numbers of the enzymes on NCBI are WP_011013497.1, WP_003855724.1, WP_003854900.1, WP_011014964.1, WP_011013816.1, WP_011014465.1, WP_011014814.1, or their similarities are 90% amino acid sequence.
  • the enhancement of enzyme activity is achieved by being selected from the following 1) to 5), or an optional combination:
  • 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 mutating the start codon of the lysC gene from GTG to ATG, and the 311th amino acid of its encoded protein is changed from T Mutation to I to achieve.
  • homoserine dehydrogenase activity is achieved by inserting the cspB promoter upstream of the start codon of the hom gene, and mutating the 378th amino acid of the protein encoded by the hom gene from G to E.
  • the enhancement of threonine synthase activity is by inserting the sod promoter upstream of the start codon of the thrC gene, and mutating the start codon of the thrC gene from GTG to ATG.
  • the enhancement of pyruvate carboxylase activity is achieved by inserting the sod promoter upstream of the start codon of the pyc gene, and mutating the 458th amino acid of the protein encoded by the pyc gene from P to S.
  • the enhancement of phosphoenolpyruvate carboxylase activity is achieved by inserting the tuf promoter upstream of the start codon of the ppc gene, and mutating the 299th amino acid of the protein encoded by the ppc gene from D to N.
  • 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 high-threonine-producing engineered bacterium, the method comprising: using genetic engineering means to enhance the genes aspB, lysC, hom, thrC, pyc, ppc and mqo, access to aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase and malate/quinone redox Strains with enhanced enzyme activity.
  • the reference sequence numbers of the genes aspB, lysC, hom, thrC, pyc, ppc and mqo on NCBI are cg0294, cg0306, cg1337, cg2437, cg0791, cg1787 and cg2192, respectively.
  • 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 enhancement of genes aspB, lysC, hom, thrC, pyc, ppc and mqo in threonine fermentation production or improvement of threonine fermentation yield.
  • aspartate aminotransferase aspartate kinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase, phosphate Enolpyruvate carboxylase and malate/quinone oxidoreductase activities to enhance the fermentative yield of threonine.
  • 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 application of the modified Corynebacterium genus microorganism or the high-threonine-yielding engineered bacterium constructed according to the above-mentioned method in the fermentative production of threonine or in improving 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 combines aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, malic acid/quinone redox
  • the bacterial strain with enhanced enzyme activity is used in conjunction with the L-threonine production of corynebacteria (such as Corynebacterium glutamicum), and the output of the bacterial strain L-threonine that enhances the above-mentioned gene expression has greater improvement than the unmodified strain, and the output can be up to 6.3g/L. It provides effective means for large-scale production of threonine and has broad application prospects.
  • the invention provides a bacterial strain for producing threonine, which combines the pyruvate-oxaloacetate metabolic node and the synthesis pathway for the production of L-threonine.
  • the present invention further strengthens the L-threonine synthesis pathway and pyruvate node of the strain, mainly including aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase
  • the expression enhancement or deregulation of carboxylase, phosphoenolpyruvate carboxylase, and malate/quinone oxidoreductase was used to explore the effect of both simultaneous expression enhancement on L-threonine.
  • the shake flask results showed that the production capacity of L-threonine was improved.
  • 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.
  • the present invention inserts the sod promoter upstream of the start codon of the aspB gene to enhance the expression of the aspB gene.
  • the present invention inserts the sod promoter upstream of the start codon of the lysC gene, mutates the start codon of the lysC gene from GTG to ATG, and mutates the 311th amino acid of the encoded protein from T to I, thereby realizing the enhancement of the lysC gene.
  • the invention inserts the cspB promoter upstream of the start codon of the hom gene, and mutates the 378th amino acid of the protein encoded by the hom gene from G to E, thereby realizing the enhancement of the hom gene.
  • the present invention inserts the sod promoter upstream of the start codon of the thrC gene, and mutates the start codon of the thrC gene from GTG to ATG to realize the enhancement of the thrC gene.
