CN116606786A - Recombinant microorganism for producing threonine and construction method and application thereof - Google Patents

Recombinant microorganism for producing threonine and construction method and application thereof Download PDF

Info

Publication number
CN116606786A
CN116606786A CN202210118708.1A CN202210118708A CN116606786A CN 116606786 A CN116606786 A CN 116606786A CN 202210118708 A CN202210118708 A CN 202210118708A CN 116606786 A CN116606786 A CN 116606786A
Authority
CN
China
Prior art keywords
microorganism
enhanced
threonine
activity
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210118708.1A
Other languages
Chinese (zh)
Inventor
康培
姚嘉琪
宫卫波
何君
李岩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Langfang Meihua Bio Technology Development Co Ltd
Original Assignee
Langfang Meihua Bio Technology Development Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Langfang Meihua Bio Technology Development Co Ltd filed Critical Langfang Meihua Bio Technology Development Co Ltd
Priority to CN202210118708.1A priority Critical patent/CN116606786A/en
Priority to PCT/CN2022/143101 priority patent/WO2023151411A1/en
Publication of CN116606786A publication Critical patent/CN116606786A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01003—Homoserine dehydrogenase (1.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01049—Glucose-6-phosphate dehydrogenase (1.1.1.49)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01009—Glyceraldehyde-3-phosphate dehydrogenase (NADP+) (1.2.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02001—Acetate kinase (2.7.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02004—Aspartate kinase (2.7.2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03001—Threonine synthase (4.2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01001—Pyruvate carboxylase (6.4.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to the technical field of microbial engineering, in particular to a recombinant microorganism for producing threonine, a construction method and application thereof. According to the invention, by constructing the strain inactivated by acetate kinase and applying the strain to threonine production, the threonine production capacity of the strain is obviously improved, and the expression of malate quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and the like are combined for strengthening, so that the threonine yield is further improved, a novel method is provided for large-scale production of threonine, and the method has higher application value.

