CN116240185B - Homoserine dehydrogenase mutant and application thereof in production of L-homoserine - Google Patents

Homoserine dehydrogenase mutant and application thereof in production of L-homoserine Download PDF

Info

Publication number
CN116240185B
CN116240185B CN202310394817.0A CN202310394817A CN116240185B CN 116240185 B CN116240185 B CN 116240185B CN 202310394817 A CN202310394817 A CN 202310394817A CN 116240185 B CN116240185 B CN 116240185B
Authority
CN
China
Prior art keywords
homoserine
mutant
corynebacterium glutamicum
homoserine dehydrogenase
coding gene
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.)
Active
Application number
CN202310394817.0A
Other languages
Chinese (zh)
Other versions
CN116240185A (en
Inventor
刘君
魏亮
徐宁
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.)
Tianjin Institute of Industrial Biotechnology of CAS
Original Assignee
Tianjin Institute of Industrial Biotechnology of CAS
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 Tianjin Institute of Industrial Biotechnology of CAS filed Critical Tianjin Institute of Industrial Biotechnology of CAS
Priority to CN202310394817.0A priority Critical patent/CN116240185B/en
Publication of CN116240185A publication Critical patent/CN116240185A/en
Application granted granted Critical
Publication of CN116240185B publication Critical patent/CN116240185B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • 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
    • 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
    • C12N9/10—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
    • 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
    • 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
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • 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
    • C12P13/12—Methionine; Cysteine; Cystine
    • 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
    • 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
    • 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
    • C12R2001/265—Micrococcus
    • C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum

Landscapes

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

Abstract

The invention belongs to the field of bioengineering, and particularly discloses a homoserine dehydrogenase mutant with improved enzyme activity and application thereof in preparation of L-homoserine or derivatives thereof. The amino acid sequence of the mutant has one mutation type in T186A, N283K, A137T/I188V compared with the wild-type homoserine dehydrogenase derived from Brevibacterium distach (Brachypodium distachyon). In a specific embodiment, the enzyme activity of the mutant is increased by 1.26-1.35 times compared with the wild type by the assay of the enzyme activity of the induced expression purification. Therefore, the beneficial mutant provided by the invention can lay a good foundation for the industrialized high-efficiency production of L-homoserine and downstream metabolites.

