WO2023142853A1 - Procédé pour la construction d'une bactérie génétiquement modifiée pour une production élevée de thréonine - Google Patents

Procédé pour la construction d'une bactérie génétiquement modifiée pour une production élevée de thréonine Download PDF

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WO2023142853A1
WO2023142853A1 PCT/CN2022/142931 CN2022142931W WO2023142853A1 WO 2023142853 A1 WO2023142853 A1 WO 2023142853A1 CN 2022142931 W CN2022142931 W CN 2022142931W WO 2023142853 A1 WO2023142853 A1 WO 2023142853A1
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threonine
activity
enhanced
iolr
enzyme
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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 genetically engineered bacterium.
  • L-threonine (L-Threonin), the chemical name is ⁇ -hydroxy- ⁇ -aminobutyric acid, the molecular formula is C 4 H 9 NO 3 , and the relative molecular mass is 119.12.
  • L-threonine is an essential amino acid, and threonine is mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc.
  • 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.
  • 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 object of the present invention is to improve the threonine-producing ability of the bacterial strain by reducing or losing the activity of the GntR family regulatory factor iolR, thereby providing a method for constructing a genetically engineered bacterium that produces high threonine (L-threonine).
  • the present invention provides a modified microorganism of the genus Corynebacterium. Compared with an unmodified microorganism, the activity of the GntR family regulatory factor iolR is reduced or lost, and the microorganism is relatively Enhanced threonine production capacity compared to unmodified microorganisms.
  • the reference sequence number of the GntR family regulator iolR on NCBI is WP_003857140.1, or an amino acid sequence with 90% similarity thereto.
  • the reduction or loss of the activity of the GntR family regulatory factor iolR in the microorganism is achieved by reducing the expression of the gene encoding the GntR family regulatory factor iolR or knocking out the endogenous gene encoding the GntR family regulatory factor iolR.
  • Methods such as mutagenesis, site-directed mutation, or homologous recombination can be used to reduce the expression of the gene encoding the GntR family regulatory factor iolR or to knock out the endogenous gene encoding the GntR family regulatory factor iolR.
  • the enzyme activity of the competitive pathway or degradation pathway related to threonine synthesis in the microorganism is reduced or lost; or,
  • the enzyme activity of the threonine synthesis pathway in the microorganism is enhanced, and the enzyme activity of the competition pathway or degradation pathway related to the threonine synthesis is reduced or lost;
  • the enzyme related to the threonine synthesis pathway is selected from at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase
  • their reference sequence numbers on NCBI are respectively WP_003855724.1, WP_011013506.1, WP_003854900.1, WP_011014183.1, WP_011014964.1, or an amino acid sequence with a similarity of 90% to the above reference sequence.
  • the enzyme of the competitive pathway related to the synthesis of threonine is selected from the group consisting of diaminopimelate dehydrogenase, threonine dehydratase, 4-hydroxy-tetrahydrodipicolinate synthase, homoserine acetyltransferase At least one; preferably, their reference sequence numbers on NCBI are respectively WP_011015254.1, WP_003862033.1, WP_011014792.1, WP_011013793.1, or an amino acid sequence with a similarity of 90% to the above reference sequence.
  • the microorganism is any one of the following 1 ⁇ 7:
  • GntR family regulator iolR The activity of GntR family regulator iolR is reduced or lost and the activities of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase are enhanced, and diaminopimelic acid Microorganisms with reduced or lost dehydrogenase activity;
  • GntR family regulator iolR The activity of GntR family regulator iolR is reduced or lost and the enzymatic activities of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase are enhanced, and threonine dehydration Microorganisms with reduced or lost enzyme activity;
  • GntR family regulator iolR The activity of GntR family regulator iolR is reduced or lost, and the enzymatic activities of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase are enhanced, and 4-hydroxy- Microorganisms with reduced or lost tetrahydrodipicolinate synthase activity;
  • the reduction in the activity of 4-hydroxy-tetrahydrodipicolinate synthase refers to the mutation of A in the initiation codon ATG of the nucleotide sequence encoding 4-hydroxy-tetrahydrodipicolinate synthase to G;
  • GntR family regulator iolR The activity of GntR family regulator iolR is reduced or lost, and the enzymatic activities of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine synthase are enhanced, and homoserine acetyl transfer Microorganisms with reduced or lost enzyme activity;
  • the reduced activity of homoserine acetyltransferase refers to the mutation of A to G in the initiation codon ATG of the nucleotide sequence encoding homoserine acetyltransferase.
  • the enhancement of the activity of enzymes related to the threonine synthesis pathway in the microorganism is achieved by being selected from the following 1) to 5), or an optional combination:
  • the attenuation includes knocking out or reducing the transcription or expression of genes or changing the amino acid and nucleotide sequences encoding corresponding enzymes.
  • the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
  • the present invention provides a method for constructing a high-threonine-producing genetically engineered bacterium, wherein the method is selected from any one of schemes i ⁇ iv:
  • Scheme i reduce or lose the activity of iolR encoding the GntR family regulatory factor in corynebacteria with amino acid production ability, and obtain the strain;
  • step B Enhancing the enzymes related to the threonine synthesis pathway in the strain of step A to obtain a strain with enhanced enzyme activity;
  • step b further weakening the protein activity of the competition pathway and degradation pathway related to threonine synthesis in the strain of step a;
  • the attenuation includes knocking out or reducing gene transcription or expression or changing the amino acid and nucleotide sequence encoding the corresponding enzyme;
  • Enhancing step 1) Enzymes related to threonine synthesis pathway in gene weakened strains to obtain enzyme activity enhanced strains;
  • the attenuation includes knocking out or reducing gene transcription or expression or changing the amino acid and nucleotide sequence encoding the corresponding enzyme;
  • the enhanced pathway is selected from the following 1) to 6), or an optional combination:
  • the enzyme related to the threonine synthesis pathway is selected from at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, threonine synthase, homoserine kinase kind;
  • the competitive pathway protein related to threonine synthesis is selected from at least one of diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, homoserine acetyltransferase, and threonine dehydratase A sort of.
  • 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 use of a strain that reduces or loses the activity of the GntR family regulator iolR in the fermentative production of threonine or in increasing the fermentative yield of threonine.
  • the fermentation yield of threonine is increased by reducing or losing the activity of the GntR family regulatory factor iolR in Corynebacterium having amino acid production ability.
  • the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
  • the present invention provides the application of the modified Corynebacterium genus microorganism or the high-threonine-yielding genetically engineered bacteria 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 improves the yield of threonine produced by strains (corynebacteria, such as corynebacterium glutamicum) by inactivating the GntR family regulatory factor iolR.
  • the production of threonine can be increased by 38.8% compared with the unmodified strain.
  • threonine yield of SMCT195 was the highest among the finally constructed strains, reaching 6.5g/L, which was 1.6 times higher than that of threonine-chassis bacteria. It provides a new way for large-scale production of threonine and has high application value.
  • the present invention first weakens or inactivates the iolR gene on the basis of Corynebacterium glutamicum ATCC 13032 to verify the influence on threonine, and obtains strains SMCT181 and SMCT182 to initially verify the influence of iolR on the yield of threonine, SMCT181 and SMCT182 Threonine was 0.4g/L and 0.2g/L respectively.
  • iolR attenuating or inactivating iolR is effective, but not as expected.
  • threonine chassis bacteria If the strain is modified to produce threonine, its synthesis pathway must first be opened up, mainly including aspartokinase-aspartate semialdehyde dehydrogenase operon (lysC fbr- asd), homoserine dehydrogenase-homoserine kinase
  • lysC fbr-thrB aspartokinase-aspartate semialdehyde dehydrogenase operon
  • homoserine dehydrogenase-homoserine kinase homoserine dehydrogenase-homoserine kinase
  • the modified strain SMCT183 was obtained by modifying lysC fbr -asd on the basis of the original strain ATCC13032, and on this basis, the Hom fb
  • the SMCT185 strain was further modified to further transform diaminopimelate dehydrogenase, threonine dehydratase, 4-hydroxy-tetrahydrodipicolinate synthase and homoserine At least one of them, a series of strains SMCT188, SMCT189, SMCT190, SMCT192, SMCT191 were obtained, the yield of threonine was further increased, and the corresponding by-products were decreased in different degrees.
  • the iolR gene was further inactivated by taking the above five strains as the starting bacteria to obtain strains SMCT193, SMCT194, SMCT195, SMCT196 and SMCT197, and the threonine production increased by 27.4%, 26.0%, 35.4%, 38.8% and 29.4%, respectively.
  • 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 adopts following technical scheme:
  • One of the technical solutions of the present invention provides a microorganism with reduced or lost activity of the GntR family regulator iolR.
  • the second technical solution of the present invention provides a GntR family regulator iolR activity reduction or loss and aspartate aminotransferase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine Amino acid synthase activity was enhanced in one less enhanced expressing microorganism.
  • the third technical solution of the present invention provides a GntR family regulatory factor iolR activity reduction or loss and aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine Microorganisms with enhanced synthase activity and reduced or lost diaminopimelate dehydrogenase activity.
  • the fourth technical solution of the present invention provides a GntR family regulatory factor iolR activity reduction or loss and aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine Microorganisms with enhanced synthase activity and reduced or lost threonine dehydratase activity.
  • the fifth technical solution of the present invention is to provide a GntR family regulator iolR activity reduction or loss and aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine A microorganism in which synthase activity is enhanced and 4-hydroxy-tetrahydrodipyridinecarboxylate synthase activity is reduced or lost.
  • the sixth technical solution of the present invention provides a GntR family regulatory factor iolR activity reduction or loss and aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase and threonine Microorganisms with enhanced synthase activity and reduced or lost homoserine acetyltransferase activity.
  • Corynebacterium is preferably Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum comprises ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (referring to NCBI Corunebacterium glutamicum evolutionary tree https://www .ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
  • GntR family regulatory factor iolR encoding gene iolR, NCBI number: cg0196, cgl0157, NCgl0154.
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Aspartate semialdehyde dehydrogenase encoding gene name asd, NCBI number: Cgl0252, Cg0307, NCgl0248.
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: Cg1337, Cgl1183, NCgl1136.
  • Threonine synthase encoding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139.
  • Homoserine kinase encoding gene thrB, NCBI number: Cgl1184, Cg1338, NCgl1137.
  • Threonine dehydratase encoding gene ilvA, NCBI number: Cgl2127, Cg2334, Ncgl2046.
  • Homoserine acetyltransferase (homoserine-O-acetyltransferase), encoding gene metX, NCBI number: Cgl0652, Cg0754, NCgl0624.
  • 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 P21/P22 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P23/P24 primer pair
  • the Psod was obtained by PCR amplification with the P25/P26 primer pair.
  • lysC g1a-T311I was amplified by PCR with the P27/P28 primer pair to obtain the dn of the downstream homology arm. Fusion PCR was performed with P21/P24 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-lysCg1a -T311I-dn was obtained by fusion PCR with P21/P28 primer pair and up-Psod, lysC g1a-T311I , dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I -asd.
  • 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 P29/P30 primer pair, and the promoter fragment PcspB was obtained by PCR amplification using the ATCC14067 genome as a template with the P31/P32 primer pair, and the ATCC13032 genome was used as a template to obtain
  • the P33/P34 primer pair was used for PCR amplification to obtain hom G378E
  • the P35/P36 primer pair was used for PCR amplification to obtain the downstream homology arm dn.
  • the fragment up-PcspB was obtained.
  • the full-length fragment up-Psod-hom G378E -dn was obtained by fusion PCR with P29/P36 primer pair and up-PcspB, hom G378E , dn as template.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P cspB -hom G378E -thrB.
  • the upstream homology arm up was obtained by PCR amplification with the P37/P38 primer pair
  • the promoter fragment Psod-thrCg1a was obtained by PCR amplification with the P39/P40 primer pair
  • PCR amplification was performed with the P41/P42 primer pair.
  • Add the downstream homology arm dn The full-length fragment up-Psod-thrCg1a-dn was obtained by fusion PCR with P37/P42 primer pair and up, Psod-thrCg1a, dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 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 iolR-1/iolR-2 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the iolR-3/iolR-4 primer pair. Fusion PCR was performed with iolR-1/iolR-4 primer pair and up and dn as templates to obtain the full-length fragment up-dn.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1 T1 competent cells, and obtained the recombinant plasmid pK18mobsacB- ⁇ iolR.
  • the upstream homology arm iolR a1g -up was obtained by PCR amplification with the iolR-5/iolR-6 primer pair, and the downstream homology arm iolR was obtained by PCR amplification with the iolR-7/iolR-8 primer pair a1g -dn.
  • the full-length fragment iolR a1g -up-dn was obtained by fusion PCR with iolR-5/iolR-8 primer pair and iolR a1g -up and iolR a1g -dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-iolR a1g .
  • a1g means that the first base of the start codon of the iolR gene (see SEQ ID NO: 3 for the iolR wild-type gene sequence) is mutated from a to g.
  • the upstream homology arm dapA a1g -up was obtained by PCR amplification with the P75/P76 primer pair, and the downstream homology arm dapA a1g -dn was obtained by PCR amplification with the P77/P78 primer pair.
  • the full-length fragment dapA a1g- up-dn was obtained by fusion PCR with primer pair P75/P78 and dapA a1g-up and dapA a1g - dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 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: 4 for the wild-type gene sequence of dapA) is mutated from a to g.
  • the upstream homology arm ilvA a1g -up was obtained by PCR amplification with the P83/P84 primer pair, and the downstream homology arm ilvA a1g -dn was obtained by PCR amplification with the P85/P86 primer pair.
  • the full-length fragment ilvA a1g- up-dn was obtained by fusion PCR using the P83/P86 primer pair and ilvA a1g-up and ilvA a1g - dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-ilvA a1g .
  • a1g means that the first base of the start codon of the ilvA gene (see SEQ ID NO: 5 for the ilvA wild-type gene sequence) is mutated from a to g.
  • the upstream homology arm metX a1g -up was obtained by PCR amplification with the P91/P92 primer pair, and the downstream homology arm metX a1g -dn was obtained by PCR amplification with the P93/P94 primer pair.
  • the full-length fragment metX a1g -up -dn was obtained by fusion PCR with P91/P94 primer pair and metX a1g -up and metX a1g -dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1 T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-metX a1g .
  • a1g means that the first base of the start codon of the metX gene (see SEQ ID NO: 6 for the metX wild-type gene sequence) is mutated from a to g.
  • the upstream homology arm up was obtained by PCR amplification with the P95/P96 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P97/P98 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR using the P95/P98 primer pair and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1 T1 competent cells, and obtained the recombinant plasmid pK18mobsacB- ⁇ metX.
  • the upstream homology arm up was obtained by PCR amplification with the P99/P100 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P101/P102 primer pair.
  • the full-length fragment up-dn was obtained by fusion PCR with primer pair P99/P102 and up and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1 T1 competent cells, and obtained the recombinant plasmid pK18mobsacB- ⁇ ddh.
  • the primers used in the construction process are shown in Table 1:
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23). Recombinant plasmids pK18mobsacB- ⁇ iolR and pK18mobsacB-iolR a1g were used to transform the competent cells by electroporation, and the transformants were screened on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was blocked 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 SMCT181 and SMCT182 respectively.
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I -asd 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 due to homologous Sex is 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), and the dilution 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 SMCT183 respectively.
  • SMCT183 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P cspB -hom G378E -thrB was used to transform the competent cells by electroporation, and the transformants were screened on the selection medium containing 15 mg/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), and the dilution 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 SMCT184 respectively.
  • SMCT184 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), and the dilution 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 SMCT185 respectively.
  • SMCT185 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23). Recombinant plasmids pK18mobsacB- ⁇ iolR and pK18mobsacB-iolR a1g were used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/L kanamycin. The gene of interest was 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), and the dilution 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 named SMCT186 and SMCT187 respectively.