Description

Recombinant microorganism for producing threonine and construction method and application thereof
Technical Field
The invention relates to the technical field of microbial engineering, in particular to a recombinant microorganism for producing threonine, a construction method and application thereof.
Background
Threonine (Thronin) has a chemical name of beta-hydroxy-alpha-aminobutyric acid and a molecular formula of C 4 H 9 NO 3 The relative molecular weight is 119.12, is an essential amino acid, and is mainly used in the aspects of medicines, chemical reagents, food reinforcing agents, feed additives and the like.
Corynebacterium glutamicum is an important producer of amino acid fermentation. In corynebacterium glutamicum, the formation of threonine from oxaloacetate requires five catalytic reactions, which are catalyzed by aspartokinase (lysC-encoded), aspartyl semialdehyde dehydrogenase (asd-encoded), homoserine dehydrogenase (hom-encoded), homoserine kinase (thrB-encoded) and threonine synthase (thrC-encoded), respectively. The current reports of threonine production using Corynebacterium glutamicum have focused mainly on their synthetic pathways, and there have been reports of the hom gene (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 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.). However, there are very few reports on metabolic engineering of threonine precursor supply and metabolic overflow of pyruvic acid during threonine synthesis.
Disclosure of Invention
The invention aims to improve the threonine producing capacity of a strain by inactivating acetate kinase, thereby providing a recombinant microorganism for producing threonine, a construction method and application thereof.
Currently, metabolic engineering for threonine synthesis using corynebacterium glutamicum is mainly focused on the threonine synthesis pathway, mainly the synthesis pathway of oxaloacetate to threonine; while pyruvic acid is an important intermediate metabolite in a microbial metabolic network, and mainly enters tricarboxylic acid circulation to provide energy and precursor substances for the growth of thalli, the metabolism overflow of pyruvic acid is caused when the upstream and downstream metabolic pathways are unbalanced, so that the waste of pyruvic acid is caused. Although the threonine precursor is oxaloacetic acid, oxaloacetic acid can be produced by pyruvate carboxylase to catalyze the production of pyruvate, and can also be produced by pyruvate into tricarboxylic acid cycle through a series of enzymatic reactions. According to the invention, in the metabolic engineering research process of threonine, the flow of oxaloacetic acid which is a precursor for synthesizing threonine from pyruvic acid can be improved by reducing the metabolic overflow of pyruvic acid, so that the synthesis of threonine is promoted, and compared with other methods for reducing the metabolic overflow of pyruvic acid, the effect of reducing or losing the activity of acetate kinase is obviously better, the metabolic overflow of pyruvic acid can be effectively reduced by reducing or losing the activity of acetate kinase, the supply of threonine synthesis precursor is improved, and the threonine synthesis capability of a strain is obviously improved.
To achieve the object of the present invention, in a first aspect, the present invention provides a modified microorganism of the genus Corynebacterium, which has reduced or lost acetate kinase activity as compared to an unmodified microorganism and which has enhanced threonine productivity as compared to an unmodified microorganism.
Preferably, the acetate kinase has a reference sequence number wp_003862874.1 on NCBI, or an amino acid sequence with 90% similarity thereto and equivalent function.
Further, the reduction or loss of acetate kinase activity in the microorganism is achieved by reducing expression of a gene encoding acetate kinase or knocking out an endogenous gene encoding acetate kinase.
Mutagenesis, site-directed mutagenesis or homologous recombination may be used to reduce expression of the acetate kinase-encoding gene or to knock out the endogenous acetate kinase-encoding gene.
Further, the microorganism has an enhanced activity of pyruvate carboxylase and/or a de-feedback inhibition compared to an unmodified microorganism.
Preferably, the reference sequence number of the pyruvate carboxylase at NCBI is WP_011013816.1, or an amino acid sequence which is 90% similar thereto and has an equivalent function.
Further, the microorganism has enhanced and/or released feedback inhibition of the activity of any one or more enzymes of the following (1) to (4) compared to the unmodified microorganism:
(1) Malate quinone oxidoreductase;
(2) Glucose-6-phosphate dehydrogenase;
(3) 6-phosphogluconate dehydrogenase;
(4) NADP dependent glyceraldehyde-3-phosphate dehydrogenase.
Preferably, the malate quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP dependent glyceraldehyde-3-phosphate dehydrogenase has reference sequence numbers WP_011014814.1, NP_600790.1, NP_600669.1, FOB 93-04945 on NCBI, or an amino acid sequence having 90% similarity thereto and having equivalent function.
Preferably, the microorganism has enhanced and/or deregulated in vivo activity of an enzyme associated with the threonine synthesis pathway compared to an unmodified microorganism; wherein the enzyme related to threonine synthesis pathway is at least one selected from aspartokinase, homoserine dehydrogenase and threonine synthase.
Preferably, the aspartokinase, homoserine dehydrogenase, threonine synthase are referenced in NCBI as WP_003855724.1, WP_003854900.1, WP_011014964.1, or amino acid sequences having 90% similarity thereto and equivalent function.
Preferably, the microorganism is any one of the following (1) to (6):
(1) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase and/or threonine synthase activity;
(2) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase and/or pyruvate carboxylase activity;
(3) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or malate quinone oxidoreductase activity;
(4) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or glucose-6-phosphate dehydrogenase activity;
(5) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or 6-phosphogluconate dehydrogenase activity;
(6) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated activity of aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from streptococcus mutans.
The enhancement of the activity of the above-mentioned enzymes is achieved by a combination selected from the following 1) to 6), or optionally:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by altering the nucleotide sequence encoding the enzyme.
Preferably, the enhancement of the activity of the enzyme is achieved by replacing the original promoter of the gene encoding the enzyme with a stronger promoter that is more active and/or mutating the start codon of the gene to ATG.
Wherein the strong promoter comprises Psod, ptuf or PcspB.
The nucleotide sequences of the promoters Psod, ptuf or PcspB are shown in SEQ ID NO.1, 2 and 3, respectively.
