A high-servne dehydrogenase mutant and application thereof

Abstract

The application discloses a homoserine dehydrogenase mutant and application thereof. 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, I397A compared with a wild-type homoserine dehydrogenase Hom from corynebacterium glutamicum. The enzyme activity of the mutant is significantly improved compared with a wild-type control through enzyme activity determination analysis of induced expression and purification. Meanwhile, the mutant can efficiently remove the feedback inhibition of L-threonine and L-isoleucine. The homoserine dehydrogenase mutant, a coding gene thereof and a host cell containing the coding gene can be applied in industry to produce L-homoserine and derivative amino acids.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and bioengineering, and specifically relates to a homoserine dehydrogenase mutant and its application in the production of homoserine or L-amino acids derived from homoserine. Background Technology

[0002] In Corynebacterium glutamicum, homoserine dehydrogenase (EC: 1.1.1.3), encoded by the hom gene, catalyzes the dehydrogenation of aspartic acid hemialdehyde to L-homoserine. L-homoserine then undergoes a series of enzymatic reactions to produce amino acids such as L-threonine, L-isoleucine, and L-methionine. It is known that homoserine dehydrogenase is subject to non-competitive feedback inhibition by L-threonine and L-isoleucine; overcoming this feedback inhibition is essential for achieving high yields of L-homoserine or amino acids derived from homoserine.

[0003] Several homoserine dehydrogenase mutants that can relieve L-threonine feedback inhibition have been reported in existing technologies. For example, Eikmanns et al. reported that the glutamate at position 378 of Hom was mutated to glycine (Eikmanns, et al. Appl. Microbial Biotechnol. 1991, 34: 617–622); Archer et al. reported that a C-terminal frameshift mutation in Hom can also relieve feedback inhibition (Archer JA, et al. Gene. 1991, 107: 53–59). CJ Pharmaceuticals of South Korea reported in CN111601886B and CN110945121B that the amino acid at position 407 of Hom was replaced by histidine and the amino acid at position 285 was replaced by isoleucine, respectively.

[0004] Therefore, obtaining homoserine dehydrogenase mutants with high enzyme activity that can effectively relieve feedback inhibition by L-threonine and L-isoleucine through screening or molecular modification remains of great significance for the efficient production of L-homoserine or its derivative amino acids by Corynebacterium glutamicum. Summary of the Invention

[0005] The purpose of this invention is to address the current state and shortcomings of the existing technology by providing a homoserine dehydrogenase mutant that relieves the feedback inhibition of L-threonine and L-isoleucine, thereby improving catalytic performance and facilitating the microbial fermentation production of L-homoserine or its derivative amino acids.

[0006] The present application is realized by the following technical idea: taking hom of corynebacterium glutamicum homoserine dehydrogenase as a template, overexpressing homoserine dehydrogenase gene hom and downstream homoserine kinase thrB gene of the same operon on a plasmid, amplifying and constructing a mutant plasmid library through a primer carrying amino acid saturation mutation, and then transforming the plasmid mutant library into a hom gene knockout chassis strain to perform L-threonine structural analog resistance plate screening, and finally screening a mutant that removes the feedback inhibition of L-threonine and L-isoleucine and has improved enzyme activity, and the mutation sites are A381D, A381L, A381I, A381P, A381V, A384R, A384D, I397C, I397D, I397G, I397S, I397R, I397V, and I397A.

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

[0008] In a first aspect, the present application provides a homoserine dehydrogenase, the amino acid sequence of which is mutated at the 381st, 384th, and / or 397th amino acid residue 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. the amino acid sequence of which is shown in SEQ ID NO. 2 and is mutated at the 381st, 384th, and / or 397th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2, or

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

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

[0014] In a second aspect, the present application provides a gene encoding the homoserine dehydrogenase of 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 application provides an expression vector comprising the encoding gene of the second aspect.

[0016] In a fourth aspect, the present application provides a host cell comprising the encoding gene of the first aspect or the expression vector of the second aspect.

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

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

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

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

[0021] In a fifth aspect, the present application provides the 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 the production of an amino acid.

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

[0023] In a sixth aspect, the present application provides a method for producing an amino acid, the method comprising the steps of:

[0024] a. culturing the host cell of the fourth aspect to produce the amino acid; and

[0025] b. isolating the amino acid from the culture medium.

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

[0027] In one specific embodiment, the host cell is a C. glutamicum with enhanced expression of lysC, an aspartate kinase gene that relieves lysine feedback inhibition, and asd, an aspartate semialdehyde dehydrogenase gene that relieves lysine feedback inhibition;

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

[0029] It should be understood that, in the scope of the present application, each of the technical features described above and each of the technical features specifically described below (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here.

