WO2023151408A1 - 高产苏氨酸菌株的构建方法 - Google Patents

高产苏氨酸菌株的构建方法 Download PDF

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WO2023151408A1
WO2023151408A1 PCT/CN2022/142847 CN2022142847W WO2023151408A1 WO 2023151408 A1 WO2023151408 A1 WO 2023151408A1 CN 2022142847 W CN2022142847 W CN 2022142847W WO 2023151408 A1 WO2023151408 A1 WO 2023151408A1
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threonine
enzyme
gene
enhanced
pathway
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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 a high-threonine-producing bacterial strain.
  • L-threonine (L-Threonin), chemical name ⁇ -hydroxy- ⁇ -aminobutyric acid, molecular formula C 4 H 9 NO 3 , relative molecular mass 119.12.
  • L-threonine is an essential amino acid, which is 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 high-threonine-producing strain.
  • the present invention improves the ability of the bacterial strain to produce L-threonine by strengthening the threonine synthesis pathway while reducing the competition pathway and/or catabolic pathway related to threonine synthesis.
  • the present invention provides a modified microorganism of the genus Corynebacterium, which has enhanced enzymatic activity related to the threonine synthesis pathway and competes with the threonine synthesis pathway compared with an unmodified microorganism. And/or the enzymatic activity related to the catabolic pathway is reduced or lost, and the microorganism has enhanced threonine production ability compared to the unmodified microorganism.
  • the enzyme related to the threonine synthesis pathway is selected from at least one of aspartate aminotransferase, aspartokinase, and threonine synthase;
  • the enzymes related to the competitive pathway and/or catabolic pathway related to threonine synthesis are selected from the group consisting of diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase, high at least one of serine-O-acetyltransferases.
  • aspartate aminotransferase Preferably, aspartate aminotransferase, aspartate kinase, threonine synthase, diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase
  • the reference sequence numbers of homoserine-O-acetyltransferase on NCBI are WP_011013497.1, WP_003855724.1, WP_011014964.1, WP_011015254.1, WP_011014792.1, WP_011014022.1, WP_00386203 3.1, WP_011013793.1, or Amino acid sequence with 90% similarity to it.
  • 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 enzymatic activity associated with threonine synthesis-related competitive pathways and/or catabolic pathways in the microorganism is achieved by being selected from the following 1)-5), or an optional combination:
  • genes related to the competitive pathway and/or catabolic pathway related to threonine synthesis are selected from at least one of ddh, dapA, tdcB, ilvA and metX.
  • Mutagenesis, site-directed mutagenesis, or homologous recombination can be used to reduce the expression of genes related to threonine synthesis-related competing pathways and/or catabolic pathways or to knock out endogenous threonine-related competing pathways and/or genes associated with catabolic pathways.
  • the microorganisms Compared with unmodified microorganisms, the microorganisms have enhanced activities of aspartate aminotransferase, aspartate kinase and threonine synthase, and diaminopimelate dehydrogenase, 4-hydroxy-tetra
  • the activities of hydrodipicolinate synthase, threonine dehydratase, and homoserine-O-acetyltransferase were reduced or lost.
  • the enhancement of aspartate aminotransferase activity is achieved by inserting the sod promoter upstream of the start codon of the coding gene aspB.
  • the enhancement of aspartokinase activity is achieved by inserting the sod promoter upstream of the start codon of the coding gene lysC, and mutating the start codon of the lysC gene from GTG to ATG, the 311st step of aspartokinase The position amino acid was mutated from T to I.
  • the enhancement of threonine synthase activity is achieved by inserting the sod promoter upstream of the initiation codon of the thrC gene, and mutating the initiation codon of the thrC gene from GTG to ATG.
  • diaminopimelate dehydrogenase activity is achieved by knocking out the ddh gene.
  • the reduction or loss of threonine dehydratase activity is achieved by knocking out the tdcB gene.
  • the reduction or loss of threonine dehydratase activity is achieved by knocking out the ilvA gene.