Preferably, the enhanced expression of the pyruvate carboxylase encoding gene, malate quinone oxidoreductase encoding gene, glucose-6-phosphate dehydrogenase encoding gene, 6-phosphogluconate dehydrogenase encoding gene, aspartokinase encoding gene, threonine synthase encoding gene is achieved by replacing the original promoter thereof with a Psod promoter;
enhanced expression of the streptococcus mutans-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase encoding gene is achieved by integrating the streptococcus mutans-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase encoding gene transcribed by the Ptuf promoter into the chromosome of the strain;
enhanced expression of the homoserine dehydrogenase encoding gene is achieved by replacing its original promoter with the PcspB promoter.
The above described release of feedback inhibition is preferably achieved by the following mutations: the elimination of feedback inhibition of the pyruvate carboxylase is realized by mutating a gene encoding the pyruvate carboxylase so that the encoded pyruvate carboxylase generates P458S mutation;
the de-feedback inhibition of glucose-6-phosphate dehydrogenase is achieved by mutating the glucose-6-phosphate dehydrogenase gene such that the encoded glucose-6-phosphate dehydrogenase is subjected to an A243T mutation;
the feedback inhibition of aspartokinase is released by mutating the aspartokinase encoding gene so that the encoded aspartokinase generates T311I mutation;
the release of feedback inhibition of homoserine dehydrogenase is achieved by mutating the gene encoding homoserine dehydrogenase such that the homoserine dehydrogenase undergoes a G378E mutation.
Preferably, the microorganism of the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum). Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 and the like (see NCBI Corunebacterium glutamicum treelets https:// www.ncbi.nlm.nih.gov/genome/469), and Corynebacterium glutamicum ATCC13032 is more preferable.
In a second aspect, the present invention provides a method for constructing a threonine-producing strain, the method comprising:
A. weakening a gene encoding acetate kinase in coryneform bacteria having amino acid productivity to obtain a gene-weakened strain; the attenuation comprises knocking out or reducing the expression of an acetate kinase encoding gene; and/or
B. Enhancing the activity of pyruvate carboxylase and/or releasing its feedback inhibition;
C. enhancing the activity of and/or releasing the feedback inhibition of any one or more of the following enzymes (1) to (4):
(1) Malate quinone oxidoreductase;
(2) Glucose-6-phosphate dehydrogenase;
(3) 6-phosphogluconate dehydrogenase;
(4) NADP dependent glyceraldehyde-3-phosphate dehydrogenase;
and/or
D. Enhancing the activity of and/or releasing feedback inhibition by an enzyme associated with a threonine synthesis pathway selected from at least one of aspartokinase, homoserine dehydrogenase, threonine synthase;
the enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by altering the nucleotide sequence encoding the enzyme.
In a third aspect, the present invention provides a method for producing threonine, the method comprising the steps of:
a) Culturing the microorganism to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
In a fourth aspect, the invention provides the use of a reduced or lost enzymatic activity of acetate kinase in threonine fermentation production or in improving threonine fermentation production.
Preferably, the reduction or loss of acetate kinase activity in the microorganism is achieved by reducing expression of a gene encoding acetate kinase or knocking out an endogenous gene encoding acetate kinase.
Further, the fermentation yield of threonine is improved by inactivating acetate kinase in corynebacteria (Corynebacterium) having an amino acid-producing ability.
Preferably, the corynebacterium of the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), which includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287, etc. (see NCBI Corunebacterium glutamicum, tree https:// www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC13032.
In a fifth aspect, the present invention provides the use of the modified coryneform microorganism or the threonine-producing strain constructed according to the above-mentioned method for threonine fermentation production or for improving threonine fermentation production.
The transformation methods of the related strains comprise transformation modes of strengthening and weakening genes and the like which are known to the person skilled in the art, and are referred to the system path engineering of the full-scope high-yield L-arginine corynebacterium crenatum [ D ]. University of Jiangnan, 2016; cui Yi metabolically engineering corynebacterium glutamicum to produce L-leucine [ D ]. Tianjin university of science and technology; xu Guodong construction of L-isoleucine-producing Strain and optimization of fermentation conditions university of Tianjin science and technology 2015.
The invention has the beneficial effects that: according to the invention, through inactivating acetate kinase, metabolic overflow of pyruvic acid is reduced, overflow metabolite is reduced, waste of pyruvic acid is reduced, more pyruvic acid flows to threonine synthesis precursor oxaloacetic acid, thus the threonine production capacity of the strain is obviously improved, and the threonine yield of the strain is obviously improved compared with that of a strain which is not modified. And the expression of the malic acid quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and the like are combined for strengthening, so that the threonine yield is further improved. The transformation can be used for fermentation production of threonine and has good application value.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The information of the protein and the coding gene thereof related to the invention is as follows:
acetate kinase, coding gene name ackA, NCBI accession number: cg3047, cgl2752, NCgl2656.
Aspartokinase, coding gene name lysC, NCBI accession number: cg0306, cgl0251, NCgl0247.
Homoserine dehydrogenase, coding gene name hom, NCBI accession number: cg1337, cgl1183, NCgl1136.
Threonine synthase, encoding gene name thrC, NCBI accession No.: cg2437, cgl2220, NCgl2139.
Pyruvate carboxylase, coding gene name pyc, NCBI accession number: cg0791, cgl0689, NCgl0659.
Malate quinone oxidoreductase, encoding gene name mqo, NCBI accession No.: cg2192, cgl2001, NCgl1926.
Glucose-6-phosphate dehydrogenase, encoding the gene name zwf, NCBI accession No.: cg1778, cgl1576, NCgl1514.
6-phosphogluconate dehydrogenase, coding gene name gnd, NCBI accession number: cg1643, cgl1452, NCgl1396.
Streptococcus mutans derived NADP dependent glyceraldehyde-3-phosphate dehydrogenase encoding the gene designation gapN, NCBI accession number: FOB93_04945.
EXAMPLE 1 construction of plasmid for genome engineering of Strain
1. Aspartokinase expression enhancing plasmid pK18mobsacB-Psod-lysC a1g-T311I Construction of (3)