  • the invention inserts the sod promoter upstream of the start codon of the pyc gene, and mutates the 458th amino acid of the protein encoded by the pyc gene from P to S, thereby realizing the enhancement of the pyc gene.
  • the invention inserts the tuf promoter upstream of the start codon of the ppc gene, and mutates the 299th amino acid of the encoded protein of the ppc gene from D to N, so as to realize the enhancement of the ppc gene.
  • the invention inserts the sod promoter upstream of the start codon of the mqo gene to enhance the mqo gene.
  • Corynebacterium preferred Corynebacterium glutamicum, most preferably Corynebacterium glutamicum ATCC 13032.
  • Aspartate aminotransferase encoding gene aspB, NCBI number: cg0294, Cgl0240, NCgl0237.
  • Aspartokinase encoding gene lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Threonine synthase encoding gene thrC, NCBI number: cg2437, Cgl2220, NCgl2139.
  • Phosphoenolpyruvate carboxylase encoding gene ppc, NCBI number: cg1787, Cgl1585, NCgl1523.
  • genes lysC and thrC are from Corynebacterium glutamicum, and the nucleotide sequences of the wild-type genes lysC and thrC are respectively shown in SEQ ID NO: 1-2.
  • 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, Psod was obtained by PCR amplification with the P105/P106 primer pair, and PCR was performed with the P107/P108 primer pair
  • the dn of the downstream homology arm was amplified, and fusion PCR was carried out with the P103/P108 primer pair and up, Psod, and dn as templates to obtain the full-length fragment Psod-aspB.
  • 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-Psod-aspB.
  • the upstream homology arm up was obtained by PCR amplification with the P21/P22 primer pair, Psod was obtained by PCR amplification with the P23/P24 primer pair, and PCR was performed with the P25/P26 primer pair Amplify the sequence mid of lysC g1a , use the P27/P28 primer pair to perform PCR amplification to obtain the downstream homology arm dn, use the P21/P28 primer pair to perform fusion PCR with up, Psod, mid, and dn as templates, and obtain the full-length fragment P sod - lysCg1a- T311I .
  • 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 genome of Corynebacterium glutamicum ATCC 13032 was used as a template, and the upstream homology arm up was obtained by PCR amplification with the P29/P30 primer pair.
  • the genome was used as a template to perform PCR amplification with the P33/P34 primer pair to obtain the mid of the hom G378 sequence, and to perform PCR amplification with the P35/P36 primer pair to obtain the downstream homology arm dn, and to use the P29/P36 primer pair to obtain up, P cspB , mid, dn was used as a template for fusion PCR to obtain the full-length fragment P cspB -hom G378E .
  • 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 cspB -hom G378E .
  • the upstream homology arm up was obtained by PCR amplification with the P37/P38 primer pair, Psod was obtained by PCR amplification with the P39/P40 primer pair, and PCR was performed with the P41/P42 primer pair
  • the dn of the downstream homology arm was amplified, and fusion PCR was carried out with the P37/P42 primer pair and up, Psod, dn as templates to obtain the full-length fragment P sod -thrC g1a .
  • 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 P13/P14 primer pair
  • the Psod was obtained by PCR amplification with the P15/P16 primer pair
  • PCR was performed with the P17/P18 primer pair Amplify the pyc P458S sequence mid
  • use the P19/P20 primer pair to perform PCR amplification to obtain the downstream homology arm dn
  • use the P13/P20 primer pair to perform fusion PCR with up, Psod, mid, and dn as templates, and obtain the full-length fragment P sod -pyc P458S .
  • 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 -pyc P458S .
  • the upstream homology arm up was obtained by PCR amplification with the P53/P54 primer pair, P tuf was obtained by PCR amplification with the P55/P56 primer pair, and P tuf was obtained by PCR with the P57/P58 primer pair.