  • homoserine-O-acetyltransferase activity is achieved by knocking out the metX 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 high-threonine-producing strain, the method comprising: using genetic engineering means to enhance genes related to the threonine synthesis pathway in corynebacteria with amino acid production capacity, and inactivating or Attenuation of genes associated with competing and/or catabolic pathways associated with threonine synthesis;
  • the gene related to the threonine synthesis pathway is selected from at least one of aspB, lysC, thrC;
  • genes related to the competitive pathway or catabolic pathway related to threonine synthesis are selected from at least one of ddh, dapA, tdcB, ilvA, metX;
  • the reference sequence numbers of the genes aspB, lysC, thrC, ddh, dapA, tdcB, ilvA, and metX on NCBI are cg0294, cg0306, cg2437, Cg2900, cg2161, cg1116, cg2334, and cg0754, respectively.
  • the enhanced pathway is selected from the following 1) to 6), or an optional combination:
  • the weakening is selected from the following 1)-5), or an optional combination:
  • the weakening method can be selected from at least one of mutagenesis, site-directed mutation, homologous recombination and the like.
  • 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 application of the modified Corynebacterium genus microorganism or the high-threonine-producing strain constructed according to the above-mentioned method in the fermentative production of threonine or in increasing the fermentative yield of threonine.
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention enhances the expression of one or more of corynebacterium (such as Corynebacterium glutamicum) aspartate aminotransferase, aspartokinase, and threonine synthase, so that the L-threonine of the bacterial strain acid production has been improved; further, diaminopimelic acid dehydrogenase inactivation, 4-hydroxy-tetrahydrodipicolinate synthase weakened, and threonine dehydratase tdcB, threonine dehydratase ilvA, high
  • One or more strains with weakened or inactivated expression of one or more serine-O-acetyltransferases have increased L-threonine production, and finally can reach 6.7g/L, thus providing an effective solution for large-scale production of threonine. Means, broad application prospects.
  • the invention provides a bacterial strain for producing threonine, which adopts a combination plan of threonine branch inactivation, catabolism inactivation and synthesis pathway enhancement to improve the ability of the bacterial strain to produce L-threonine.
  • the present invention strengthens the L-threonine synthesis pathway of the starting bacterial strain (such as Corynebacterium glutamicum ATCC 13032), reduces branch expression, and reduces product decomposition, mainly including aspartate aminotransferase, aspartate kinase, Threonine synthase, diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydratase, homoserine-O-acetyltransferase expression enhancement, weakening, inactivation or degradation regulation to explore its effect on threonine production.
  • the shake flask results showed that the production capacity of L-threonine was improved.
  • Inactivation or weakening during the transformation process includes promoter replacement, ribosome binding site changes, point mutations, and sequence deletions.
  • Expression enhancement during transformation includes promoter replacement and ribosome binding site changes. , copy number increase, plasmid overexpression and other means, and the above means are all means 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, and mutates the start codon of the lysC gene from GTG to ATG, and the 311th amino acid of its encoded protein (aspartokinase) is mutated from T to I, Enhancement of the lysC gene was achieved.
  • 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 knocks out the ddh gene to realize the inactivation of the ddh gene.
  • the present invention mutates the start codon of dapA gene from ATG to GTG to realize the weakening of dapA gene.
  • the invention knocks out the tdcB gene to realize the inactivation of the tdcB gene.
  • the invention knocks out the ilvA gene to realize the inactivation of the ilvA gene.
  • the invention knocks out the metX gene to realize the inactivation of the metX 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.
  • Threonine dehydratase encoding gene tdcB, NCBI number: cg1116, Cgl0978, NCgl0939.
  • Threonine dehydratase encoding gene ilvA, NCBI number: cg2334, Cgl2127, Ncgl2046.
  • Homoserine-O-acetyltransferase encoding gene metX, NCBI number: cg0754, Cgl0652, NCgl0624.
  • genes lysC, thrC, and dapA are from Corynebacterium glutamicum, and the nucleotide sequences of the wild-type genes lysC, thrC, and dapA are respectively shown in SEQ ID NO: 1-3.
  • 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 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 plasmid construction method is the same as above, and the primers used are P99, P100, P101, and P102.
  • the upstream homology arm up was obtained by PCR amplification with the P99/P100 primer pair
  • the downstream homology arm dn was obtained by PCR amplification with the P101/P102 primer pair.
  • the primer pair used up and dn as templates for fusion PCR to obtain the full-length fragment ⁇ ddh.
  • 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- ⁇ ddh.
  • the upstream homology arm up was obtained by PCR amplification with the P75/P76 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P75/P78 primer pair.
  • the primer pair was used for fusion PCR with up and dn as templates to obtain the full-length fragment dapA a1g .