PCR amplification was performed using ATCC13032 genome as a template and the P21/P22 primer pairThe upstream homology arm up is amplified by a P23/P24 primer pair to obtain a promoter fragment Psod, and amplified by a P25/P26 primer pair to obtain lysC a1g-T311I And (3) carrying out PCR amplification by using a P27/P28 primer pair to obtain a downstream homologous arm dn. And carrying out fusion PCR by taking the P21/P24 primer pair and up and Psod as templates to obtain a fragment up-Psod. With the P21/P28 primer pair with up-Psod, lysC a1g-T311I Fusion PCR is carried out by taking dn as a template to obtain a full-length fragment up-Psod-lysC a1g-T311I Dn. pK18mobsacB was digested with BamHI/HindIII. The up-Psod-lysC after enzyme digestion a1g-T311I Assembling the dn and the pK18mobsacB by using a seamless cloning kit, transforming the Trans1T1 competent cells to obtain a recombinant plasmid pK18mobsacB-Psod-lysC a1g-T311I 。
2. Homoserine dehydrogenase expression enhancing plasmid pK18mobsacB-PcspB-hom G378E Construction of (3)
The plasmid construction method is described in 1 above, and the primers used are P29, P30, P31, P32, P33, P34, P35 and P36.
3. Threonine synthase expression enhancing plasmid pK18mobsacB-Psod-thrC a1g Construction of (3)
Plasmid construction methods refer to 1 above, and the primers used are P37, P38, P39, P40, P41, and P42.
4. Pyruvate carboxylase expression enhancing plasmid pK18mobsacB-Psod-pyc P458S Construction of (3)
The plasmid construction method is described in the above 1, and the primers are P13, P14, P15, P16, P17, P18, P19 and P20.
5. Construction of acetate kinase inactivating plasmid pK18 mobsacB-DeltaackA
The ATCC13032 genome is used as a template, the P165/P166 primer pair is used for PCR amplification to obtain an upstream homology arm up, and the P167/P168 primer pair is used for PCR amplification to obtain a downstream homology arm dn. Fusion PCR was performed using the P165/P168 primer pair and up/dn as a template to obtain full-length fragment up-dn. pK18mobsacB was digested with BamHI/HindIII. And assembling the up-dn and the pK18mobsacB after enzyme digestion by using a seamless cloning kit, and transforming the Trans1T1 competent cells to obtain a recombinant plasmid pK18 mobsacB-delta ackA.
6. Construction of Malate quinone oxidoreductase expression enhancing plasmid pK18mobsacB-Psod-mqo
Plasmid construction methods referring to 1 above, the primers used were P169, P170, P171, P172, P173, P174.
7. Glucose-6-phosphate dehydrogenase expression enhancing plasmid pK18mobsacB-Psod-zwf A243T Construction of (3)
The plasmid construction method is described in 1 above, and the primers used are P129, P130, P131, P132, P133, P134, P135 and P136.
8. Construction of 6-phosphogluconate dehydrogenase expression enhancing plasmid pK18mobsacB-Psod-gnd
The plasmid construction method is described in 1 above, and the primers used are P123, P124, P125, P126, P127 and P128.
9. Construction of Streptococcus mutans-derived NADP-dependent glyceraldehyde-3-phosphate dehydrogenase expression-enhancing plasmid pK18mobsacB-Ptuf-gapN
PCR amplification was performed with the ATCC13032 genome as a template, the P137/P138 primer pair to obtain an upstream homology arm up, the P139/P140 primer pair to obtain a promoter fragment Ptuf, the P141/P142 primer pair as a template to obtain gapN, and the ATCC13032 genome as a template, the P143/P144 primer pair to obtain a downstream homology arm dn. And (3) performing fusion PCR by using the P137/P140 primer pair and using up and Ptuf as templates to obtain fragments up-Ptuf. Fusion PCR was performed using the P137/P144 primer pair and up-Ptuf, gapN, dn as a template to obtain the full-length fragment up-Ptuf-gapN-dn. pK18mobsacB was digested with BamHI/HindIII. Assembling the digested up-Ptuf-gapN-dn and pK18mobsacB with a seamless cloning kit, and transforming the competent cells of Trans1T1 to obtain a recombinant plasmid pK18mobsacB-Ptuf-gapN.
The primers used in the above plasmid construction procedure are shown in Table 1.
TABLE 1 primer sequences
EXAMPLE 2 construction of genome-engineered Strain
1. Construction of aspartokinase-enhanced expression Strain
ATCC13032 competent cells were prepared according to the classical method of corynebacterium glutamicum (c.glutamicum Handbook, charter 23). Recombinant plasmid pK18mobsacB-Psod-lysC a1g-T311I The competent cells were transformed by electroporation and transformants were selected on selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The obtained transformant was cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. The genome of the colonies grown on the sucrose medium did not carry the inserted vector sequence. The desired mutant strain was designated SMCT106 by amplifying the desired fragment by PCR and performing nucleotide sequencing analysis, and in this strain, the start codon of the lysC gene was mutated from GTG to ATG, the amino acid 311 encoded by it was changed from threonine to isoleucine, and the promoter of the lysC gene was replaced with Psod promoter, as compared with the ATCC13032 strain.
2. Construction of homoserine dehydrogenase expression-enhancing Strain
Method for constructing Strain referring to the above 1, plasmid pK18mobsacB-PcspB-hom was prepared by starting with SMCT106 G378E Introducing into SMCT106, and performing homoserine dehydrogenase expression enhancement transformation to obtain modified strain named SMCT107, wherein hom gene of the strain is mutated compared with that of the strain SMCT106Resulting in a mutation of the encoded protein to produce G378E, while the hom gene promoter is replaced with the PcspB promoter from ATCC 14067.
3. Construction of threonine synthase expression-enhanced strains
Method for constructing Strain referring to the above 1, plasmid pK18mobsacB-Psod-thrC was prepared from SMCT107 as starting strain a1g The modified strain obtained by introducing threonine synthase expression enhancement into the SMCT107 is named as SMCT108, compared with the strain SMCT107, the thrC gene of the strain is mutated to cause the mutation of the initiation codon of the thrC gene from GTG to ATG, and meanwhile, the promoter of the thrC gene is replaced by Psod promoter.
4. Construction of pyruvate carboxylase expression-enhancing Strain
Method for constructing Strain referring to the above 1, plasmid pK18mobsacB-Psod-pyc was prepared using SMCT108 as starting strain P458S The modified strain obtained by introducing the modified strain into the SMCT108 and carrying out the enhancement of the expression of the pyruvate carboxylase is named as SMCT109, compared with the strain SMCT108, the mutation of the P458S of the coded protein is caused by the mutation of the pyc gene of the strain, and meanwhile, the promoter of the pyc gene is replaced by the Psod promoter.
5. Construction of Malate quinone oxidoreductase expression-enhanced Strain
Referring to the above 1, the modified strain obtained by introducing the plasmid pK18mobsacB-Psod-mqo into SMCT109 using SMCT109 as a starting strain and performing malate dehydrogenase expression enhancement modification was named as SMCT111, and compared with the strain SMCT109, the promoter of the mqo gene of the strain was replaced with the Psod promoter.