  • SMCT185 competent cells were prepared according to the classical method of glutamicum (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), and the dilution 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 SMCT188.
  • SMCT185 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-dapA 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), and the dilution 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 SMCT189 respectively.
  • SMCT185 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-ilvA 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), and the dilution 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 SMCT190 respectively.
  • SMCT185 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-metX 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), and the dilution 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. Through PCR amplification of the target sequence and nucleotide sequencing analysis, the target mutant strains were named SMCT191, respectively.
  • SMCT185 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ metX 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), and the dilution 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 mutant strains were named SMCT192.
  • the construction method was the same as above, using SMCT185 as the starting bacterium, inactivating the metX gene, and the constructed strains were named SMCT192.
  • SMCT188, SMCT189, SMCT190, SMCT191 and SMCT192 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Charpter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ iolR 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.
  • 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), and the dilution 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 named SMCT193, SMCT194, SMCT195, SMCT196, SMCT197 respectively.
  • strain genotype SMCT181 ATCC13032, ⁇ iolR SMCT182 ATCC13032, iolR a1g SMCT183 ATCC13032, P sod -lysC g1a-T311I -asd SMCT184 ATCC13032,P sod -lysC g1a-T311I -asd,P cspB -hom G378E -thrB SMCT185 ATCC13032, P sod -lysC g1a-T311I -asd, P cspB -hom G378E -thrB, P sod -thrC g1a SMCT187 ATCC13032, P sod -lysC g1a-T311I -asd, P cspB -hom G378E -thrB, P sod -thrC g1a SMCT187 ATCC13032,
  • 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 SMCT181, SMCT182, SMCT183, SMCT184, SMCT185, SMCT186, SMCT187, SMCT188, SMCT189, SMCT190, SMCT191, SMCT192, SMCT193, SMCT194, SMCT195, SMCT196 and SMCT197 slant seeds 1 loop to 20mL In the 500mL Erlenmeyer flask of the seed medium, shake culture 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.
  • iolR was inactivated, and a series of iolR inactivated strains (SMCT193, SMCT194, SMCT195, SMCT196 and SMCT197) were constructed. 35.4%, 38.8%, and 29.4%. It was shown again that the modification of iolR was an effective site for improving threonine production.
  • iolR when iolR is modified in combination with other sites, the yield of threonine is improved compared with that of iolR alone, indicating that iolR interacts with other sites (such as aspartokinase, aspartate semialdehyde dehydrogenation Expression enhancement and deregulation of enzymes, homoserine kinase, homoserine dehydrogenase, threonine synthase and diaminopimelate dehydrogenase, 4-hydroxy-tetrahydrodipicolinate synthase, threonine dehydration enzyme, homoserine acetyltransferase expression down-regulation) can effectively increase the production of threonine.
  • sites such as aspartokinase, aspartate semialdehyde dehydrogenation Expression enhancement and deregulation of enzymes, homoserine kinase, homoserine dehydrogenase, threonine synthase

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Abstract

L'invention concerne un procédé de construction d'une bactérie génétiquement modifiée pour la production élevée de thréonine. La capacité de la souche à produire de la thréonine est améliorée au moyen de la réduction de l'activité du facteur de régulation transcriptionnel iolR de la famille GntR, et le rendement de thréonine produit par la souche peut être augmenté d'au plus 38,8 % comparé à une souche non modifiée. En outre, la voie de synthèse de la thréonine est encore renforcée, et/ou la voie en concurrence et la voie de dégradation liées à la synthèse de la thréonine sont éliminées ou affaiblies, afin d'améliorer le rendement de la thréonine et de réduire, à des degrés divers, les sous-produits L-lysine, L-isoleucine et L-méthionine. Le procédé fournit une nouvelle manière de production à grande échelle de thréonine et présente une grande valeur d'application.
PCT/CN2022/142931 2022-01-29 2022-12-28 Procédé pour la construction d'une bactérie génétiquement modifiée pour une production élevée de thréonine WO2023142853A1 (fr)

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