  • the ppc D299N sequence mid was obtained by PCR amplification, and the downstream homology arm dn was obtained by PCR amplification with the P59/P60 primer pair, and the fusion PCR was carried out with the up, P tuf , mid, and dn templates by the P53/P60 primer pair to obtain the full-length Fragment Ptuf -ppc D299N .
  • 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 tuf -ppc D299N .
  • the upstream homology arm up was obtained by PCR amplification with the P169/P170 primer pair, the Psod was obtained by PCR amplification with the P171/P172 primer pair, and PCR was performed with the P173/P174 primer pair
  • the dn of the downstream homology arm was amplified, and fusion PCR was performed with the P169/P174 primer pair and up, Psod, dn as templates to obtain the full-length fragment P sod -mqo.
  • 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.
  • Embodiment 2 Construction of Genome Modification Strain
  • ATCC13032 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-Psod-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 .
  • 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 strains were obtained and named SMCT241 respectively.
  • SMCT241 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • 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.
  • 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 strains were obtained and named SMCT242 respectively.
  • SMCT242 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P cspB -hom G378E 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 strains were obtained and named SMCT243 respectively.
  • SMCT243 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • 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 strains were obtained and named SMCT244 respectively.
  • SMCT244 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -pyc P458S 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 strains were obtained and named SMCT245 respectively.
  • SMCT245 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P tuf -ppc D299N 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 strains were obtained and named SMCT246 respectively.
  • the strain construction method is the same as above, using SMCT246 as the starting bacterium to transform the malic acid/quinone oxidoreductase expression-enhanced strain, and the obtained transformed strain is named SMCT247.
  • SMCT246 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -mqo 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 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 strains were obtained and named SMCT247 respectively.
  • 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 ring of SMCT241, SMCT242, SMCT243, SMCT244, SMCT245, SMCT246, SMCT247 slant seeds and put them into a 500mL Erlenmeyer flask containing 20mL of seed medium, and shake at 30°C and 220r/min for 16h.
  • 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.
  • At least one of aspartate aminotransferase, aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, malate/quinone oxidoreductase When expression enhancement was combined, the production of L-threonine was improved.

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Abstract