  • 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-dapA a1g .
  • a1g means that the first base of the initiation codon of the dapA gene (see SEQ ID NO: 3 for the wild-type gene sequence of dapA) is mutated from a to g. 6. Construction of recombinant plasmid pK18mobsacB- ⁇ tdcB inactivation scheme of threonine dehydratase tdcB
  • the upstream homology arm up was obtained by PCR amplification with the P145/P146 primer pair
  • the downstream homology arm dn was obtained by PCR amplification with the P145/P148 primer pair.
  • the primer pair used up and dn as templates for fusion PCR to obtain the full-length fragment ⁇ tdcB.
  • 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- ⁇ tdcB.
  • the upstream homology arm up was obtained by PCR amplification with the P87/P88 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P89/P90 primer pair.
  • 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- ⁇ ilvA.
  • the upstream homology arm up was obtained by PCR amplification with the P95/P96 primer pair
  • the downstream homology arm dn was obtained by PCR amplification with the P95/P98 primer pair.
  • the primer pair used up and dn as templates for fusion PCR to obtain the full-length fragment ⁇ metX.
  • 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- ⁇ metX.
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (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 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 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 SMCT256 respectively.
  • SMCT256 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 SMCT257 respectively.
  • SMCT257 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ ddh was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/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 strains were obtained and named SMCT258 respectively.
  • SMCT258 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23). Recombinant plasmid pK18mobsacB- ⁇ tdcB. The competent cells were transformed by electroporation, and transformants were screened on a selection medium containing 15 mg/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 strains were obtained and named SMCT259 respectively.
  • SMCT259 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23). Recombinant plasmid pK18mobsacB- ⁇ ilvA. The competent cells were transformed by electroporation, and transformants were screened on a selection medium containing 15 mg/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 strains were obtained and named SMCT260 respectively.
  • SMCT260 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23). Recombinant plasmid pK18mobsacB- ⁇ metX. The competent cells were transformed by electroporation, and transformants were screened on a selection medium containing 15 mg/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 strains were obtained and named SMCT261 respectively.
  • 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 ring of SMCT241, SMCT242, SMCT256, SMCT257, SMCT258, SMCT259, SMCT260, SMCT261 slant seeds and transfer them to a 500mL Erlenmeyer flask containing 20mL of seed medium, shake and culture for 16h at 30°C and 220r/min .
  • 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.
  • SMCT258 twenty three 3.9 SMCT259 twenty two 4.3 SMCT260 twenty three 5.5 SMCT261 twenty two 6.7
  • the L-threonine output of bacterial strains with enhanced expression of one or more of aspartate aminotransferase, aspartate kinase, and threonine synthase has been improved; Inactivation of acid dehydrogenase; weakening of 4-hydroxy-tetrahydrodipicolinate synthase; weakening of one or more expressions of threonine dehydratase tdcB, threonine dehydratase ilvA, homoserine-O-acetyltransferase or
  • the L-threonine yield of the inactivated strain was increased, and the highest L-threonine yield was 6.7g/L.

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Abstract

本发明提供一种高产苏氨酸菌株的构建方法。本发明通过将棒杆菌(如谷氨酸棒状杆菌)的天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶中的一种或多种表达加强，使菌株的L-苏氨酸产量有所提高；进一步地，将菌株的二氨基庚二酸脱氢酶失活，4-羟基-四氢二吡啶甲酸合酶弱化，且苏氨酸脱水酶、高丝氨酸-O-乙酰转移酶中的一种或多种表达弱化或失活，L-苏氨酸产量有所提高，最终可达6.7g/L，从而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，涉及一种高产苏氨酸菌株的构建方法。
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、饲料添加剂等多个领域。
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L-苏氨酸的报道主要集中在其合成路径中，支路失活、分解代谢失活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菌株的构建方法。
为了实现本发明目的，本发明通过加强苏氨酸合成路径同时降低与苏氨酸合成相关的竞争途径和/或分解代谢途径使菌株生产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，其与苏氨酸合成途径相关的酶活性增强，且与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的酶活性降低或丧失，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。
其中，与苏氨酸合成途径相关的酶选自天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶中的至少一种；
所述与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的酶选自二氨基庚二酸脱氢酶、4-羟基-四氢二吡啶甲酸合酶、苏氨酸脱水酶、高丝氨酸-O-乙酰转移酶中的至少一种。优选地，天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶、二氨基庚二酸脱氢酶、4-羟基-四氢二吡啶甲酸合酶、苏氨酸脱水酶、高丝氨酸-O-乙酰转移酶在NCBI上的参考序列编号分别为WP_011013497.1、WP_003855724.1、WP_011014964.1、WP_011015254.1、WP_011014792.1、WP_011014022.1、WP_003862033.1、WP_011013793.1，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)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强。
所述微生物体内与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的酶活性降低或丧失是通过选自以下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；
其中，与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因选自ddh、dapA、tdcB、ilvA、metX中的至少一种。
可以采用诱变、定点突变或同源重组的方法来降低与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因的表达或敲除内源的与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因。
所述微生物与未修饰的微生物相比，其体内天冬氨酸氨基转移酶、天冬氨酸激酶和苏氨酸合酶活性增强，且二氨基庚二酸脱氢酶、4-羟基-四氢二吡啶甲酸合酶、苏氨酸脱水酶和高丝氨酸-O-乙酰转移酶活性降低或丧失。
进一步地，天冬氨酸氨基转移酶活性的增强是通过在编码基因aspB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编码基因lysC起始密码子上游插入sod启动子，并将lysC基因的起始密码子由GTG突变为ATG、天冬氨酸激酶的第311位氨基酸由T突变为I来实现的。
进一步地，苏氨酸合酶活性的增强是通过在编码基因thrC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实现的。
进一步地，二氨基庚二酸脱氢酶活性降低或丧失是通过敲除ddh基因来实现的。
进一步地，4-羟基-四氢二吡啶甲酸合酶活性降低或丧失是通过将dapA基因起始密码子由ATG突变为GTG。
进一步地，苏氨酸脱水酶活性降低或丧失是通过敲除tdcB基因来实现的。
进一步地，苏氨酸脱水酶活性降低或丧失是通过敲除ilvA基因来实现的。
进一步地，高丝氨酸-O-乙酰转移酶活性降低或丧失是通过敲除metX基因来实现的。
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，并失活或弱化与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因；
其中，所述与苏氨酸合成途径相关的基因选自aspB、lysC、thrC中的至少一种；
所述与苏氨酸合成相关的竞争途径或分解代谢途径相关的基因选自ddh、dapA、tdcB、ilvA、metX中的至少一种；
优选地，基因aspB、lysC、thrC、ddh、dapA、tdcB、ilvA、metX在NCBI上的参考序列编号分别为cg0294、cg0306、cg2437、Cg2900、cg2161、cg1116、cg2334、cg0754。
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)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强；
本发明中，所述弱化选自以下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。
弱化的方法可选自诱变、定点突变、同源重组等中的至少一种。
第三方面，本发明提供一种生产苏氨酸的方法，所述方法包括如下步骤：
a)培养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高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。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明通过将棒杆菌(如谷氨酸棒状杆菌)天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶中的一种或多种表达加强，使菌株的L-苏氨酸产量有所提高；进一步地，将二氨基庚二酸脱氢酶失活，4-羟基-四氢二吡啶甲酸合酶弱化，且苏氨酸脱水酶tdcB、苏氨酸脱水酶ilvA、高丝氨酸-O-乙酰转移酶中的一种或多种表达弱化或失活的菌株，L-苏氨酸产量有所提高，最终可达6.7g/L，从而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，采用苏氨酸支路失活、分解代谢失活与合成途径加强的组合方案，来提高菌株生产L-苏氨酸的能力。菌株改造过程及效果：
本发明对出发菌株(如谷氨酸棒状杆菌ATCC 13032)的L-苏氨酸合成路径强化和降低支路表达、降低产物分解，主要包括天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶、二氨基庚二酸脱氢酶、4-羟基-四氢二吡啶甲酸合酶、苏氨酸脱水酶，高丝氨酸-O-乙酰转移酶表达强化、弱化、失活或解调控，以探索其对苏氨酸产量的影响。摇瓶结果显示L-苏氨酸的生产能力有所提升。
