Homoserine dehydrogenase mutant and application thereof

Abstract

The invention discloses a homoserine dehydrogenase mutant and application of the homoserine dehydrogenase mutant. Compared with wild homoserine dehydrogenase Hom from corynebacterium glutamicum, the amino acid sequence of the mutant has one mutation type of A381D, A381L, A381I, A381P, A381V, A384R, A384D, I397C, I397D, I397G, I397S, I397R, I397V and I397A. Through enzyme activity determination and analysis of induced expression and purification, the enzyme activity of the mutant is remarkably improved compared with that of a wild control. Meanwhile, the feedback inhibition of L-threonine and L-isoleucine can be efficiently relieved. The homoserine dehydrogenase mutant, the coding gene of the homoserine dehydrogenase mutant and the host cell containing the coding gene provided by the invention can be industrially applied to produce L-homoserine and derived amino acids thereof.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and bioengineering, and particularly relates to a homoserine dehydrogenase mutant and application thereof in the production of homoserine or L-amino acid derived from homoserine. Background Art

[0002] In Corynebacterium glutamicum, homoserine dehydrogenase (EC: 1.1.1.3), encoded by the hom gene, catalyzes the dehydrogenation of aspartate semialdehyde to generate L-homoserine, which is then converted into amino acids such as L-threonine, L-isoleucine, and L-methionine through a series of enzyme reactions. It is known in the prior art that homoserine dehydrogenase is subject to non-competitive feedback inhibition by L-threonine and L-isoleucine, and the removal of feedback inhibition is necessary to achieve high yields of L-homoserine or amino acids derived from homoserine.

[0003] The prior art has reported a number of homoserine dehydrogenase mutants that can relieve L-threonine feedback inhibition, such as Eikmanns et al. reported that the 378th glutamic acid of Hom was mutated to glycine (Eikmanns, et al. Appl. Microbial Biotechnol. 1991, 34: 617-622); Archer et al. reported that the C-terminal frameshift mutation of Hom can also relieve feedback inhibition (Archer JA, et al. Gene. 1991, 107: 53-59). Korean CJ Corporation reported in CN111601886B and CN110945121B that the 407th amino acid of Hom was replaced by histidine and the 285th amino acid was replaced by isoleucine.

[0004] Therefore, obtaining homoserine dehydrogenase mutants that can effectively relieve the feedback inhibition of L-threonine and L-isoleucine and have high enzyme activity by screening or molecularly modifying homoserine dehydrogenase is still of great significance for the efficient production of L-homoserine or its derived amino acids by Corynebacterium glutamicum. Summary of the invention

[0005] The purpose of the present invention is to provide a homoserine dehydrogenase mutant in view of the current status and shortcomings of the prior art, so that the feedback inhibition of L-threonine and L-isoleucine is relieved, the catalytic performance is improved, and the microbial fermentation production of L-homoserine or its derived amino acids is facilitated.

[0006] The present invention is realized by the following technical ideas: using homoserine dehydrogenase Hom of Corynebacterium glutamicum as a template, overexpressing a homoserine dehydrogenase gene hom and a downstream homoserine kinase thrB gene of the same operon on a plasmid, amplifying and constructing a mutant plasmid library through primers carrying amino acid saturation mutations, and then transforming the plasmid mutant library into a genome knockout hom gene chassis strain for L-threonine structural analogue resistance plate screening, and finally screening to obtain mutants that relieve feedback inhibition of L-threonine and L-isoleucine and improve enzyme activity, wherein the mutation sites are A381D, A381L, A381I, A381P, A381V, A384R, A384D, I397C, I397D, I397G, I397S, I397R, I397V and I397A, respectively.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a homoserine dehydrogenase, wherein the amino acid sequence of the homoserine dehydrogenase is mutated at the amino acid residues at positions 381, 384, and / or 397 corresponding to the amino acid sequence shown in SEQ ID NO.2.

[0009] In a preferred embodiment, the homoserine dehydrogenase is derived from Corynebacterium glutamicum.