本发明提供一种高产苏氨酸工程菌的构建方法。本发明将天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶、苹果酸/醌氧化还原酶活性增强的菌株联合用于棒杆菌(如谷氨酸棒状杆菌)的L-苏氨酸生产，增强上述基因表达的菌株L-苏氨酸的产量较未改造菌株有较大提高，产量可达6.3g/L。为大规模生产苏氨酸提供有效手段，应用前景广阔。

Description

高产苏氨酸工程菌的构建方法 技术领域
本发明属于微生物工程技术领域，具体地说，涉及一种高产苏氨酸工程菌的构建方法。
背景技术
L-苏氨酸(L-Threonin)，化学名称β-羟基-α-氨基丁酸，分子式C4H9NO3，相对分子质量为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、饲料添加剂等诸多方面。
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编码)以及苏氨酸合酶(thrC编码)。Hermann Sahm等人一直致力于高产苏氨酸的谷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L-苏氨酸的报道主要集中在其合成路径中，丙酮酸-草酰乙酸代谢节点与合成途径组合等方面的报道较少，且现有报道仅对L-苏氨酸合成路径做了初步研究，并未形成系统。
发明内容
本发明的目的是提供一种高产苏氨酸工程菌的构建方法。
为了实现本发明目的，本发明通过加强苏氨酸合成路径中的酶同时加强前体的供应使菌株生产L-苏氨酸的能力得到提升。
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，其天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶和苹果酸/醌氧化还原酶的活性增强，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，天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶和苹果酸/醌氧化还原酶在NCBI上的参考序列编号分别为WP_011013497.1、WP_003855724.1、WP_003854900.1、WP_011014964.1、WP_011013816.1、WP_011014465.1、WP_011014814.1，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)～5)，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。
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，并将lysC基因起始密码子由GTG突变为ATG、其编码蛋白的第311位氨基酸由T突变为I来实现的。
进一步地，高丝氨酸脱氢酶活性的增强是通过在hom基因起始密码子上游插入cspB启动子，并将hom基因编码蛋白的第378位氨基酸由G突变为E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thrC基因起始密 码子由GTG突变为ATG。
进一步地，丙酮酸羧化酶活性的增强是通过在pyc基因起始密码子上游插入sod启动子，并将pyc基因编码蛋白的第458位氨基酸由P突变为S来实现的。
进一步地，磷酸烯醇丙酮酸羧化酶活性的增强是通过在ppc基因起始密码子上游插入tuf启动子，并将ppc基因编码蛋白的第299位氨基酸由D突变为N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。
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，本发明提供高产苏氨酸工程菌的构建方法，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基因aspB、lysC、hom、thrC、pyc、ppc和mqo，获得天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶和苹果酸/醌氧化还原酶活性增强的菌株。
优选地，基因aspB、lysC、hom、thrC、pyc、ppc和mqo在NCBI上的参考序列编号分别为cg0294、cg0306、cg1337、cg2437、cg0791、cg1787和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)通过对酶的编码基因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。
第四方面，本发明提供基因aspB、lysC、hom、thrC、pyc、ppc和mqo的增强在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
进一步地，通过增强具有氨基酸生产能力的棒杆菌(Corynebacterium)中的天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶和苹果酸/醌氧化还原酶的活性来提高苏氨酸的发酵产量。
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高产苏氨酸工程菌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：
本发明将天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶、苹果酸/醌氧化还原酶活性增强的菌株联合用于棒杆菌(如谷氨酸棒状杆菌)的L-苏氨酸生产，增强上述基因表达的菌株L-苏氨酸的产量较未改造菌株有较大提高，产量可达6.3g/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菌株，将丙酮酸-草酰乙酸代谢节点与合成途径组合用于L-苏氨酸的生产。菌株改造过程及效果：
本发明对菌株的L-苏氨酸合成路径和丙酮酸节点进一步强化，主要包括天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶、苹果酸/醌氧化还原酶表达强化或解调控，以探索两者同时表达强化后对L-苏氨酸的影响。摇瓶结果显示L-苏氨酸的生产能力有所提升。
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。
具体地，
本发明在aspB基因起始密码子上游插入sod启动子，实现对aspB基因的表达增强。
本发明在lysC基因起始密码子上游插入sod启动子，并将lysC基因起始密码子由GTG突变为ATG、其编码蛋白的第311位氨基酸由T突变为I，实现对lysC基因的增强。
本发明在hom基因起始密码子上游插入cspB启动子，并将hom基因编码蛋白的第378位氨基酸由G突变为E，实现对hom基因的增强。
本发明在thrC基因起始密码子上游插入sod启动子，并将thrC基因起始密码子由GTG突变为ATG，实现对thrC基因的增强。
本发明在pyc基因起始密码子上游插入sod启动子，并将pyc基因编码蛋白的第458位氨基酸由P突变为S，实现对pyc基因的增强。
本发明在ppc基因起始密码子上游插入tuf启动子，并将ppc基因编码蛋白的第299位氨基酸由D突变为N，实现对ppc基因的增强。
本发明在mqo基因起始密码子上游插入sod启动子，实现对mqo基因的增强。