改造过程中的失活或弱化包括启动子的替换，核糖体结合位点的改变、点突变、序列的缺失等手段，改造过程中的表达强化包括启动子的替换，核糖体结合位点的改变、拷贝数的增加、质粒过表达等手段，且以上手段均为本领域技术人员公知手段。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(天冬氨酸激酶)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。
本发明敲除ddh基因，实现对ddh基因的失活。
本发明将dapA基因起始密码子由ATG突变为GTG，实现对dapA基因的弱化。
本发明敲除tdcB基因，实现对tdcB基因的失活。
本发明敲除ilvA基因，实现对ilvA基因的失活。
本发明敲除metX基因，实现对metX基因的失活。
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。
苏氨酸合酶，编码基因thrC，NCBI编号：cg2437、Cgl2220、NCgl2139。
二氨基庚二酸脱氢酶，编码基因ddh，NCBI编号：Cg2900、Cgl2617、NCgl2528。
4-羟基-四氢二吡啶甲酸合酶，编码基因dapA，NCBI编号：cg2161、Cgl1971、NCgl1896。
苏氨酸脱水酶，编码基因tdcB，NCBI编号：cg1116、Cgl0978、NCgl0939。
苏氨酸脱水酶，编码基因ilvA，NCBI编号：cg2334、Cgl2127、Ncgl2046。
高丝氨酸-O-乙酰转移酶，编码基因metX，NCBI编号：cg0754、Cgl0652、NCgl0624。
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、dapA来自谷氨酸棒状杆菌，野生型基因lysC、thrC、dapA的核苷酸序列分别如SEQ ID NO:1-3所示。
以下实施例中使用的实验材料如下：
Figure PCTCN2022142847-appb-000001
Figure PCTCN2022142847-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 PCTCN2022142847-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-T1Phage 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、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。
4、二氨基庚二酸脱氢酶失活质粒pK18mobsacB-△ddh的构建
质粒构建方法同上，所用引物为P99、P100、P101、P102。
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P99/P100引物对进行PCR扩增得到上游同源臂up，以P101/P102引物对进行PCR扩增得到下游同源臂dn，以P99/P102引物对以up、dn为模板进行融合PCR，获得全长片段△ddh。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-△ddh。
5、4-羟基-四氢二吡啶甲酸合酶弱化方案重组质粒pK18mobsacB-dapA a1g的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P75/P76引物对进行PCR扩增得到上游同源臂up，以P77/P78引物对进行PCR扩增得到下游同源臂dn，以P75/P78引物对以up、dn为模板进行融合PCR，获得全长片段dapA a1g。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-dapA a1g。
其中，a1g表示dapA基因(dapA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。6、苏氨酸脱水酶tdcB失活方案重组质粒pK18mobsacB-△tdcB的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P145/P146引物对进行PCR扩增得到上游同源臂up，以P147/P148引物对进行PCR扩增得到下游同源臂dn，以P145/P148引物对以up、dn为模板进行融合PCR，获得全长片段△tdcB。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-△tdcB。
7、苏氨酸脱水酶ilvA失活方案重组质粒pK18mobsacB-△ilvA的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P87/P88引物对进行PCR扩增得到上游同源臂up，以P89/P90引物对进行PCR扩增得到下游同源臂dn，以P87/P90引物对以up、dn为模板进行融合PCR，获得全长片段△ilvA。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-△ilvA。
8、高丝氨酸-O-乙酰转移酶失活方案重组质粒pK18mobsacB-△metX的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P95/P96引物对进行PCR扩增得到上游同源臂up，以P97/P98引物对进行PCR扩增得到下游同源臂dn，以P95/P98引物对以up、dn为模板进行融合PCR，获得全长片段△metX。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-△metX。
质粒构建过程中所用引物如表1所示：
表1
Figure PCTCN2022142847-appb-000004
Figure PCTCN2022142847-appb-000005
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-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、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SMCT242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SMCT256。
4、二氨基庚二酸脱氢酶失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT256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SMCT257。
5、4-羟基-四氢二吡啶甲酸合酶表达弱化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT257感受态细胞。重组质粒pK18mobsacB-△ddh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SMCT258。
6、苏氨酸脱水酶tdcB失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT258感受态细胞。重组质粒pK18mobsacB-△tdcB。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SMCT259。
7、苏氨酸脱水酶ilvA失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT259感受态细胞。重组质粒pK18mobsacB-△ilvA。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SMCT260。
8、高丝氨酸-O-乙酰转移酶失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT260感受态细胞。重组质粒pK18mobsacB-△metX。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SMCT261。