6. Construction of glucose-6-phosphate dehydrogenase expression-enhancing Strain
Method for constructing Strain referring to the above 1, plasmid pK18mobsacB-Psod-zwf was prepared by starting with SMCT109 A243T The strain was introduced into SMCT109, and the glucose-6-phosphate dehydrogenase was modified to enhance the expression, and the modified strain was designated as SMCT112, and compared with the strain SMCT109, the zwf gene of the strain was mutated to produce A243T mutation in the encoded protein, and the promoter of the zwf gene was replaced with the Psod promoter.
7. Construction of 6-phosphogluconate dehydrogenase expression-enhancing Strain
Referring to the above 1, the modified strain obtained by introducing the plasmid pK18mobsacB-Psod-gnd into SMCT109 using SMCT109 as a starting strain and performing the modification of enhancing the expression of 6-phosphogluconate dehydrogenase was designated as SMCT113, and the gnd gene promoter of the modified strain was replaced with Psod promoter as compared with the strain SMCT 109.
8. Construction of an NADP-dependent glyceraldehyde-3-phosphate dehydrogenase expression-enhancing Strain derived from Streptococcus mutans
Method for constructing a Strain referring to the above 1, modification of the enhanced expression of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase was performed using SMCT109 as a starting strain, plasmid pK18mobsacB-Ptuf-gapN was introduced into SMCT109, and the obtained modified strain was designated as SMCT114, and compared with the strain SMCT109, the strain was inserted with the gapN gene transcribed by Ptuf after Cgl1705 of the chromosome.
9. Construction of acetate kinase inactivated Strain
Referring to the above 1, the strains were modified by introducing plasmid pK18mobsacB- ΔackA into the starting bacteria and inactivating acetate kinase, respectively, using SMCT108, SMCT109, SMCT111, SMCT112, SMCT113 and SMCT114 as starting bacteria, and the obtained modified strains were named as SMCT119, SMCT110, SMCT115, SMCT116, SMCT117 and SMCT118, and the ackA gene was knocked out as compared with the corresponding starting bacteria.
Genotype information of the strain obtained above is shown in table 2.
TABLE 2 genotype information for strains
Strain name Genotype of the type
SMCT106 ATCC13032,P sod -lysC a1g-T311I
SMCT107 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E
SMCT108 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g
SMCT109 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S
SMCT110 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,ΔackA
SMCT111 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,P sod -mqo
SMCT112 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,P sod -zwf A243T
SMCT113 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod- pyc P458S ,P sod -gnd
SMCT114 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,Ps od -pyc P458S ,P tuf -gapN
SMCT115 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,P sod -mqo,ΔackA
SMCT116 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,P sod -zwf A243T ,ΔackA
SMCT117 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,P sod -gnd,ΔackA
SMCT118 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,P sod -pyc P458S ,P tuf -gapN,ΔackA
SMCT119 ATCC13032,P sod -lysC a1g-T311I ,P cspB -hom G378E ,P sod -thrC a1g ,ΔackA
Example 3 shake flask fermentation verification of strains
Shake flask fermentation verification was performed on each strain constructed in example 2, specifically as follows:
1. culture medium
Seed activation medium: BHI 3.7%, agar 2%, pH 7.
Seed culture medium: 5g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 16g/L of ammonium sulfate, 8g/L of urea, 10.4g/L of monopotassium phosphate, 21.4g/L of dipotassium phosphate, 5mg/L of biotin, 3g/L of magnesium sulfate, 50g/L of glucose and pH value of 7.2.
Fermentation medium: corn steep liquor 50mL/L, glucose 30g/L, ammonium sulfate 4g/L, MOPS 30g/L, monopotassium 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, and pH 7.2.
2. Engineering bacterium shake flask fermentation production of L-threonine
(1) Seed culture: the slant seed 1 of the selected strains of SMCT108, SMCT109, SMCT110, SMCT111, SMCT112, SMCT113, SMCT114, SMCT115, SMCT116, SMCT117, SMCT118 and SMCT119 is looped into a 500mL triangular flask filled with 20mL seed culture medium, and is cultured for 16h at 30 ℃ under 220r/min in an oscillating way, so as to obtain seed liquid.
(2) Fermentation culture: 2mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 20mL of a fermentation medium, and subjected to shaking culture at 33℃and 220r/min for 24 hours to obtain a fermentation broth.
(3) 1mL of the fermentation broth was centrifuged (12000 rpm,2 min), and the supernatant was collected, and the L-threonine in the fermentation broths of the engineering bacteria and the control bacteria was detected by HPLC.
The results of the shake flask fermentation of threonine are shown in Table 3.
TABLE 3 fermentation test results
As can be seen from the results in Table 3, the yields of various threonine-producing bacteria were improved by 10% to 30% after the inactivation of acetate kinase. Meanwhile, SMCT108 is taken as a starting bacterium, threonine yields of modified bacteria for further enhancing expression of propyl pyruvate carboxylase, malate quinone oxidoreductase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and mutans streptococcus NADP-dependent glyceraldehyde-3-phosphate dehydrogenase are further improved, the modification of the sites is beneficial to threonine production, meanwhile, acetate kinase is inactivated on the basis of the modification, the threonine yields are further improved, and the improvement amplitude is obviously higher than that of the inactivated acetate kinase of SMCT108, and the modification combination of the modification of the genes and the inactivation of the acetate kinase is more beneficial to threonine production.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> gallery plum blossom biotechnology development Co., ltd
<120> a recombinant microorganism for threonine production, construction method and application thereof
<130> KHP211124469.0
<160> 65
<170> SIPOSequenceListing 1.0
<210> 1
<211> 192
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60
ttcgttgcaa tatcaacaaa aaggcctatc attgggaggt gtcgcaccaa gtacttttgc 120
gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180
gattttttac cc 192
<210> 2
<211> 200
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tggccgttac cctgcgaatg tccacagggt agctggtagt ttgaaaatca acgccgttgc 60
ccttaggatt cagtaactgg cacattttgt aatgcgctag atctgtgtgc tcagtcttcc 120
aggctgctta tcacagtgaa agcaaaacca attcgtggct gcgaaagtcg tagccaccac 180
gaagtccagg aggacataca 200
<210> 3
<211> 260
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
acctgcgttt ataaagaaat gtaaacgtga tcggatcgat ataaaagaaa cagtttgtac 60
tcaggtttga agcattttct ccaattcgcc tggcaaaaat ctcaattgtc gcttacagtt 120
tttctcaacg acaggctgct aagctgctag ttcggtggcc tagtgagtgg cgtttacttg 180
gataaaagta atcccatgtc gtgatcagcc attttgggtt gtttccatag catccaaagg 240
tttcgtcttt cgatacctat 260
<210> 4