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：
天冬氨酸氨基转移酶，编码基因aspB，NCBI编号：cg0294、Cgl0240、NCgl0237。
天冬氨酸激酶，编码基因lysC，NCBI编号：cg0306、Cgl0251、NCgl0247。
高丝氨酸脱氢酶，编码基因hom，NCBI编号：cg1337、Cgl1183、NCgl1136。
苏氨酸合酶，编码基因thrC，NCBI编号：cg2437、Cgl2220、NCgl2139。
丙酮酸羧化酶，编码基因pyc，NCBI编号：cg0791、Cgl0689、NCgl0659。
磷酸烯醇丙酮酸羧化酶，编码基因ppc，NCBI编号：cg1787、Cgl1585、NCgl1523。
苹果酸/醌氧化还原酶，编码基因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来自谷氨酸棒状杆菌，野生型基因lysC、thrC的核苷酸序列分别如SEQ ID NO:1-2所示。
以下实施例中使用的实验材料如下：
Figure PCTCN2022142849-appb-000001
Figure PCTCN2022142849-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 PCTCN2022142849-appb-000003
Figure PCTCN2022142849-appb-000004
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。
实施例1 菌株基因组改造质粒的构建
1、天冬氨酸氨基转移酶方案重组质粒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Psod，以P107/P108引物对进行PCR扩增得到下游同源臂dn，以P103/P108引物对以up、Psod、dn为模板进行融合PCR，获得全长片段Psod-aspB。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-Psod-aspB。
2、天冬氨酸激酶表达强化方案重组质粒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Psod，以P25/P26引物对进行PCR扩增得到lysC g1a序列mid，以P27/P28引物对进行PCR扩增得到下游同源臂dn，以P21/P28引物对以up、Psod、mid、dn为模板进行融合PCR，获得全长片段P sod-lysC g1a- T311I。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、高丝氨酸脱氢酶表达强化方案重组质粒pK18mobsacB-P cspB-hom G378E的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P29/P30引物对进行PCR扩增得到上游同源臂up，以ATCC14067基因组为模板以P31/P32引物对进行PCR扩增得到P cspB，以ATCC13032基因组为模板以P33/P34引物对进行PCR扩增得到hom G378序列mid，以P35/P36引物对进行PCR扩增得到下游同源臂dn，以P29/P36引物对以up、P cspB、mid、dn为模板进行融合PCR，获得全长片段P cspB-hom G378E。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 cspB-hom G378E。
4、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Psod，以P41/P42引物对进行PCR扩增得到下游同源臂dn，以P37/P42引物对以up、Psod、dn为模板进行融合PCR，获得全长片段P sod-thrC g1a。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。
5、丙酮酸羧化酶表达强化方案重组质粒pK18mobsacB-P sod-pyc P458S的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P13/P14引物对进行PCR扩增得到上游同源臂up，以P15/P16引物对进行PCR扩增得到Psod，以P17/P18引物对进行PCR扩增得到pyc P458S序列mid，以P19/P20引物对进行PCR扩增得到下游同源臂dn，以P13/P20引物对以up、Psod、mid、dn为模板进行融合PCR，获得全长片段P sod-pyc P458S。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-pyc P458S。
6、磷酸烯醇式丙酮酸羧化酶表达强化方案重组质粒pK18mobsacB-P tuf-ppc D299N的构建
以谷氨酸棒状杆菌ATCC 13032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P tuf，以P57/P58引物对进行PCR扩增得到ppc D299N序列mid，以P59/P60引物对进行PCR扩增得到下游同源臂dn，以P53/P60引物对以up、P tuf、mid、dn为模板进行融合PCR，获得全长片段P tuf-ppc D299N。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 tuf-ppc D299N。
7、天冬氨酸氨基转移酶表达强化方案重组质粒pK18mobsacB-P sod-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Psod，以P173/P174引物对进行PCR扩增得到下游同源臂dn，以P169/P174引物对以up、Psod、dn为模板进行融合PCR，获得全长片段P sod-mqo。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。
质粒构建过程中所用引物如表1所示：
表1
Figure PCTCN2022142849-appb-000005
Figure PCTCN2022142849-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2 基因组改造菌株的构建
1、天冬氨酸氨基转移酶加强菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC13032感受态细胞。重组质粒pK18mobsacB-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，获得目的突变菌株分别命名为SMCT241。
2、天冬氨酸激酶强化表达及解调控菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT241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，获得目的突变菌株分别命名为SMCT242。
3、高丝氨酸脱氢酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT242感受态细胞。重组质粒pK18mobsacB-P cspB-hom G378E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，获得目的突变菌株分别命名为SMCT243。
4、苏氨酸合酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT243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，获得目的突变菌株分别命名为SMCT244。
5、丙酮酸羧化酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT244感受态细胞。重组质粒pK18mobsacB-P sod-pyc P458S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，获得目的突变菌株分别命名为SMCT245。
6、磷酸烯醇丙酮酸羧化酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT245感受态细胞。重组质粒pK18mobsacB-P tuf-ppc D299N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，获得目的突变菌株分别命名为SMCT246。
7、苹果酸/醌氧化还原酶表达强化菌株的构建
菌株构建方法同上，以SMCT246为出发菌，进行苹果酸/醌氧化还原酶表达强化菌株的改造，获得的改造菌株命名为SMCT247。
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT246感受态细胞。重组质粒pK18mobsacB-P sod-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，获得目的突变菌株分别命名为SMCT247。
获得的菌株如表2所示：
表2
Figure PCTCN2022142849-appb-000007
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SMCT241、SMCT242、SMCT243、SMCT244、SMCT245、SMCT246、SMCT247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 L-苏氨酸摇瓶发酵结果
菌株编号 OD 562 L-苏氨酸(g/L)
ATCC13032 25 —
SMCT241 26 0.8
SMCT242 25 1.8
SMCT243 24 2.6
SMCT244 23 3.6
SMCT245 23 4.1
SMCT246 25 5.7
SMCT247 24 6.3
从表3可以看出，天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶、苹果酸/醌氧化还原酶一种或多种表达加强的菌株的L-苏氨酸产量有所提高，L-苏氨酸最高产量为5.9g/L。天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶、苹果酸/醌氧化还原酶至少一个表达强化相组合时，其L-苏氨酸的产量均有提升。
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 PCTCN2022142849-appb-000008
Figure PCTCN2022142849-appb-000009

Claims (7)

  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，其天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶和苹果酸/醌氧化还原酶的活性增强，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，酶的活性的增强是由选自以下1)～6)，或任选的组合实现的：
    1)通过导入具有所述酶的编码基因的质粒而增强；
    2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
    3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
    4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
    5)通过对酶的氨基酸序列进行改变而增强；
    6)通过对酶的编码基因的核苷酸序列进行改变而增强。
  3. 根据权利要求1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，并将lysC基因起始密码子由GTG突变为ATG、其编码蛋白的第311位氨基酸由T突变为I来实现的；和/或
    高丝氨酸脱氢酶活性的增强是通过在hom基因起始密码子上游插入cspB启动子，并将hom基因编码蛋白的第378位氨基酸由G突变为E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thrC基因起始密码子由GTG突变为ATG；和/或
    丙酮酸羧化酶活性的增强是通过在pyc基因起始密码子上游插入sod启动子，并将pyc基因编码蛋白的第458位氨基酸由P突变为S来实现的；和/或
    磷酸烯醇丙酮酸羧化酶活性的增强是通过在ppc基因起始密码子上游插入tuf启动子，并将ppc基因编码蛋白的第299位氨基酸由D突变为N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。
  4. 根据权利要求1-3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)。
  5. 高产苏氨酸工程菌的构建方法，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基因aspB、lysC、hom、thrC、pyc、ppc和mqo，获得天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、丙酮酸羧化酶、磷酸烯醇丙酮酸羧化酶和苹果酸/醌氧化还原酶活性增强的菌株；
    所述增强的途径选自以下1)～6)，或任选的组合：
    1)通过导入具有所述酶的编码基因的质粒而增强；
    2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
    3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
    4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
    5)通过对酶的氨基酸序列进行改变而增强；
    6)通过对酶的编码基因的核苷酸序列进行改变而增强。
  6. 根据权利要求5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)。
  7. 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1-4任一项所述的微生物或权利要求5-6任一项所述方法构建的工程菌，以获得所述微生物或工程菌的培养物；
    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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