[0010] In a specific embodiment, the homoserine dehydrogenase is:

[0011] a. Its amino acid sequence is as shown in SEQ ID NO.2 and the amino acid residues at positions 381, 384, and / or 397 of the amino acid sequence shown in SEQ ID NO.2 are mutated, or

[0012] b. A homoserine dehydrogenase derived from a having the sequence defined by a and having one or more amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue substituted, deleted or added at positions other than position 381, 384, and / or 397, and having the homoserine dehydrogenase function defined by a.

[0013] In a preferred embodiment, the amino acid sequence of the homoserine dehydrogenase corresponds to Asp or Leu or Ile or Pro or Val at amino acid residue 381 of the amino acid sequence shown in SEQ ID NO.2, and / or Arg or Asp at amino acid residue 384, and / or Cys or Asp or Gly or Ser or Arg or Val or Ala at amino acid residue 397.

[0014] In a second aspect, the present invention provides a gene encoding the homoserine dehydrogenase described in the first aspect;

[0015] Preferably, the nucleotide sequence of the gene is obtained by mutating the nucleotide sequence shown in SEQ ID NO. 3. In a third aspect, the present invention provides an expression vector comprising the coding gene described in the second aspect.

[0016] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains the encoding gene described in the first aspect or the expression vector described in the second aspect.

[0017] In a preferred embodiment, the host cell is from the genus Escherichia, Coryneb acterium, Brevibacterium, Bacillus, Serratia or Vibrio.

[0018] In a preferred embodiment, the host cell is Escherichia coli (E. Coli) or Corynebacterium glutamicum (Corynebacterium glutamicum).

[0019] In a preferred embodiment, the encoding gene of the second aspect or the expression vector of the third aspect is integrated into the chromosome of the host cell.

[0020] In a preferred embodiment, the host cell expresses the homoserine dehydrogenase described in the first aspect.

[0021] In a fifth aspect, the present invention provides use of the homoserine dehydrogenase of the first aspect, or the encoding gene of the second aspect, or the expression vector of the third aspect, or the host cell of the fourth aspect in producing amino acids.

[0022] Preferably, the amino acid is L-threonine, L-isoleucine, L-homoserine, L-glycine, L-methionine and derivatives thereof.

[0023] In a sixth aspect, the present invention provides a method for preparing an amino acid, the method comprising the following steps:

[0024] a. Cultivating the host cell according to the fourth aspect to produce amino acids; and

[0025] b. The step of separating amino acids from the culture medium.

[0026] Preferably, the amino acid is L-threonine, L-isoleucine, L-homoserine, L-glycine, L-methionine and derivatives thereof.

[0027] In a specific embodiment, the host cell is Corynebacterium glutamicum which enhances the expression of aspartate kinase gene lysC and aspartate semialdehyde dehydrogenase gene asd that relieve lysine feedback inhibition;

[0028] Preferably, the aspartate kinase that relieves lysine feedback inhibition is lysC I293Y Mutant.

[0029] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0030] Beneficial effects of the present invention:

[0031] 1. The homoserine dehydrogenase mutant provided by the present invention, its encoding gene and the host cell comprising the encoding gene can be used in industry to produce L-homoserine and amino acids derived from L-homoserine, such as L-threonine, L-isoleucine, L-glycine, L-methionine and their derivatives;

[0032] 2. The homoserine dehydrogenase mutant provided by the present invention is a homoserine dehydrogenase with high specific activity and effective relief of feedback inhibition of L-threonine and L-isoleucine. Therefore, the homoserine dehydrogenase mutant of the present invention, its encoding gene and the host cell comprising the encoding gene can not only efficiently produce L-threonine and L-isoleucine, but also effectively relieve feedback inhibition of L-threonine and L-isoleucine, and has broad application prospects in industry;

[0033] 3. The homoserine dehydrogenase mutants and their encoding genes provided by the present invention are helpful to clarify and understand the biosynthetic pathway of L-homoserine and its derived amino acids and the related mechanism of feedback inhibition, thereby providing a theoretical basis and materials for further transforming related proteins or host cells using genetic engineering methods. DETAILED DESCRIPTION

[0034] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0035] Terminology used in this article

[0036] As used herein, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0037] As used herein, "about" means that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0038] As used herein, the definition of "or" is only alternatives and "and / or", but unless it is explicitly stated that there are only alternatives or the alternatives are mutually exclusive, the term "or" in a claim means "and / or".

[0039] The selected / optional / preferred "numerical range" used herein includes both the numerical endpoints at both ends of the range and all the natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

[0040] As used herein, the terms "wild-type" and "naturally occurring" refer to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, can be isolated from a source in nature and has not been intentionally modified by humans in the laboratory is naturally occurring.

[0041] The term "coding gene" as used herein refers to a polymer composed of nucleotides, also referred to as a polynucleotide. A coding gene can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and restored by standard molecular biology methods (e.g., using a cloning vector) and sequences and their component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, a "coding gene" refers to a nucleotide polymer removed from other nucleotides (separate fragments or entire fragments), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0042] As used herein, the terms "protein", "enzyme" are used interchangeably and have the meanings generally understood by those of ordinary skill in the art. They are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0043] The term "expression" of the present invention includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0044] The term "mutant" of the present invention refers to a polynucleotide or polypeptide comprising a change (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position. In a specific embodiment, the "mutant" in the present disclosure is a mutant having homoserine dehydrogenase.

[0045] The term "amino acid mutation" or "nucleotide mutation" of the present invention includes "substitution, duplication, deletion or addition of one or more amino acids or nucleotides". In the present disclosure, the term "mutation" refers to a change in a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0046] The term "expression cassette" of the present invention refers to a type of expression element comprising a transcriptional regulatory element and a target gene, and utilizing the transcriptional regulatory element to regulate the expression of the target gene. In the present invention, the transcriptional regulatory element comprises a promoter. In the present invention, the target gene is specifically a gene encoding homoserine dehydrogenase.

[0047] The term "vector" of the present invention refers to a DNA construct containing a DNA sequence operably linked to an appropriate control sequence to express a target gene in a suitable host. "Expression vector", "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) a transcription subunit that is suitable for transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0048] Term " host cell " of the present invention means to be easy to use any cell type that comprises homoserine dehydrogenase mutant of the present invention.In one embodiment, host cell refers to prokaryotic microorganism, and particularly, host cell derives from the amino acid whose microorganism that is suitable for fermentation production, for example Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia.As preferably, host cell is the Corynebacterium glutamicum that derives from Corynebacterium.

[0049] The term "transformation" herein has the meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0050] The host cells herein can be cultured according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0051] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0052] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0053] Example 1: Design of feedback inhibition-removing mutation sites for Corynebacterium glutamicum homoserine dehydrogenase

[0054] The C-terminal regulatory domain of homoserine dehydrogenase Hom of Corynebacterium glutamicum has been confirmed to be the main binding domain of inhibitors L-threonine and L-isoleucine. The amino acid sequence of homoserine dehydrogenase Hom of Corynebacterium glutamicum is shown in SEQ ID NO.2, and its coding gene sequence is shown in SEQ ID NO.3.

[0055] The present invention detects by size exclusion chromatography that the homoserine dehydrogenase Hom of Corynebacterium glutamicum is a tetramer structure. Based on the homoserine dehydrogenase (PDBcode: 3MTJ, sequence identity 38%) from the tetramer homologous protein Thiobacillus denitrificans, homology modeling and molecular docking of L-threonine effectors are performed, and prediction shows that L-threonine is bound to the tetramer interface and 369-399 amino acid residues are the main binding region. The present invention speculates that the conservative amino acids involved in the interface interaction in the main binding region play an important role in feedback inhibition, and the amino acid conservation is analyzed by analyzing the interface amino acid sites and based on the multiple sequence alignment of 1000 homologous sequences. 9 amino acid sites located at the tetramer interface in the main binding region and conserved> 80% are selected as mutation sites, namely D375, L380, A381, A384, I392, S393, L394, I397 and Q399.

[0056] Example 2. Screening of feedback inhibition mutants of homoserine dehydrogenase in Corynebacterium glutamicum

[0057] First, a screening method for homoserine dehydrogenase mutants that relieve feedback inhibition was designed. The homoserine dehydrogenase gene hom and the downstream homoserine kinase thrB gene of the same operon were overexpressed on a plasmid, and a mutant plasmid library was constructed by amplifying primers carrying amino acid saturation mutations. The plasmid mutant library was then transformed into a genome knockout hom gene chassis strain for L-threonine structural analog resistance plate screening.

[0058] The plasmids for overexpressing hom and thrB genes were constructed as follows: Based on the pEC-ccdB plasmid, a derivative of the pEC-XK99E plasmid (GenBank: AY219683.1), the hom and thrB genes were overexpressed. trc The promoter controls gene expression. The pEC-ccdB plasmid was modified based on the pEC-XK99E plasmid, maintaining the original core skeleton. First, in order to achieve efficient cloning, the BsaI restriction site on the plasmid was removed by point mutation, and the ampicillin expression frame was added. The commonly used efficient cloning screening marker ccdB expression frame was added to its original cloning site. trcThe RBS sequence required for gene expression is added behind the promoter, and the plasmid sequence is shown in SEQ ID NO.1. The plasmid pEC-ccdB is used as a template, and PEC-F and PEC-R are used as primers to amplify the plasmid skeleton. According to the reported genome sequence of Corynebacterium glutamicum ATCC13032 (GenBank: BA000036.3), the ATCC13032 genome is used as a template, and homthrB-F and homthrB-R are used as primers to amplify the hom and thrB gene fragments. After the above-mentioned plasmid skeleton and target gene fragment are recovered, they are connected and transformed into E.coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.) through the one-step recombination kit of Novozyme, and then coated on LB solid medium containing 25 μg / mL kanamycin, cultured overnight at 37°C, and the obtained transformants are PCR verified using plasmid universal primers PTRC99C-F and PBV220-R, and sent to a sequencing company for sequencing verification. The correct recombinant plasmid is named pEC-homthrB. LB medium: 5g / L yeast powder, 10g / L peptone, 10g / L NaCl. To prepare solid culture medium, add 15g / L agar powder.

[0059] The single-site saturation mutation library plasmids of 9 sites were constructed as follows: pEC-homthrB was used as a template, and XXX-F (such as 375-F) and PEC-2 of the corresponding sites were used as primers, and XXX-R (such as 375-R) and PEC-1 were used as primers to amplify two fragments of each site. After the fragments were recovered, they were connected through the one-step recombination kit of Novozyme and transformed into E. coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.), and spread on LB solid medium containing 25 μg / mL kanamycin. The transformants needed to be greater than 300 clones. 2 mL of sterile water was added to the plate of each library, and the colonies were collected with a coating rod. The collected bacterial solution was centrifuged and the plasmid library was extracted to obtain the saturation mutation library plasmids of 9 sites. The primers used in this embodiment are shown in Table 1.

[0060] Table 1 shows the primers

[0061]

[0062]

[0063] Plate screening of the library: First, a screening chassis was constructed, and the hom gene on the genome of the lysine-producing bacteria ZCgLJ6 of Corynebacterium glutamicum (see CN113201514A) was knocked out to obtain the LCgL1 strain. According to the method reported in the conventional literature, the competent cells of the LCgL1 strain were prepared, and the saturated mutation library plasmids of the 9 sites constructed above were respectively transferred into the competent cells of LCgL1, 1 mL of TSB medium preheated at 46°C was added, incubated at 46°C for 6 min, incubated at 30°C for 2 h, and the bacterial solution was washed once with MM liquid medium, and the bacterial solution with 300-1000 clones grown on the MM structure-free plate was coated on the MM solid medium containing 25 μg / mL kanamycin and 3g / L L-threonine structural analog α-amino-β-hydroxyvaleric acid (AHV), and cultured at 30°C for 72 h. The components of TSB culture medium are (g / L): glucose, 5g / L; yeast powder, 5g / L; soy peptone, 9g / L; urea, 3g / L; succinic acid, 0.5g / L; K2HPO4·3H2O, 1g / L; MgSO4·7H2O, 0.1g / L; biotin, 0.01mg / L; vitamin B1, 0.1mg / L; MOPS, 20g / L, adjust the pH to 7.2. MM medium: 5g / L glucose, 1g / L KH2PO4, 5g / L (NH4)2SO4, 0.4g / L MgSO4·7H2O, 0.5g / L NaCl, 2g / Lurea, 200μg / L biotin, 100μg / L vitaminB1, 100μg / L vitamin B5, 0.03g / L vitamin B3,0.09mg / LNa2B4O7·10H2O,0.04mg / L(NH4)6Mo7O 24 ·4H2O,0.01mg / L ZnSO4·7H2O,0.01mg / LCuSO4·5H2O,0.01mg / L MnCl2·4H2O,1mg / LFeCl3·6H2O,0.01mg / L CaCl2, adjust pH to 7.2; prepare solid culture medium and add 15g / L agar powder. The results showed that only A381, A384 and I397 sites could grow clones. The obtained transformants were verified by PCR using plasmid universal primers PTRC99C-F and PBV220-R. The PCR products were sent to a sequencing company for sequencing, and after clone separation and purification, a total of 14 mutants were obtained, as shown in Table 2.

[0064] Example 3. Evaluation of L-threonine production of Corynebacterium glutamicum homoserine dehydrogenase mutants

[0065] The L-threonine production of the strain was evaluated using a 24-well plate. The strain was first inoculated into 800 μL TSB liquid medium per well and cultured for 6-8 h. The culture was then inoculated as a seed into 800 μL fermentation medium supplemented with 25 μg / mL kanamycin and 0.5 mM IPTG per well of a 24-well plate. The initial OD 600 The control was about 0.06 (detected by microplate reader), cultured at 30℃ for 18h, the speed of the well plate shaker was 800rpm, and the relative humidity was 90%. The L-threonine production was detected after the fermentation. The experiment was set up in 3 parallels. The fermentation medium components were: glucose, 80g / L; yeast powder, 1g / L; soy peptone, 1g / L; NaCl, 1g / L; ammonium sulfate, 1g / L; urea, 8g / L; K2HPO4·3H2O, 1g / L; MgSO4·7H2O, 0.45g / L; FeSO4·7H2O, 0.05g / L; biotin, 0.4mg / L; vitamin B1, 0.1mg / L; MOPS, 40g / L; initial pH 7.2. The L-threonine content was detected by the HPLC method of common amino acids.

[0066] The results are shown in Table 2. The L-threonine production of the 14 mutants was higher than that of the wild type WT, which was 1.75-4.29 times higher than that of the WT, indicating that the mutations at the above sites can relieve the feedback inhibition of L-threonine and L-isoleucine, thereby significantly increasing the L-threonine production.

[0067] Table 2. L-threonine production of the wild type and 14 mutants

[0068]

[0069]

[0070] Example 4. Evaluation of the application of Corynebacterium glutamicum homoserine dehydrogenase mutants to improve the production of various amino acids

[0071] In order to further verify the application effect of the homoserine dehydrogenase mutant of the present invention in the production of aspartate family amino acids that depend on the key enzyme Hom, the aspartate kinase gene lysC I293Y and the expression of aspartate semialdehyde dehydrogenase gene asd, improve the supply of precursors, and enhance the synthesis of various amino acids. In this example, A381P, A381V, A384D, I397R and I397V were selected for further verification.

[0072] The verification strains were constructed as follows: First, the wild-type and mutant overexpression plasmids were constructed. (1) Based on the pEC-homthrB plasmid, lysC I293Y-asd operon expression, hom gene is wild type four gene overexpression plasmid. Ppyc-F and Ppyc-R were used as primers to contain P pyc -13 promoter (see CN113755492A) as a template to amplify P pyc -13 promoter fragment; LA-F and LA-R were used as primers and the genome of ZCgLJ6 strain was used as template to amplify lysC I293Y -asd gene fragment; pEC1-F and pEC1-R were used as primers and pEC-homthrB plasmid was used as template to amplify the plasmid backbone. After the above three fragments were recovered, they were connected through the one-step recombination kit of Novozymes and transformed into E. coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.), and coated on LB solid medium containing 25μg / mL kanamycin. The transformants were verified by PCR and sequencing to obtain the four-gene overexpression plasmid pEC-HTLA ​​with the hom gene as wild type. (2) The corresponding mutations were introduced respectively. Each site was amplified with Bone-F and Bone-R, HT-F and XXX-R (such as 381V-R), XXX-F (such as 381V-F) and HT-R using pEC-HTLA ​​as template. Mutations were introduced by primers. The above three fragments were recovered and connected by the one-step recombination kit of Novozyme, and then transformed into E. coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.), and respectively coated on LB solid medium containing 25 μg / mL kanamycin. The transformants were verified by PCR and sequencing to obtain mutant plasmids pEC-H A381P TLA, pEC-H A381V TLA, pEC-H A384D TLA, pEC-H I397R TLA and pEC-H I397V TLA. (3) The above wild-type plasmids and mutant plasmids were transformed into LCgL1 strains to obtain CgH-WT, CgH-G378E, CgH-A381V, CgH-A381P, CgH-A384D, CgH-I397R and CgH-I397V strains. The primers used in this example are shown in Table 3.

[0073] Table 3. Primers for constructing wild-type and mutant overexpression plasmids

[0074]

[0075] Fermentation evaluation of strains: The same well plate fermentation method as in Example 3 was used to detect the contents of various amino acids by HPLC. The results are shown in Table 4. Compared with the wild-type control strain, the introduction of the hom gene to release feedback inhibition mutations A381P, A381V, A384D, I397R and I397V can simultaneously increase the production of L-threonine, L-homoserine, L-isoleucine and glycine. Among them, the increase in L-threonine production was 0.39-1.99 times; the increase in L-homoserine production was 1.71-14.81 times; the increase in L-isoleucine production was 7-63%; and the increase in glycine production was 2.29-3.88 times. The above results show that these mutants can be used for the production of aspartic acid family amino acids and their derivatives that rely on the key enzyme Hom.

[0076] Table 4. Comparison of wild type and mutants for the production of aspartate family amino acids and their derivatives

[0077]

[0078] Example 5. Characterization of the feedback inhibition-resistant enzymatic properties of the homoserine dehydrogenase mutant of Corynebacterium glutamicum

[0079] In order to further characterize the feedback inhibition relief level of the mutants, the WT, A384D and I397V mutants were recombinantly expressed and purified in Escherichia coli, and their feedback inhibition relief effects were characterized.

[0080] Recombinant expression and purification of enzymes: pET-28a protein expression plasmid and Escherichia coli Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.) were used as hosts to express recombinant proteins. Based on the histidine tag, conventional nickel columns and gravity columns were used for purification and desalting to obtain wild-type and mutant pure enzymes. The recombinant expression plasmid was constructed by using 28a-F and 28a-R primers and pET-28a plasmid as template to amplify the plasmid backbone; using hom-F and hom-R primers, plasmids containing the corresponding wild type or mutant (A384D and I397V) were used as templates to amplify the corresponding hom gene fragments; the above two fragments were recovered and connected through the one-step recombination kit of Novogene and then transformed into E.coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.), and were respectively coated on LB solid culture medium containing 25μg / mL kanamycin and 10μg / L chloramphenicol. The transformants were verified by PCR and sequencing to obtain expression plasmids pET-WT, pET-A384D and I397V, respectively. The above expression plasmids were transformed into Escherichia coli Transetta (DE3) to obtain recombinant expression strains. The recombinant expression strain was cultured overnight in LB medium, and the inoculum was transferred to 20 mL LB liquid medium supplemented with 25 μg / L kanamycin and 10 μg / L chloramphenicol at a rate of 1%. The culture was carried out in a 250 mL Erlenmeyer flask at 37°C and 220 rpm in a shaking incubator until the OD 600 The final concentration of IPTG was 0.5 mM, and the mixture was cultured at 16°C and 220 rpm for 16 h to induce the expression of recombinant protein.

[0081] Table 5. Primers for constructing homoserine dehydrogenase recombinant expression plasmid

[0082]

[0083] The enzyme activity assay system for homoserine dehydrogenase was: 100 mM Tris–HCl (pH 8.0), 60 mM L-homoserine, 1.2 mM NADP + , enzyme amount is about 4μg and the corresponding concentration of inhibitor. OD is measured by microplate reader 340nm The change of absorbance value, the reaction temperature is 30℃. The enzyme activity of each mutant or wild type without adding inhibitors is set to 100%, and the residual relative enzyme activity after adding inhibitors is calculated respectively. At the same time, the specific enzyme activity of wild type and mutants without adding inhibitors is measured respectively. The enzyme activity unit is defined as follows: the amount of enzyme required to produce 1 micromole of NADPH in 1 minute is 1U. The specific enzyme activity refers to the catalytic activity of the enzyme contained in each milligram of pure enzyme. The experiments were set up in 3 parallels.

[0084] The residual relative enzyme activity of the wild-type and mutant enzymes under different concentrations and types of inhibition conditions is shown in Table 6. The wild-type WT enzyme can completely inhibit the enzyme activity by adding 10mM inhibitor, while the mutant still has 80% or more residual activity after adding 10mM inhibitor, more than 50% residual activity after adding 25mM inhibitor, and more than 40% residual activity after adding 50mM inhibitor, indicating that the A384D and I397V mutants can effectively relieve the feedback inhibition of L-threonine and L-isoleucine. The specific enzyme activity of the wild type without adding inhibitors is 1.06±0.06U / mg, and the A384D and I397V mutants are 1.87±0.04U / mg and 2.05±0.05U / mg, respectively. The mutants are 76.8% and 94.3% higher than the wild-type control, respectively, indicating that the above mutations significantly improve the enzyme activity and are more conducive to the efficient production of downstream metabolites.

[0085] Table 6. Residual relative enzyme activities of wild-type and mutant enzymes under different concentrations and types of inhibition

[0086]

[0087]

[0088] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A homoserine dehydrogenase, characterized in that The homoserine dehydrogenase is: a. Its amino acid sequence is as shown in SEQ ID NO.2 and the amino acid residue at position 381 and / or position 384 and / or position 397 of the amino acid sequence shown in SEQ ID NO.2 is mutated, or b. A homoserine dehydrogenase derived from a having the sequence defined by a and having one or several amino acid residues, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue substitution, deletion or addition at positions other than position 381 and / or 384 and / or 397, and having the homoserine dehydrogenase function defined by a.

2. The homoserine dehydrogenase according to claim 1, characterized in that The amino acid sequence of the homoserine dehydrogenase corresponds to the amino acid sequence shown in SEQ ID NO.2, wherein the 381st amino acid residue is Asp or Leu or Ile or Pro or Val, and / or the 384th amino acid residue is Arg or Asp, and / or the 397th amino acid residue is Cys or Asp or Gly or Ser or Arg or Val or Ala.

3. The gene encoding homoserine dehydrogenase according to any one of claims 1-2.

4. The coding gene according to claim 3, characterized in that The nucleotide sequence is obtained by performing mutation on the basis of the nucleotide sequence shown in SEQ ID No.

3.

5. An expression cassette comprising the gene encoding homoserine dehydrogenase according to claim 3 or 4.

6. An expression vector comprising the coding gene according to claim 3 or 4.

7. A host cell, comprising the homoserine dehydrogenase according to claim 1 or 2, or the coding gene according to claim 3 or 4, or the expression cassette according to claim 5, or the expression vector according to claim 6; Preferably, the host cell is from the genus Corynebacterium, Escherichia, Brevibacterium, Bacillus, Serratia or Vibrio; Further preferably, the host cell is Corynebacterium glutamicum or Escherichia coli.

8. The host cell according to claim 7, characterized in that The host cell has enhanced expression of the aspartate kinase gene lysC and the aspartate semialdehyde dehydrogenase gene asd that relieve lysine feedback inhibition; Preferably, the aspartate kinase that relieves lysine feedback inhibition is lysC I293Y Mutant.

9. Use of the homoserine dehydrogenase according to claim 1 or 2, or the encoding gene according to claim 3 or 4, or the expression cassette according to claim 5, or the expression vector according to claim 6, or the host cell according to claim 7 in producing amino acids; Preferably, the amino acid is L-threonine, L-isoleucine, L-homoserine, L-glycine, L-methionine and derivatives thereof.

10. A method for preparing an amino acid, the method comprising the following steps: a. Cultivating the host cell according to claim 7 to produce L-threonine and other aspartate family amino acids; and b. a step of separating the amino acids from the culture broth; Preferably, the amino acid is L-threonine, L-isoleucine, L-homoserine, L-glycine, L-methionine and derivatives thereof.

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