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aattcgagct cggtacccgg ggatccagcg acaggacaag cactgg 46
<210> 5
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
cccggaataa ttggcagcta tgtgcacctt tcgatctacg 40
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgtagatcga aaggtgcaca tagctgccaa ttattccggg 40
<210> 7
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tttctgtacg accagggcca tgggtaaaaa atcctttcgt a 41
<210> 8
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
tacgaaagga ttttttaccc atggccctgg tcgtacagaa a 41
<210> 9
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
tcggaacgag ggcaggtgaa ggtgatgtcg gtggtgccgt ct 42
<210> 10
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
agacggcacc accgacatca ccttcacctg ccctcgttcc ga 42
<210> 11
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gtaaaacgac ggccagtgcc aagcttagcc tggtaagagg aaacgt 46
<210> 12
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
aattcgagct cggtacccgg ggatccctgc gggcagatcc ttttga 46
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
atttctttat aaacgcaggt catatctacc aaaactacgc 40
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gcgtagtttt ggtagatatg acctgcgttt ataaagaaat 40
<210> 15
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gtatatctcc ttctgcagga ataggtatcg aaagacgaaa 40
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tttcgtcttt cgatacctat tcctgcagaa ggagatatac 40
<210> 17
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tagccaattc agccaaaacc cccacgcgat cttccacatc c 41
<210> 18
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ggatgtggaa gatcgcgtgg gggttttggc tgaattggct a 41
<210> 19
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gtaaaacgac ggccagtgcc aagcttgctg gctcttgccg tcgata 46
<210> 20
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
attcgagctc ggtacccggg gatccgccgt tgatcattgt tcttca 46
<210> 21
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
cccggaataa ttggcagcta ggatataacc ctatcccaag 40
<210> 22
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
cttgggatag ggttatatcc tagctgccaa ttattccggg 40
<210> 23
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
acgcgtcgaa atgtagtcca tgggtaaaaa atcctttcgt a 41
<210> 24
<211> 41
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tacgaaagga ttttttaccc atggactaca tttcgacgcg t 41
<210> 25
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gtaaaacgac ggccagtgcc aagcttgaat acgcggattc cctcgc 46
<210> 26
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
aattcgagct cggtacccgg ggatcctgac agttgctgat ctggct 46
<210> 27
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
cccggaataa ttggcagcta tagagtaatt attcctttca 40
<210> 28
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tgaaaggaat aattactcta tagctgccaa ttattccggg 40
<210> 29
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gaagatgtgt gagtcgacac gggtaaaaaa tcctttcgta 40
<210> 30
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
tacgaaagga ttttttaccc gtgtcgactc acacatcttc 40
<210> 31
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ggtggagcct gaaggaggtg cgagtgatcg gcaatgaatc cgg 43
<210> 32
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ccggattcat tgccgatcac tcgcacctcc ttcaggctcc acc 43
<210> 33
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gtaaaacgac ggccagtgcc aagcttcgcg gcagacggag tctggg 46
<210> 34
<211> 52
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
cgagctcggt acccggggat ccacccgggt gtggcgcgca agaagatgcc ag 52
<210> 35
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
taaatgttgt acgcggacca gaacaagatt ccgccgtgga ccacgc 46
<210> 36
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
ggcggaatct tgttctggtc cgcgtacaac atttacatac acc 43
<210> 37
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gtaaaacgac ggccagtgcc aagcttagca aggtgttaga gcaaattttc g 51
<210> 38
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
aattcgagct cggtacccgg ggatcctccg tatgctccca aacctc 46
<210> 39
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
cccggaataa ttggcagcta gttcaacttc cttttatctc 40
<210> 40
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gagataaaag gaagttgaac tagctgccaa ttattccggg 40
<210> 41
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ttcttcgggg aatctgacat gggtaaaaaa tcctttcgta 40
<210> 42
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
tacgaaagga ttttttaccc atgtcagatt ccccgaagaa 40
<210> 43
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gtaaaacgac ggccagtgcc aagcttcctt caagaactta ggggtc 46
<210> 44
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
catgattacg aattcgagct cggtacccgg ggatccgatg aggctttggc tctgcg 56
<210> 45
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
agcccggaat aattggcagc tagatggtag tgtcacgatc ct 42
<210> 46
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
aggatcgtga cactaccatc tagctgccaa ttattccggg ct 42
<210> 47
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
gggtcgtgtt tgtgctcatg ggtaaaaaat cctttcgta 39
<210> 48
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
tacgaaagga ttttttaccc atgagcacaa acacgacccc ct 42
<210> 49
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
cacccaagcc aatatcttca gtcatggtga tctggacgtg gtca 44
<210> 50
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
tgaccacgtc cagatcacca tgactgaaga tattggcttg ggtg 44
<210> 51
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
tcacgacgtt gtaaaacgac ggccagtgcc aagcttcgaa tcacgatggc gttt 54
<210> 52
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
acgaattcga gctcggtacc cggggatccc gatgtgggtg acacatgggg tgccgtca 58
<210> 53
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
ggaaacctac gaaaggattt tttacccatg actaatggag ataatctcgc acag 54
<210> 54
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
ctgtgcgaga ttatctccat tagtcatggg taaaaaatcc tttcgtaggt ttcc 54
<210> 55
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
gtaaaatcgc cactaccccc aaatggttag ctgccaatta ttccgggctt gtga 54
<210> 56
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
tcacaagccc ggaataattg gcagctaacc atttgggggt agtggcgatt ttac 54
<210> 57
<211> 57
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
gttgtaaaac gacggccagt gccaagcttc atggtgcgca gtgtggttcg tgcgacg 57
<210> 58
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
acgaattcga gctcggtacc cggggatcct gtttacctga cactcaagcc ccgtgcac 58
<210> 59
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
gccgatttca agatatctaa caagccgctt agtctgagat aatctgggtc agtggt 56
<210> 60
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
accactgacc cagattatct cagactaagc ggcttgttag atatcttgaa atcggc 56
<210> 61
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
ttgacataat ttttatattg ttttgtcatt tactgaatcc taagggcaac ggcgttga 58
<210> 62
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
tcaacgccgt tgcccttagg attcagtaaa tgacaaaaca atataaaaat tatgtcaa 58
<210> 63
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
agatgaagta ggtgggtgaa tatagctgtt atttgatatc aaatacgacg gattta 56
<210> 64
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
taaatccgtc gtatttgata tcaaataaca gctatattca cccacctact tcatct 56
<210> 65
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
ttgtaaaacg acggccagtg ccaagcttga ttggaatcgg catgggtgtt ctgcgt 56