[0030] Advantages of the present application:

[0031] 1. The high serine dehydrogenase mutant, the encoding gene thereof, and the host cell comprising the encoding gene provided by the present application 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 derivatives thereof.

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

[0033] 3. The high serine dehydrogenase mutant and the encoding gene thereof provided by the present application help to clarify and understand the biosynthetic pathway of L-homoserine and amino acids derived therefrom and the related mechanism of feedback inhibition, thereby providing a theoretical basis and materials for further modification of related proteins or host cells by genetic engineering means. DETAILED DESCRIPTION

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

[0035] As used herein

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

[0037] As used herein, "about" means a value includes the standard deviation of error of the apparatus or method used to determine the value.

[0038] As used herein, the definition of "or" is the inclusive, and not the exclusive or. That is, "A or B" means "A, B, or both A and B." The term "and / or" as used herein refers to and preferably covers a mixture, one or all, of the enumerated elements.

[0039] As used herein, the term "numerical range" of the alternatives / preferred "numerical range" includes both the numerical endpoints of the range and all the natural numbers falling within the range, intermediate to the aforesaid numerical endpoints.

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

[0041] As used herein, the term "coding gene" refers to a polymer composed of nucleotides, also referred to as a polynucleotide. A coding gene can be in the form of an isolated fragment or can be a component of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in quantity or concentration, identifiable, manipulatable, and restorable by standard molecular biology methods (e.g., using a cloning vector) to the sequence and its 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" is substituted for "T". In other words, "coding gene" refers to a nucleotide polymer removed from other nucleotides (isolated fragment or entire fragment) or can be a component or constituent 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" and "enzyme" are used interchangeably and have the meaning commonly understood by one of ordinary skill in the art. They are used interchangeably herein and are polymers of amino acids of any length. The polymers can be linear or branched, they can comprise modified amino acids, and they can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0043] The term "expression" as used herein includes any step involved in the production of a 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 an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type", or "compared to", polynucleotide or polypeptide, wherein substitution refers to the replacement of 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 after the nucleotide or amino acid occupying a position, which is adjacent and immediately follows. 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 an alteration of 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 the expression of the target gene is regulated by the transcriptional regulatory element. In the present invention, the transcriptional regulatory element comprises a promoter. In the present invention, the target gene is specifically a coding gene of homoserine dehydrogenase.

[0047] The term "vector" of the present invention refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences for expression of a gene of interest in a suitable host. "Expression vector", "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector can include, for example, a transcriptional unit comprising i) a collection of genetic elements having a regulatory effect on gene expression, such as promoters and enhancers; ii) a structure or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important and any vector can be used, including plasmids, viruses, bacteriophages, 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, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0048] The term "host cell" of the present application means any cell type which is susceptible to transformation with any cell type comprising the high serine dehydrogenase mutant of the present application. In one embodiment, the host cell refers to a prokaryotic microorganism, in particular, the host cell is derived from a microorganism suitable for fermentative production of amino acids, such as Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia. Preferably, the host cell is Corynebacterium glutamicum derived from Corynebacterium.

[0049] The term "transformation" herein has the meaning commonly understood by one of ordinary skill in the art, i.e., the process of introducing foreign DNA into a host. The methods of transformation include 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 culture of the host cell herein can be performed according to the 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, etc. can be appropriately adjusted according to the actual situation.

[0051] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[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, for which no specific conditions are indicated, are generally carried out according to the 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: Designing the feedback inhibition relief mutation site of Corynebacterium glutamicum homoserine dehydrogenase

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

[0055] The present application finds that hom of corynebacterium glutamicum homoserine dehydrogenase is a tetramer structure through size exclusion chromatography detection. Based on the homoserine dehydrogenase from Thiobacillus denitrificans (PDB code: 3MTJ, sequence identity 38%) which is a homologous protein of the tetramer, homologous modeling and molecular docking of L-threonine effector are performed, and the prediction shows that L-threonine is combined at the interface of the tetramer and the amino acid residues 369-399 are the main binding region. The present application speculates that the conserved amino acids involved in the interface interaction in the main binding region play an important role in feedback inhibition, and by analyzing the interface amino acid sites and analyzing the amino acid conservation based on 1000 homologous sequences, 9 amino acid sites in the main binding region which are located at the interface of the tetramer and have a conservation of >80% are selected as mutation sites, namely D375, L380, A381, A384, I392, S393, L394, I397 and Q399.