获得的菌株如表2所示：
表2
菌株名称 基因型
SMCT241 ATCC13032,P sod-aspB
SMCT242 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I
SMCT256 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I,P sod-thrC g1a
SMCT257 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I,P sod-thrC g1a,△ddh
SMCT258 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I,P sod-thrC g1a,△ddh,dapA  a1g
SMCT259 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I,P sod-thrC g1a,△ddh,dapA  a1g,△tdcB
SMCT260 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I,P sod-thrC g1a,△ddh,dapA  a1g,△tdcB,△ilvA
SMCT261 ATCC13032,P sod-aspB,P sod-lysC g1a-T311I,P sod-thrC g1a,△ddh,dapA  a1g,△tdcB,△ilvA,△metX
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、SMCT256、SMCT257、SMCT258、SMCT259、SMCT260、SMCT261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
SMCT256 24 2.5
SMCT257 23 3.3
SMCT258 23 3.9
SMCT259 22 4.3
SMCT260 23 5.5
SMCT261 22 6.7
从表3可以看出，天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶一种或多种表达加强的菌株的L-苏氨酸产量有所提高；二氨基庚二酸脱氢酶失活；4-羟基-四氢二吡啶甲酸合酶弱化；苏氨酸脱水酶tdcB、苏氨酸脱水酶ilvA、高丝氨酸-O-乙酰转移酶一种或多种表达弱化或失活的菌株的L-苏氨酸产量有所提高，L-苏氨酸最高产量为6.7g/L。
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 PCTCN2022142847-appb-000006
Figure PCTCN2022142847-appb-000007

Claims (10)

  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，且与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的酶活性降低或丧失，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；
    其中，与苏氨酸合成途径相关的酶选自天冬氨酸氨基转移酶、天冬氨酸激酶、苏氨酸合酶中的至少一种；
    所述与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的酶选自二氨基庚二酸脱氢酶、4-羟基-四氢二吡啶甲酸合酶、苏氨酸脱水酶、高丝氨酸-O-乙酰转移酶中的至少一种。
  2. 根据权利要求1所述的微生物，其特征在于，与苏氨酸合成途径相关的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)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强；
    所述微生物体内与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的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；
    其中，与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因选自ddh、dapA、tdcB、ilvA、metX中的至少一种。
  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与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因的表达或敲除内源的与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因。
  4. 根据权利要求1所述的微生物，其特征在于，所述微生物与未修饰的微生物相比，其体内天冬氨酸氨基转移酶、天冬氨酸激酶和/或苏氨酸合酶活性增强，且二氨基庚二酸脱氢酶、4-羟基-四氢二吡啶甲酸合酶、苏氨酸脱水酶和/或高丝氨酸-O-乙酰转移酶活性降低或丧失。
  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编码基因aspB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编码基因lysC起始密码子上游插入sod启动子，并将lysC基因的起始密码子由GTG突变为ATG、天冬氨酸激酶的第311位氨基酸由T突变为I来实现的；和/或
    苏氨酸合酶活性的增强是通过在编码基因thrC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实现的；和/或
    二氨基庚二酸脱氢酶活性降低或丧失是通过敲除ddh基因来实现的；和/或
    4-羟基-四氢二吡啶甲酸合酶活性降低或丧失是通过将dapA基因起始密码子由ATG突变为GTG；和/或
    苏氨酸脱水酶活性降低或丧失是通过敲除tdcB基因来实现的；和/或
    苏氨酸脱水酶活性降低或丧失是通过敲除ilvA基因来实现的；和/或
    高丝氨酸-O-乙酰转移酶活性降低或丧失是通过敲除metX基因来实现的。
  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. 高产苏氨酸菌株的构建方法，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，并失活或弱化与苏氨酸合成相关的竞争途径和/或分解代谢途径相关的基因；
    其中，所述与苏氨酸合成途径相关的基因选自aspB、lysC、thrC中的至少一种；
    所述与苏氨酸合成相关的竞争途径或分解代谢途径相关的基因选自ddh、dapA、tdcB、ilvA、metX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)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强；
    所述弱化选自以下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. 根据权利要求7所述的方法，其特征在于，弱化的方法选自诱变、定点突变、同源重组中的至少一种。
  9. 根据权利要求7或8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)。
  10. 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7-9中任一项所述方法获得的高产苏氨酸菌株，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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