Claims (10)

1. A modified coryneform microorganism, characterized in that the activity of acetate kinase is reduced or lost as compared to an unmodified microorganism, and the microorganism has an enhanced threonine-producing ability as compared to an unmodified microorganism.
2. The microorganism of claim 1, wherein the reduction or loss of acetate kinase activity in the microorganism is achieved by reducing expression of a gene encoding acetate kinase or knocking out an endogenous gene encoding acetate kinase.
3. The microorganism of claim 2, wherein the expression of the gene encoding acetate kinase is reduced or the endogenous gene encoding acetate kinase is knocked out by mutagenesis, site-directed mutagenesis or homologous recombination.
4. The microorganism of claim 1, wherein the microorganism has increased pyruvate carboxylase activity and/or is free from feedback inhibition compared to an unmodified microorganism;
preferably, the microorganism has an enhanced and/or deregulated activity of any one or more of the following enzymes (1) to (4) compared to the unmodified microorganism:
(1) Malate quinone oxidoreductase;
(2) Glucose-6-phosphate dehydrogenase;
(3) 6-phosphogluconate dehydrogenase;
(4) NADP dependent glyceraldehyde-3-phosphate dehydrogenase.
5. The microorganism according to any one of claims 1 to 4, wherein the activity of an enzyme associated with the threonine synthesis pathway in vivo is enhanced and/or feedback inhibition is released compared to an unmodified microorganism;
wherein the enzyme related to threonine synthesis pathway is at least one selected from aspartokinase, homoserine dehydrogenase and threonine synthase.
6. The microorganism according to claim 5, wherein the microorganism is any one of the following (1) to (6):
(1) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase and/or threonine synthase activity;
(2) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase and/or pyruvate carboxylase activity;
(3) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or malate quinone oxidoreductase activity;
(4) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or glucose-6-phosphate dehydrogenase activity;
(5) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or 6-phosphogluconate dehydrogenase activity;
(6) a microorganism having reduced or lost acetate kinase activity and enhanced and/or deregulated activity of aspartokinase, homoserine dehydrogenase, threonine synthase, pyruvate carboxylase and/or NADP-dependent glyceraldehyde-3-phosphate dehydrogenase derived from streptococcus mutans.
7. The microorganism according to any of claims 4 to 6, wherein the enhancement of the activity of the enzyme is achieved by a member selected from the group consisting of 1) to 6), or optionally a combination of:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by altering the nucleotide sequence encoding the enzyme.
8. The microorganism according to any of claims 1 to 7, characterized in that the microorganism is corynebacterium glutamicum (Corynebacterium glutamicum).
9. A method for constructing a threonine-producing strain, the method comprising:
A. weakening a gene encoding acetate kinase in coryneform bacteria having amino acid productivity to obtain a gene-weakened strain; the attenuation comprises knocking out or reducing the expression of an acetate kinase encoding gene; and/or
B. Enhancing the activity of pyruvate carboxylase and/or releasing its feedback inhibition; and/or
C. Enhancing the activity of and/or releasing the feedback inhibition of any one or more of the following enzymes (1) to (4):
(1) Malate quinone oxidoreductase;
(2) Glucose-6-phosphate dehydrogenase;
(3) 6-phosphogluconate dehydrogenase;
(4) NADP dependent glyceraldehyde-3-phosphate dehydrogenase;
and/or
D. Enhancing the activity of and/or releasing feedback inhibition by an enzyme associated with a threonine synthesis pathway selected from at least one of aspartokinase, homoserine dehydrogenase, threonine synthase;
the enhanced pathway is selected from the following 1) to 6), or an optional combination:
1) Enhanced by introducing a plasmid having a gene encoding the enzyme;
2) Enhancement by increasing the copy number of the gene encoding the enzyme on the chromosome;
3) Enhanced by altering the promoter sequence of the gene encoding the enzyme on the chromosome;
4) Enhanced by operably linking a strong promoter to a gene encoding said enzyme;
5) Enhancement by modification of the amino acid sequence of the enzyme;
6) Enhanced by altering the nucleotide sequence encoding the enzyme.
10. A method for producing threonine, characterized in that the method comprises the steps of:
a) Culturing the microorganism of any one of claims 1-8 to obtain a culture of the microorganism;
b) Collecting the threonine produced from the culture obtained in step a).
CN202210118708.1A 2022-02-08 2022-02-08 Recombinant microorganism for producing threonine and construction method and application thereof Pending CN116606786A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202210118708.1A CN116606786A (en) 2022-02-08 2022-02-08 Recombinant microorganism for producing threonine and construction method and application thereof
PCT/CN2022/143101 WO2023151411A1 (en) 2022-02-08 2022-12-29 Recombinant microorganism for producing threonine, and construction method therefor and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210118708.1A CN116606786A (en) 2022-02-08 2022-02-08 Recombinant microorganism for producing threonine and construction method and application thereof