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

[0057] Firstly, a screening method of feedback inhibition mutants of homoserine dehydrogenase is designed. The homoserine dehydrogenase gene hom and the downstream homoserine kinase thrB gene in the same operon are overexpressed on a plasmid, the primer carrying amino acid saturation mutation is amplified and a mutant plasmid library is constructed, and then the plasmid mutant library is transformed into a genome knockout hom gene chassis strain to perform L-threonine structural analog resistance plate screening.

[0058] The plasmid overexpressing hom and thrB genes is constructed as follows: on the basis of the pEC-XK99E plasmid (GenBank: AY219683.1), the overexpression of hom and thrB genes is performed, and the P trc The promoter controls the expression of the gene. The pEC-ccdB plasmid is obtained by modifying the pEC-XK99E plasmid, and the original core skeleton is maintained. Firstly, in order to realize efficient cloning, the BsaI enzyme cutting site on the plasmid is removed by point mutation, the expression frame of ampicillin is increased, and the commonly used efficient cloning screening marker ccdB expression frame is added at the original cloning site. The P trcThe RBS sequence required for gene expression was added behind the promoter, and the plasmid sequence is shown in SEQ ID NO. 1. The plasmid backbone was amplified using pEC-ccdB as the template and PEC-F and PEC-R as primers. According to the reported Corynebacterium glutamicum ATCC13032 genome sequence (GenBank: BA000036.3), the hom and thrB gene fragments were amplified using the ATCC13032 genome as the template and homthrB-F and homthrB-R as primers. After recovering the plasmid backbone and the target gene fragment, the ligation was performed using the Novagen One-step recombination kit, and then the product was transformed into E. coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.). The obtained transformants were coated on LB solid medium containing 25 μg / mL kanamycin and incubated at 37°C overnight. The transformants were verified by PCR using the universal primers PTRC99C-F and PBV220-R, and were sent to a sequencing company for sequencing verification. The correct recombinant plasmid was named pEC-homthrB. The LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, and 15 g / L agar powder were added to prepare the solid culture medium.

[0059] The plasmid construction of the single-site saturation mutation library of 9 sites is as follows: pEC-homthrB as the template, XXX-F (such as 375-F) and PEC-2 as primers and XXX-R (such as 375-R) and PEC-1 as primers were used to amplify two fragments of each site, and then the fragments were recovered and ligated using the Novagen One-step recombination kit. The product was transformed into E. coli Trans-T1 (Beijing Quanshijin Biotechnology Co., Ltd.), and the transformants were coated on LB solid medium containing 25 μg / mL kanamycin. More than 300 colonies were required. 2 mL of sterile water was added to each plate of the library, and the colonies were collected using a spreader. 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 example are shown in Table 1.

[0060] Table 1 is the primer

[0061]

[0062]

[0063] Plate screening of the library: first, a screening chassis was constructed by knocking out the hom gene on the genome of lysine-producing Corynebacterium glutamicum ZCgLJ6 (see CN113201514A) to obtain LCgL1 strain. According to the method reported in the conventional literature, the competent cells of LCgL1 strain were prepared, and the above-constructed saturated mutation library plasmid of 9 sites was respectively transferred into the LCgL1 competent cells, 1 mL of 46°C preheated TSB medium was added, 46°C incubation for 6 min, 30°C incubation for 2 h, and the bacterial liquid was washed once with MM liquid medium. The bacterial liquid that could grow 300-1000 clones on the MM non-structural analogue plate was coated on the MM solid medium containing 25 μg / mL kanamycin and 3 g / L L-threonine structural analogue α-amino-β-hydroxyvaleric acid (AHV), and cultured at 30°C for 72 h. The composition of TSB medium is (g / L): glucose, 5 g / L; yeast powder, 5 g / L; soybean peptone, 9 g / L; urea, 3 g / L; succinic acid, 0.5 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.1 g / L; biotin, 0.01 mg / L; vitamin B1, 0.1 mg / L; MOPS, 20 g / L, pH adjusted to 7.2. MM medium: 5 g / L glucose, 1 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.4 g / L MgSO4·7H2O, 0.5 g / L NaCl, 2 g / L urea, 200 μg / L biotin, 100 μg / L vitamin B1, 100 μg / L vitamin B5, 0.03 g / L vitamin B3, 0.09 mg / L Na2B4O7·10H2O, 0.04 mg / L (NH4)6Mo7O 24 ·4H2O, 0.01 mg / L ZnSO4·7H2O, 0.01 mg / L CuSO4·5H2O, 0.01 mg / L MnCl2·4H2O, 1 mg / L FeCl3·6H2O, 0.01 mg / L CaCl2, pH adjusted to 7.2; the solid medium was prepared by adding 15 g / L agar powder. The results showed that only A381, A384 and I397 sites could grow clones, the obtained transformants were verified by PCR using universal primers PTRC99C-F and PBV220-R, the PCR products were sent to a sequencing company for sequencing, and 14 kinds of mutants were obtained after separation and purification of the clones, as shown in Table 2.

[0064] Example 3. L-threonine production evaluation of high methionine dehydrogenase mutants of Corynebacterium glutamicum

[0065] The L-threonine production of the strains was evaluated using 24-well plates. The strains were first inoculated into 800 μL TSB liquid medium in each well and incubated for 6-8 h, and the culture was used as seed to inoculate 800 μL fermentation medium containing 25 μg / mL kanamycin and 0.5 mM IPTG in each well of a 24-well plate, and the initial OD 600 The control was about 0.06 (determined by a microplate reader), and the culture was incubated at 30°C for 18 h at a shaking speed of 800 rpm and a relative humidity of 90%. The L-threonine production was determined after the fermentation was completed. Three parallel experiments were set. The fermentation medium contained the following components: glucose, 80 g / L; yeast powder, 1 g / L; soybean peptone, 1 g / L; NaCl, 1 g / L; ammonium sulfate, 1 g / L; urea, 8 g / L; K2HPO4·3H2O, 1 g / L; MgSO4·7H2O, 0.45 g / L; FeSO4·7H2O, 0.05 g / L; biotin, 0.4 mg / L; vitamin B1, 0.1 mg / L; MOPS, 40 g / L; and the initial pH was 7.2. The L-threonine content was determined by a common HPLC method for 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, and was increased by 1.75-4.29 times compared with WT, indicating that the mutation 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. Application evaluation of the Corynebacterium glutamicum homoserine dehydrogenase mutant for increasing the production of various amino acids

[0071] To further verify the application effect of the homoserine dehydrogenase mutant of the application in the production of aspartate family amino acids that depend on the key enzyme Hom, the expression of the aspartokinase gene lysC I293Y and the aspartate semialdehyde dehydrogenase gene asd was enhanced to increase 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 strain was constructed as follows: first, the overexpression plasmid of the wild type and the mutant was constructed. (1) On the basis of the pEC-homthrB plasmid, the lysC I293YThe four-gene overexpression plasmid with wild-type hom gene was obtained by amplifying the P pyc The plasmid with P-13 promoter (see CN113755492A) as the template was used as the template, and the P pyc The lysC gene fragment was amplified by using LA-F and LA-R as primers and the genome of the ZCgLJ6 strain as the template. I293Y The asd gene fragment was amplified by using pEC1-F and pEC1-R as primers and the pEC-homthrB plasmid as the template. The three fragments above were recovered and connected by using a One-step Repligation Kit from Novagen, and then transformed into E. coli Trans-T1 (Beijing Quansijin Biotechnology Co., Ltd.). The transformants were coated on LB solid medium containing 25 μg / mL kanamycin, and verified by PCR and sequencing to obtain the four-gene overexpression plasmid pEC-HTLA with wild-type hom gene. A381P TLA, pEC-H A381V TLA, pEC-H A384D TLA, pEC-H I397R TLA and pEC-H I397V TLA.

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

[0074]

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

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

[0080] Recombinant expression and purification of enzymes: The pET-28a protein expression plasmid and E. coli Transetta (DE3) (Beijing Zixingjin Biotechnology Co., Ltd.) host were used to express recombinant proteins. Based on the histidine tag, the conventional nickel column and gravity column were used for purification and desalination to obtain wild-type and mutant pure enzymes. For recombinant expression plasmid construction, the 28a-F and 28a-R primers were used to amplify the plasmid backbone with the pET-28a plasmid as the template; the hom-F and hom-R primers were used to amplify the corresponding hom gene fragment with the plasmid containing the corresponding wild-type or mutant (A384D and I397V) as the template; after recovering the above two fragments, the Novagen one-step recombination kit was used for ligation, and then the ligation product was transformed into E. coli Trans-T1 (Beijing Zixingjin Biotechnology Co., Ltd.). The transformants were coated on LB solid medium containing 25 μg / mL kanamycin and 10 μg / L chloramphenicol. The transformants were verified by PCR and sequencing to obtain the expression plasmids pET-WT, pET-A384D and I397V, respectively. The above expression plasmids were transformed into E. coli Transetta (DE3) to obtain recombinant expression strains. The recombinant expression strains were cultured overnight in LB medium, inoculated into 20 mL of LB liquid medium containing 25 μg / L kanamycin and 10 μg / L chloramphenicol at a 1% inoculation amount, and cultured in a 250 mL flask at 37°C, 220 rpm on a shaker until the OD 600 was 0.8-1, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated at 16°C, 220 rpm for 16 h to induce expression of the recombinant protein. The primers used in this example are shown in Table 5.

[0081] Table 5. Construction primers for recombinant expression plasmids of homoserine dehydrogenase

[0082]

[0083] The enzyme activity determination system of homoserine dehydrogenase was: 100 mM Tris-HCl (pH 8.0), 60 mM L-homoserine, 1.2 mM NADP + , about 4 μg of enzyme, and corresponding concentrations of inhibitors. The changes in OD 340nm absorbance were measured by a microplate reader, and the reaction temperature was 30°C. The enzyme activity of each mutant or wild-type without adding inhibitors was set to 100%, and the residual relative enzyme activity after adding inhibitors was calculated, respectively. The specific enzyme activity of wild-type and mutants without adding inhibitors was also determined. The enzyme activity unit was defined as follows: the amount of enzyme required to produce 1 micromole of NADPH per minute was 1 U. The specific enzyme activity referred to the enzyme catalytic activity contained in each milligram of pure enzyme. Each experiment was set in triplicate.

[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 is completely inhibited by the addition of 10 mM inhibitor, while the mutants still have 80% and above activity after the addition of 10 mM inhibitor, 50% and above activity after the addition of 25 mM inhibitor, and 40% and above activity after the addition of 50 mM inhibitor, indicating that the A384D and I397V mutants can efficiently relieve the feedback inhibition of L-threonine and L-isoleucine. The specific enzyme activity of the wild type without the addition of inhibitor is 1.06 ± 0.06 U / mg, and that of the A384D and I397V mutants is 1.87 ± 0.04 U / mg and 2.05 ± 0.05 U / mg, respectively, which is 76.8% and 94.3% higher than the wild type, 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 activity of wild type and mutant enzymes under different concentrations and types of inhibition conditions

[0086]

[0087]

[0088] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A homoserine dehydrogenase, characterized in that, The amino acid sequence of the homoserine dehydrogenase is the amino acid residue at position 381 of the amino acid sequence shown in SEQ ID NO. 2 is mutated to Asp or Leu or lie or Pro or Val, or the amino acid residue at position 384 is mutated to Arg or Asp, or the amino acid residue at position 397 is mutated to Cys or Asp or Gly or Ser or Arg or Val or Ala.

2. The gene encoding the homoserine dehydrogenase according to claim 1.

3. The genetic code of claim 2, wherein, The nucleotide sequence thereof is obtained by mutating the nucleotide sequence shown in SEQ ID NO.

3.

4. An expression cassette containing the gene encoding the homoserine dehydrogenase according to claim 2 or 3.

5. An expression vector comprising the gene encoding according to claim 2 or 3.

6. A host cell containing the homoserine dehydrogenase according to claim 1 or the gene encoding according to claim 2 or 3 or the expression cassette according to claim 4 or the expression vector according to claim 5.

7. The host cell of claim 6, wherein, The host cell is from the genus Corynebacterium Corynebacterium , Escherichia Escherichia , Brevibacterium Brevibacterium , Bacillus Bacillus , Serratia Serratia or Vibrio Vibrio .

8. The host cell of claim 7, wherein, The host cell is Corynebacterium glutamicum ( Corynebacterium glutamicum ) or Escherichia coli ( E.Coli ).

9. The host cell of any of claims 6-8, wherein, Expression of an aspartokinase gene that enhances relief of lysine feedback inhibition in the host cell lysC and an aspartate semialdehyde dehydrogenase gene asd ​ 10. Use of the homoserine dehydrogenase according to claim 1 or the gene encoding according to claim 2 or 3 or the expression cassette according to claim 4 or the expression vector according to claim 5 or the host cell according to any one of claims 6 to 9 for the production of amino acids.

11. Use according to claim 10, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The amino acids are L-threonine, L-isoleucine, L-homoserine, L-glycine, L-methionine and derivatives thereof.

12. A method for producing amino acids, comprising the steps of: a. culturing the host cell according to any one of claims 6 to 9 to produce aspartate family amino acids such as L-threonine; and b. isolating these amino acids from the culture broth.

13. The method of claim 12, wherein, The amino acids are L-threonine, L-isoleucine, L-homoserine, L-glycine, L-methionine and derivatives thereof.

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