Description

Homoserine dehydrogenase mutant and application thereof in production of L-homoserine
Technical Field
The invention belongs to the field of bioengineering, and particularly discloses a homoserine dehydrogenase mutant with improved enzyme activity and application thereof in preparation of L-homoserine or derivatives thereof.
Background
L-homoserine is a small number of non-protein amino acids which are not produced on a large scale, is an unnecessary chiral amino acid, and is an important precursor of amino acids of the aspartic acid family such as threonine, methionine and lysine. Has important application in pharmacy, cosmetics, agriculture and spice. L-homoserine has been considered as a synthetic precursor of L-glufosinate due to its chemical skeleton identical to that of the pesticide L-glufosinate, and has better economical efficiency than the chemical method.
In addition, L-homoserine and its derivatives are potential platforms for the production of many important compounds, including 1, 4-butanediol, isobutanol, 2, 4-dihydroxybutyrate, 1, 3-propanediol.
In microorganisms such as E.coli and Corynebacterium glutamicum, L-homoserine biosynthesis starts from the aspartate pathway. Glucose passes through glycolysis pathway, pentose phosphate pathway, TCA cycle, etc. to produce oxaloacetate, which then enters the aspartic acid bypass pathway. The aspartate precursors then finally produce L-homoserine by catalysis of aspartokinase (Aspartokinase, EC 2.7.2.4), aspartate semialdehyde dehydrogenase (ASPARTATE-SEMIALDEHYDE DEHYDROGENASE, EC 1.2.1.11), homoserine dehydrogenase (Homoserine dehydrogenase, EC 1.1.1.3), and the like. Among them, homoserine dehydrogenase is a common key enzyme in the synthesis pathway of amino acids of the aspartate family such as homoserine, threonine, lysine, isoleucine, methionine, etc., and its catalytic activity is often subject to feedback inhibition or repression by end products or intermediate metabolites.
Therefore, the key enzyme mutant with excellent catalytic performance is obtained by screening or molecular modification of homoserine dehydrogenase, and has very important significance for efficient production of amino acids in the aspartate family.
Disclosure of Invention
Based on the above-mentioned needs, it is a primary object of the present invention to provide homoserine dehydrogenase mutants which have improved catalytic properties and are useful for microbial fermentation production of metabolites such as L-homoserine.
The invention is realized by adopting the following technical thought that a B.discochyon wild Hom fragment is used as a template, an error-prone PCR random mutation method is utilized to obtain a Hom coding gene mutation library, and a vector construction strategy based on Goldengate technology is adopted to subclone the Hom coding gene mutation library onto an expression vector, so that a recombinant plasmid library containing homoserine dehydrogenase Hom mutant coding genes is obtained. Then the recombinant plasmid library is introduced into BL21 (DE 3) competent cells, and mutants with improved enzyme activity are finally obtained through preliminary screening of a 96-well plate, secondary screening of a test tube and shaking of a bottle, and mutation sites of the mutants are respectively T186A, N283K, A T/I188V.
The invention provides a homoserine dehydrogenase mutant derived from wild-type brachypodium distachyon (Brachypodiumdistachyon), which is characterized in that, relative to the amino acid sequence shown in SEQ ID No.2, only the T186 th site is mutated from threonine T to alanine A or the 283 th site is mutated from asparagine N to one site mutation in lysine K relative to the amino acid sequence shown in SEQ ID No. 2; there is only a combination of mutations from alanine a to threonine T at position 137 and from isoleucine I to valine V at position 188. The invention further provides a coding gene of the homoserine dehydrogenase mutant. In a preferred embodiment, the nucleotide sequence of the homoserine dehydrogenase Hom encoding gene is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1. The invention also provides an expression vector and a host cell containing the homoserine dehydrogenase Hom mutant coding gene. In a preferred embodiment, the expression vector includes, but is not limited to, pEC-K99E, which is produced by constructing a recombinant plasmid containing the gene encoding the Hom mutant and introducing it into a chassis cell of a microorganism for fermentative production of L-homoserine, downstream derivatives, and the like. In a specific embodiment, the microbial chassis cell may be selected from the group consisting of corynebacterium, enterobacter, preferably escherichia coli (ESCHERICHIA COLI), corynebacterium glutamicum (Corynebacterium glutamicum).
The invention also provides application of the homoserine dehydrogenase mutant in fermentation production of L-homoserine and derivatives. An engineering bacterium chassis with a certain L-homoserine production capacity is selected for application performance test, and in a specific embodiment, the engineering bacterium Cg-HSE is characterized in that a self-high serine degradation pathway gene thrB is knocked out from corynebacterium glutamicum Corynebacterium glutamicumATCC 13032, and an aspartokinase gene LysC is overexpressed.
The present invention provides a process for producing L-homoserine or a derivative thereof, comprising fermenting a recombinant microorganism containing the coding gene to produce L-homoserine and/or a derivative thereof, wherein the microorganism is produced by using a chassis engineering bacterium having L-homoserine producing ability as a starting bacterium.
Preferably, the microorganism is Escherichia coli or Corynebacterium glutamicum having L-homoserine productivity, more preferably Escherichia coli or Corynebacterium glutamicum having L-homoserine productivity.
Further preferably, the corynebacterium glutamicum has knocked out the self-homoserine degradation pathway gene thrB while overexpressing aspartokinase LysC; particularly preferably, the starting corynebacterium glutamicum is Corynebacterium glutamicumATCC to 13032.
The invention has the beneficial effects that the homoserine dehydrogenase mutant with the catalytic activity improved to different degrees is obtained by carrying out mutation and screening on the homoserine dehydrogenase encoding gene Hom from B.discochyon. Therefore, the homoserine dehydrogenase mutant provided by the invention can lay a good foundation for producing L-homoserine and other downstream aspartic acid family metabolites by efficient fermentation, and has better industrial application prospects compared with non-mutant homoserine dehydrogenase.
Drawings
FIG. 1, homoserine dehydrogenase BdHSD and mutant enzyme activity assays.
FIG. 2 effect of expression of different homoserine dehydrogenase mutants on homoserine production.
Description of the embodiments
For the purpose of providing a better understanding of the invention, reference should be made to the following detailed description of the invention, taken in conjunction with the accompanying drawings, which are not to be taken in a limiting sense. The experimental methods used in the examples, unless otherwise specified, are conventional methods well known to those skilled in the art. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 construction of Hom mutant library of homoserine dehydrogenase
Coding sequence of homoserine dehydrogenase Hom gene of Breviburnum (SEQ ID NO: 1):
ATGCCCCCACAGGCTCAAGCAAAATCAGTATTGCCTGTAGTACTACTTGGCTGCGGCGGTGTTGGTCGCCACCTGCTGCGCCACATCGTGTCTTGTCGTCCGCTGCATGCAAATCAGGGCGTGAGCATCCGCGTGGTCGGCGTCAGCGATAGTTCCAGCTTGTTGTTAGCGGCGGACGACCTCAGAGCGGGCGCGGGTCTGGACGACGCGCTGCTGGGAGATTTATGCGCAGCAAAGTCGGCGGGTTCCCCGCTGTCCTCGCTGCTGGCGCGTGGTCAGTGTCAGCTGTTCAACAAGCCGGAGGCAATGTCTAAAGTGATCGACGCTGCGACGATGCTGGGCCGCACCACCGGTCTGGTTCTGGTGGACTGTTCTGCCACCTATGATACCATCGGTGTGTTAAAAGACGCAGTTGACCAGGGTTGCTGCGTTGTGCTGGCGAACAAGAAACCGCTGACGAGCAGCTACGAAGATTTCCAAAAGCTGACGAGCAATTTTCGTCAAATTCGATTTGAGTCCACCGTTGGTGCGGGTTTACCAGTTATTGCGAGCGTTACCCGTATCATCGCCAGCGGCGACCCGATTTCACGTATTGTGGGCAGCCTCAGCGGCACCCTGGGCTACGTTATGTCCGAGCTGGAGGATGGTAAACGCTTCAGCGAGGTGGTGAAAACCGCAAAGAGCTTGGGTTACACCGAGCCGGACCCGCGTGATGACTTGTCAGGGATGGATGTTGCACGCAAAGCCCTTATTcTGGCTCGTCTGCTTGGCCGTCAGATTTCCATGGAAGACATCAACGTGGAATCCCTGTACCCGAGCGAACTCGGTCCGGAAGTCATCAGCACCAATGACTTCCTGGAATCCGGCTTGGCTCAACTGGATGAATCTATTGAGGAACGTGTGAAGGCTGCTAGCCTGCGTGGTAATGTTCTGCGTTATGTGGGTGTCATCGAGAGCACGGGCTGCCAGGTTGGCTTGCAAGAAGTTCCGAAAGATAGCGCGTTGGGTCGTCTGATGGGTTCCGACAACGTGGTGGAAATTTATAGCCGTTGCTACGAGAACAGCCCGCTGGTTATCCAGGGTGCGGGTGCGGGCAACGACACTACTGCCGCGGGCGTCCTGGCTGATATCGTTGATCTGCAAGATTTGTTTCAAAAGCGCGTT.
which encodes the wild-type amino acid sequence (SEQ ID NO: 2):
MPPQAQAKSVLPVVLLGCGGVGRHLLRHIVSCRPLHANQGVSIRVVGVSDSSSLLLAADDLRAGAGLDDALLGDLCAAKSAGSPLSSLLARGQCQLFNKPEAMSKVIDAATMLGRTTGLVLVDCSATYDTIGVLKDAVDQGCCVVLANKKPLTSSYEDFQKLTSNFRQIRFESTVGAGLPVIASVTRIIASGDPISRIVGSLSGTLGYVMSELEDGKRFSEVVKTAKSLGYTEPDPRDDLSGMDVARKALILARLLGRQISMEDINVESLYPSELGPEVISTNDFLESGLAQLDESIEERVKAASLRGNVLRYVGVIESTGCQVGLQEVPKDSALGRLMGSDNVVEIYSRCYENSPLVIQGAGAGNDTTAAGVLADIVDLQDLFQKRV.
The invention adopts EASYTAQ DNA polymerase (Beijing TransGen Biotech, china) with low fidelity, takes wild B.discochyon genome as a template, and utilizes primers P1 (5'-CACCAGGTCTCAGGAGATATACATATGCCCCCACAGGCTCAAGCAA-3') and P2 (5'-CACCAGGTCTCAGGTGAACGCGCTTTTGAAACAAATCTTGCAGATC-3') to obtain the Hom coding gene mutation library by an error-prone PCR method. The fidelity in the PCR amplification process is further reduced by adding magnesium ions and manganese ions with certain concentration into a PCR reaction system, and the obtained Hom coding gene is controlled to contain 2-3 point mutations. The error-prone PCR system adopted by the invention is as follows: 5. mu.L of 10X EasyTaq buffer, 0.2. Mu.M upstream primer P1, 0.2. Mu.M upstream primer P2, 200. Mu.M dNTPs, 0.8. 0.8 mM MnCl 2、6 mM MgSO4, 50 ng template DNA, 1. Mu. LEASYTAQ DNA polymerase, and sterile water was added to make up to a 50. Mu.L system. The PCR reaction procedure was: pre-denaturation at 95 ℃ 3 min; denaturation at 95℃of 30 s; annealing at 58 ℃ for 30 s; extending at 72 ℃ for 2 min times, and circulating for 35 times; extending at 72deg.C for 5 min, and preserving at 4deg.C.
The High-FIDELITY DNA polymerase of Phusion with High fidelity is adopted, the pET21b plasmid is used as a template, and the primers P3 (5'-CACCAGGTCTCACACCACCACCACCACCACTGAGATC-3') and P4 (5'-CACCAGGTCTCACTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTG-3') are utilized to obtain the pET21b plasmid skeleton by a common PCR method. 10. mu.L of 5 XPhusion HF buffer, 0.5. Mu.M upstream primer P3, 0.5. Mu.M upstream primer P4, 200. Mu.M dNTPs, 3% DMSO, 50 ng template DNA, 0.5. Mu.L Phusion High-FIDELITY DNA polymerase (Thermo Scientific, USA), and sterile water was added to make up to a 50. Mu.L system. The PCR reaction procedure was: pre-denaturation at 98 ℃ of 1 min; denaturation at 98℃of 10 s; annealing at 62 ℃ for 20 s; extending at 72 ℃ for 3 min times, and circulating for 35 times; extending at 72deg.C for 5 min, and preserving at 4deg.C.
And connecting the Hom coding gene mutation library obtained by the PCR with a pET21b plasmid skeleton based on Goldengate method to obtain recombinant expression plasmid pET21b-Hom mut containing different mutants of the Hom coding gene. Goldengate connection system is: t4 DNA LIGASE buffer 1.5. Mu.L, T4 DNA LIGASE. Mu.L, bsaI 1. Mu.L, 10 XBSA 1.5. Mu.L, plasmid backbone, fragments (molar ratio 1:1, total 10. Mu.L) in total 15. Mu.L. PCR ligation conditions: 37℃for 3min, 22℃for 4 min (40 cycles); 30min at 22 ℃;80 ℃ for 5min; 10min at 4 ℃. And then the connection system is introduced into competent cells of escherichia coli DH5 alpha according to a conventional escherichia coli heat shock conversion method to obtain a recombinant plasmid library.
Example 2 screening identification of a homoserine dehydrogenase variant library
Transformed into E.coli BL21 (DE 3). And (3) picking single colonies, culturing overnight at 37 ℃ in a 96-well plate, transferring to a new LB culture medium, adding 0.4 mmol/L IPTG, and inducing for 16-20 h at 16 ℃. After centrifugation, adding lysis buffer containing lysozyme for incubation for 3 hours, and centrifuging and collecting supernatant to obtain crude enzyme solution. The enzyme mutants were subjected to high throughput evaluation and screening by measuring the rate of change of NADPH content in the reaction system. Through multiple rounds of screening, 10 mutants with significantly improved crude enzyme activity were obtained in total. The results of subsequent protein purification and enzyme activity measurement of 10 mutants show that mut-5, mut-8 and mut-10 show higher enzyme activity, and the specific enzyme activity reaches 10.3U/mg, 9.7U/mg and 9.6U/mg, which are respectively improved by 35.5%,27.6% and 26.3% compared with the wild type.
The LB medium comprises the following components: 1% yeast extract, 2% tryptone, 1% NaCl.
Example 3 method for measuring enzyme Activity of homoserine dehydrogenase mutant
The cells inducing expression were collected, the medium was removed by centrifugation, and the cells were resuspended in pellet using lysis buffer pre-chilled at 10 mL (20: 20 mM Na 2HPO3, 200 mM NaCl,pH 7.5). Using an ultrasonic cytoclasis instrument to break cells, setting 200W power, and carrying out ultrasonic treatment on the cells by 2 s intermittent 1 s and min; the supernatant was then collected for subsequent protein purification and enzyme activity assays by centrifugation at 8000 Xg in a high-speed cryocentrifuge, 10 min.
Homoserine dehydrogenase activity was determined by detecting the change in absorbance at 340 nm, with a molar extinction coefficient of 6220M −1 cm−1. The reaction system was 100 mmol/L Tris buffer (pH 7.5), 80 mmol/L L-Hse,400 mmol/L NaCL and 1 mmol/L NAD (P) +. The enzyme activity unit of homoserine dehydrogenase is defined as the amount of enzyme consumed per 1. Mu. Mol of NAD (P) H produced per unit time.
As a result of screening, determination and analysis of the activity of homoserine dehydrogenase, the specific enzyme activities of the T186A, N K mutant and the A137T/I188V mutant reach 10.3U/mg, 9.7U/mg and 9.6U/mg, which are respectively improved by 35.5%,27.6% and 26.3% compared with the wild type (shown in figure 1).
Example 4 expression of homoserine dehydrogenase mutant engineering bacteria to increase fermentation production of L-homoserine
Based on the homoserine dehydrogenase mutant obtained in example 3, a microbial cell factory was constructed for fermentative production of L-homoserine and downstream derived products. The preferred plasmid in this example is a E.coli-Corynebacterium glutamicum pEC-XK99E shuttle inducible expression vector, which itself contains a strong promoter (including the operator lacO) that when added to an inducer such as IPTG or lactose will cause the repressor protein to leave the operator and initiate gene expression. The Hom coding gene mutant fragment obtained by PCR is connected with pEC-XK99E plasmid skeleton based on Goldengate method to construct expression plasmids pEC-XK99E-WT, pEC-XK99E-T186A, pEC-XK99E-N283K and pEC-XK99E-A137T/I188V. The expression plasmids were then respectively electrotransformed into Corynebacterium glutamicum H101, and homoserine producing strains H101-BdHSDwt, H101-BdHSD1, H101-BdHSD2, and H101-BdHSD3 were constructed. In a specific embodiment, the engineering bacterium H101 is characterized in that the self-homoserine degradation pathway gene thrB is knocked out in corynebacterium glutamicum Corynebacterium glutamicumATCC 13032, and aspartokinase LysC is overexpressed.
The engineering strains obtained above were inoculated into triangular flasks containing 20 mL LBHIS medium, respectively, and cultured for 16: 16 h to mid-log phase. Cells were collected by centrifugation, suspended in fresh fermentation medium, and inoculated into 500 mL flasks containing 25 mL fermentation medium with an initial OD 600 of 1.0. The LBHIS medium is: yeast powder 5 g/L, peptone 10 g/L, sodium chloride 10 g/L, brain heart infusion 18.5 g/L, sorbitol 91 g/L.
Seed culture medium: 30 g/L of corn steep liquor, 5 g/L of urea, 0.5 g/L of magnesium sulfate, 5 g/L of ammonium sulfate, 0.25 g/L of monopotassium phosphate, 25 g/L of glucose and 0.4 g/L of threonine.
Fermentation medium: corn steep liquor 70 g/L, feSO 4-citrate solution 0.55 ml/L (FeSO 4-7H2O 20 g/L, citric acid 18.14 g/L), ammonium sulfate 35 g/L, magnesium sulfate 0.5 g/L,85% phosphoric acid 0.225 ml/L, potassium dihydrogen phosphate 0.25 g/L, dextrose monohydrate 50 g/L, vitamin solution 7 ml/L (containing biotin 300 mg/L, vitamin B1500 mg/L, calcium pantothenate salt 2 g/L, nicotinamide 600 mg/L), urea 5 g/L, threonine 0.4 g/L.
As shown in FIG. 2, after 36H fermentation culture, the homoserine yield of the strain expressing the BdHSD mutant is obviously improved, wherein the L-homoserine yield of the recombinant strain H101-BdHSD1 containing the BdHSD T A mutant is highest and reaches 3.2 g/L, and the L-homoserine content of the strain is improved by 28% compared with that of the control strain H101-BdHSDwt. Compared with the homoserine dehydrogenase without mutation, the homoserine dehydrogenase Hom mutant can be expressed in chassis engineering bacteria, so that the capacity of producing L-homoserine by fermenting the strain can be obviously improved.

Claims (11)

1. A homoserine dehydrogenase Hom mutant derived from brachypodium distachyon (Brachypodium distachyon), characterized in that the amino acid sequence of the mutant is mutated from threonine T to alanine a at position 186 only, relative to the amino acid sequence shown in SEQ ID No. 2; or only the 283 th asparagine N to lysine K; or there is only a combined mutation from alanine A to threonine T at position 137 and from isoleucine I to valine V at position 188.
2. The homoserine dehydrogenase Hom mutant encoding gene according to claim 1.
3. The coding gene according to claim 2, wherein the nucleotide sequence is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1.
4. An expression vector comprising the coding gene according to claim 2 or 3.
5. A recombinant microorganism comprising the coding gene according to claim 2 or 3.
6. The recombinant microorganism according to claim 5, which is E.coli or Corynebacterium glutamicum.
7. The use of homoserine dehydrogenase Hom mutant according to claim 1 or its coding gene for the preparation of L-homoserine.
8. A process for producing L-homoserine, which comprises fermenting a recombinant microorganism comprising the coding gene according to claim 2 or 3, wherein the microorganism comprises a chassis-engineering bacterium having L-homoserine-producing ability as a starting bacterium.
9. The method according to claim 8, wherein the microorganism is Escherichia coli or Corynebacterium glutamicum having L-homoserine producing ability.
10. The method according to claim 9, wherein the corynebacterium glutamicum is a starting corynebacterium glutamicum from which the native homoserine degradation pathway gene thrB has been knocked out, while overexpressing aspartokinase LysC.
11. The method of claim 10, wherein the starting corynebacterium glutamicum is Corynebacterium glutamicum ATCC 13032.
CN202310394817.0A 2023-04-13 2023-04-13 Homoserine dehydrogenase mutant and application thereof in production of L-homoserine Active CN116240185B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310394817.0A CN116240185B (en) 2023-04-13 2023-04-13 Homoserine dehydrogenase mutant and application thereof in production of L-homoserine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310394817.0A CN116240185B (en) 2023-04-13 2023-04-13 Homoserine dehydrogenase mutant and application thereof in production of L-homoserine

Publications (2)

Publication Number Publication Date
CN116240185A CN116240185A (en) 2023-06-09
CN116240185B true CN116240185B (en) 2024-06-14

Family

ID=86628016

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310394817.0A Active CN116240185B (en) 2023-04-13 2023-04-13 Homoserine dehydrogenase mutant and application thereof in production of L-homoserine

Country Status (1)

Country Link
CN (1) CN116240185B (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR122021002092B1 (en) * 2012-05-28 2022-11-29 Evogene Ltd METHOD FOR INCREASE GROWTH RATE, BIOMASS, VIGOR, NITROGEN USE EFFICIENCY, AND/OR ABIOTICAL STRESS TOLERANCE, AND/OR REDUCED TIME TO EMERGENCY OF INFLORESCENCE OF A PLANT
CN106978405B (en) * 2016-01-18 2021-03-12 中国科学院天津工业生物技术研究所 Aspartokinase/homoserine dehydrogenase mutant and application thereof
CN112779204B (en) * 2021-01-26 2022-08-09 天津科技大学 Genetically engineered bacterium for producing L-homoserine and application thereof
CN112501144B (en) * 2021-02-02 2021-04-20 中国科学院天津工业生物技术研究所 Homoserine acetyltransferase mutant and application thereof in production of O-acetylhomoserine
CN115725537A (en) * 2022-07-11 2023-03-03 中国科学院天津工业生物技术研究所 Aspartokinase mutant and application thereof in producing L-homoserine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
微生物产赖氨酸的研究进展;刘晓飞;高学军;刘营;敖金霞;王青竹;;东北农业大学学报;20100125(第01期);全文 *
谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除;饶志明等;中国生物工程杂志;20061231;第27卷(第1期);第59-63页 *

Also Published As

Publication number Publication date
CN116240185A (en) 2023-06-09

Similar Documents

Publication Publication Date Title
Zhang et al. Production of L-alanine by metabolically engineered Escherichia coli
US5000000A (en) Ethanol production by Escherichia coli strains co-expressing Zymomonas PDC and ADH genes
CN107690482B (en) Modified microorganism for optimized production of 2,4-dihydroxybutyric acid
EP3006556B1 (en) Recombinant escherichia coli for producing succinic acid and application thereof
JP5264778B2 (en) Method for producing fermentation products from carbon sources containing glycerol using corynebacteria
KR101083136B1 (en) Microorganisms for producing l-amino acids and process for producing l-amino acids using them
US8900838B2 (en) Method for the preparation of 1,3-propanediol from sucrose
Neuner et al. Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering
US10415068B2 (en) Microorganism for production of putrescine and methods for production of putrescine using the same
CN115725537A (en) Aspartokinase mutant and application thereof in producing L-homoserine
DK2430152T3 (en) A microorganism with increased L-lysine productivity and method for producing L-lysine using the same
KR101285945B1 (en) Corynebacterium sp. Having Improved L-lysine Production and Process for Preparing the L-lysine Employing the Same
CN116240185B (en) Homoserine dehydrogenase mutant and application thereof in production of L-homoserine
US9605280B2 (en) Escherichia coli containing mutated lpdA gene and application thereof
CN113788881B (en) Cysteine transporter mutant and application thereof in production of L-cysteine
EP2430152B1 (en) Microorganism with enhanced l-lysine productivity and method for producing l-lysine using the same
US20230392112A1 (en) Genetically modified microorganism for producing 3-hydroxyadipic acid and/or alpha-hydromuconic acid, and method for producing chemical product
CN113122563A (en) Method for constructing R-3-aminobutyric acid production strain
CN117645985B (en) Acetylglucosamine-6 phosphate phosphatase mutant and application thereof
CN116376857A (en) 3-phosphoglycerate dehydrogenase mutant with improved enzyme activity and thermal stability
CN116355060A (en) Homoserine transporter mutant and preparation method and application thereof
CN113278567A (en) Method for improving strain to promote recombinant protein production
松原充 Fermentative production of 1-propanol from biomass using recombinant Escherichia coli
WO2023142881A1 (en) Method for constructing threonine-producing strain
CN117660269A (en) Recombinant escherichia coli for producing malonic acid as well as preparation method 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
GR01 Patent grant
GR01 Patent grant