Publications (1)

Publication Number Publication Date
CN116606786A true CN116606786A (en) 2023-08-18

Family

ID=87563556

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210118708.1A Pending CN116606786A (en) 2022-02-08 2022-02-08 Recombinant microorganism for producing threonine and construction method and application thereof

Country Status (2)

Country Link
CN (1) CN116606786A (en)
WO (1) WO2023151411A1 (en)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150131880A (en) * 2014-05-16 2015-11-25 삼성전자주식회사 A microorganism having enhanced productivity of succinate and a method for producing succinate using the same
CN113322218B (en) * 2020-02-28 2022-11-22 廊坊梅花生物技术开发有限公司 Recombinant corynebacterium glutamicum and method for producing L-threonine

Also Published As

Publication number Publication date
WO2023151411A1 (en) 2023-08-17

Similar Documents

Publication Publication Date Title
JP6679803B2 (en) New promoter and its use
CN113322218B (en) Recombinant corynebacterium glutamicum and method for producing L-threonine
JPH10165180A (en) Production of l-lysine
KR101285945B1 (en) Corynebacterium sp. Having Improved L-lysine Production and Process for Preparing the L-lysine Employing the Same
KR101429815B1 (en) Corynebacterium sp. microorganism having enhanced L-threonine productivity by regulation of GntK activity and a method of producing L-threonine using the same
JP2009507505A (en) Amino acid production method using microorganisms
KR101485222B1 (en) Corynebacterium sp. microorganism having enhanced L-threonine productivity by regulation of dapA activity and a method of producing L-threonine using the same
KR100564805B1 (en) Corynebaterium sp. transformed with a heterogenous fructokinase gene and method for producing L-lysine using the same
CN116606786A (en) Recombinant microorganism for producing threonine and construction method and application thereof
CN116555136A (en) Modified corynebacterium microorganism and construction method and application thereof
CN116555131A (en) Recombinant microorganism and construction method and application thereof
CN116622596A (en) Modified corynebacterium microorganism, construction method thereof and application thereof in threonine production
CN116606785A (en) Modified corynebacterium microorganism and application and construction method thereof
CN116536227A (en) Threonine-producing modified corynebacterium microorganism, construction method and application thereof
CN116536226A (en) Construction method of threonine producing engineering bacteria
KR102377745B1 (en) Novel promoter and use thereof
CN116555251A (en) Recombinant microorganism for producing threonine and application thereof
CN116622599A (en) Construction method of high-threonine-producing strain
CN116555132A (en) Modified corynebacterium microorganism and application and construction method for producing threonine thereof
WO2023151406A1 (en) Method for constructing threonine-producing strain
CN116555134A (en) Construction method of threonine producing strain
CN116555365A (en) Modified corynebacterium microorganism, construction method and application thereof
WO2023151407A1 (en) Construction method for threonine-producing strain
CN116622597A (en) Construction method of engineering bacteria for high threonine production
CN116536310A (en) Promoter, threonine-producing recombinant microorganism and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination