Aspartate kinase mutant and its application in producing L-homoserine

Abstract

The present invention belongs to the field of bioengineering, and specifically discloses an aspartate kinase mutant with improved enzyme activity and its application in the preparation of L-homoserine or its derivatives. The amino acid sequence of the mutant is compared to the wild-type aspartate kinase derived from Saccharomyces cerevisiae, and there is one mutation type among V299A, N317S, N485S, Q107R / N317S, T114S / Q510R, and T155A / K390E. In a specific implementation, the enzyme activity assay analysis of the induced expression and purification showed that the enzyme activity of the mutant was 1.7 to 3.8 times higher than that of the wild type. Therefore, the beneficial mutants provided by the present invention can lay a good foundation for the industrialized and efficient production of L-homoserine and downstream metabolites.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically discloses an aspartate kinase mutant with improved enzyme activity and an application thereof in preparing L-homoserine or a derivative thereof. Background Art

[0002] L-homoserine is an important non-protein amino acid and a precursor of methionine, lysine, and threonine. It participates in numerous physiological and biochemical reactions and metabolic processes in the body, possessing significant physiological functions and applications. Homoserine and its derivatives possess extensive biological activity and can be used as antifungal agents to effectively inhibit sickle cell formation. The derivative, homoserine methyl ester, is a key intermediate in the anticancer compound Chinese bittersweet alkaloid. Homoserine is also the primary raw material for the synthesis of the novel pesticide L-phosphinothricin ammonium. Glufosinate ammonium, a systemic herbicide, is in increasing demand annually due to its broad-spectrum and high herbicidal activity. The urgent need for these drugs has fueled a surge in demand for L-homoserine, a key intermediate. Furthermore, homoserine has potential applications as a fertilizer additive for crops and a feed additive for livestock. For example, in chick diets, homoserine exhibits similar biological activity to threonine and can be used as a threonine substitute to improve growth performance in chicks when threonine is deficient. At the same time, homoserine can be converted into acrylic acid, 3-hydroxypropionyl-CoA, 3-hydroxypropionate, poly-3-hydroxypropionate and 1,2-propylene glycol as an intermediate. In summary, homoserine has broad application prospects as an intermediate or raw material.

[0003] Currently, the main methods for synthesizing L-homoserine are chemical and enzymatic methods. The chemical method uses L-methionine or aspartic acid as raw materials, requires the use of iodide and large amounts of organic solvents, and generates sulfides, which are associated with serious environmental and safety issues. The enzymatic method uses pyruvate and aldehyde compounds as raw materials, and produces homoserine under the co-catalysis of aldolase, formate dehydrogenase, and L-amino acid dehydrogenase complexes. This is costly and requires the use of toxic raw materials formaldehyde and formic acid, as well as expensive coenzymes. In recent years, the production of L-homoserine by microbial fermentation has attracted increasing attention, offering a safe, cost-effective, and environmentally friendly alternative production strategy.

[0004] In microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae, L-homoserine biosynthesis begins from the aspartate shunt pathway. Glucose undergoes glycolysis, the pentose phosphate pathway, and the TCA cycle to generate the oxaloacetate precursor, which then enters the aspartate shunt pathway. The aspartate precursor is then catalyzed sequentially by aspartate kinase (EC: 2.7.2.4), aspartate-semialdehyde dehydrogenase (EC: 1.2.1.11), and homoserine dehydrogenase (EC: 1.1.1.3) to ultimately produce L-homoserine. Aspartate kinase is the first common key enzyme in the biosynthesis pathways of aspartate family amino acids, including homoserine, threonine, lysine, isoleucine, and methionine. It directs the central carbon metabolic flux into the aspartate shunt pathway. Its catalytic activity is often subject to feedback inhibition or repression by end products or intermediate metabolites. Therefore, obtaining key enzyme mutants with excellent catalytic properties by screening or molecular modification of aspartate kinase is of great significance for the efficient production of aspartate family amino acids. Summary of the Invention

[0005] Based on the above needs, the primary purpose of the present invention is to provide an aspartate kinase mutant so that its catalytic performance is improved, which is conducive to the production of metabolites such as L-homoserine by microbial fermentation.

[0006] The present invention provides a wild-type Saccharomyces cerevisiae The S288C aspartate kinase LysC mutant is characterized in that, relative to the amino acid sequence shown in SEQ ID No. 2, only one of the mutations is present: valine V is mutated to alanine A at position 299, asparagine N is mutated to serine S at position 317, or asparagine N is mutated to serine S at position 485; only a combination of mutations is present: glutamine Q is mutated to arginine R at position 107 and asparagine N is mutated to serine S at position 317; only a combination of mutations is present: threonine T is mutated to serine S at position 114 and glutamine Q is mutated to arginine R at position 510; and only a combination of mutations is present: threonine T is mutated to alanine A at position 155 and lysine K is mutated to glutamate E at position 390.

[0007] The present invention further provides a gene encoding the aspartate kinase LysC mutant. In a preferred embodiment, the nucleotide sequence of the aspartate kinase LysC encoding gene is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1. The present invention also provides an expression vector and a host cell containing the aspartate kinase LysC mutant encoding gene. In a preferred embodiment, the expression vector includes but is not limited to pACYC184 and pXMJ19, and a recombinant plasmid containing the LysC mutant encoding gene is constructed and introduced into microbial chassis cells for fermentation production of L-homoserine and downstream derivatives. The derivatives generally refer to the downstream metabolites of its metabolic pathway, such as threonine, leucine, isoleucine, etc. (such as Figure 1 Although lysine is not a derivative thereof, it can also be obtained by the method of the present invention because the LysC enzyme is located upstream in the metabolic pathway.

[0008] In a specific embodiment, the microbial chassis cells can be selected from the genus Corynebacterium, Enterobacter or Saccharomyces, preferably Escherichia coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ).

[0009] The present invention also provides the use of aspartate kinase mutants in the fermentation production of L-homoserine and its derivatives. A chassis engineering bacterium with a certain L-homoserine production capacity was selected for application performance testing. In a specific embodiment, the engineered bacterium Ec-Hom is characterized by Escherichia coli Escherichia coli Knockout of homoserine degradation pathway genes in MG1655 thrB (ProteinID: NP_414544) and metA (Protein ID: NP_418437), and overexpressed aspartate kinase / homoserine dehydrogenase genes thrA (Protein ID: NP_414543); In another specific embodiment, the engineered bacteria Cg-Hom is characterized by Corynebacterium glutamicum Corynebacterium glutamicum ATCC13032 knocks out its own homoserine degradation pathway genes thrB (Protein ID: CAF19888), while overexpressing the homoserine dehydrogenase gene hom (Protein ID: CAF19887).

[0010] The present invention is achieved through the following technical approach: using the Saccharomyces cerevisiae genome as a template, an error-prone PCR random mutagenesis method was used to generate a mutant library of the LysC encoding gene. This library was then subcloned into an expression vector using a Golden Gate-based vector construction strategy to generate a recombinant plasmid library containing genes encoding aspartate kinase LysC mutants. The recombinant plasmid library was then introduced into Escherichia coli (which already contained a homoserine biosensor, PhomBio) knocked out for the homoserine degradation pathway. Through primary screening in 96-well plates and secondary screening in test tubes and shake flasks, mutants with enhanced enzyme activity were identified. The mutations at these sites were V299A, N317S, N485S, Q107R / N317S, T114S / Q510R, and T155A / K390E, respectively.

[0011] The present invention has the beneficial effect of obtaining aspartate kinase mutants with varying degrees of enhanced catalytic activity by mutating and screening the LysC gene encoding aspartate kinase from Saccharomyces cerevisiae. Therefore, the several aspartate kinase mutants provided by the present invention lay a good foundation for the efficient fermentation production of L-homoserine and other downstream aspartate family metabolites, and have better prospects for industrial application than unmutated aspartate kinases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the microbial L-homoserine biosynthesis pathway.

[0013] Figure 2 Aspartate kinase mutant enzyme activity assay.

[0014] Figure 3 Analysis of L-homoserine production by engineered bacteria. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples and with reference to the accompanying drawings, but it should not be understood as limiting the present invention. The experimental methods used in the examples are conventional methods well known to those skilled in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0016] Example 1 Construction of Aspartate Kinase LysC Variant Library

[0017] The present invention uses the low-fidelity EasyTaq DNA polymerase (Beijing TransGen Biotech, China) to Saccharomyces cerevisiaeThe S288C genome was used as a template and primers P1 (5'-caccaggtctcagaccatgccaatggatttccaacctacatcaag-3') and P2 (5'-caccaggtctcagatctcagtggtggtggtggtggtgaattc-3') were used to obtain the gene by error-prone PCR. lysC Encoding gene mutation library. By adding a certain concentration of magnesium ions and manganese ions to the PCR reaction system, the fidelity of the PCR amplification process is further reduced and the obtained lysC The coding gene contains 2-3 point mutations. The error-prone PCR system used in this invention is: 5 μL 10×EasyTaq buffer, 0.2 μM upstream primer P1, 0.2 μM upstream primer P2, 200 μM dNTPs, 0.8 mM MnCl2, 6 mM MgSO4, 50 ng template DNA, 1 μL EasyTaq DNA polymerase, and sterile water to make up to 50 μL. The PCR reaction procedure is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 2 min, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0018] The pACYC plasmid backbone was generated by conventional PCR using the high-fidelity Phusion High-Fidelity DNA Polymerase, using the pACYC184 plasmid as a template and primers P3 (5'-caccaggtctcagatccggctgctaacaaagcc-3') and P4 (5'-caccaggtctcaggtctgtttcctgtgtgaaattgttatccg-3'). The reaction mixture consisted of 10 μL of 5× Phusion HF buffer, 0.5 μM upstream primer P1, 0.5 μM upstream primer P2, 200 μM dNTPs, 3% DMSO, 50 ng of template DNA, and 0.5 μL of Phusion High-Fidelity DNA Polymerase (Thermo Scientific, USA). Sterile water was added to make up to 50 μL. The PCR reaction program was as follows: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s; annealing at 62°C for 20 s; extension at 72°C for 3 min, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0019] Based on the Goldengate method, the PCR-derived lysC The coding gene mutation library was connected to the pACYC184 plasmid backbone to obtain lysCRecombinant expression plasmid pACYC184-lysC encoding different gene mutants mut The GoldenGate ligation system consisted of 1.5 μL of T4 DNA Ligase buffer, 1 μL of T4 DNA Ligase, 1 μL of BsaI, 1.5 μL of 10× BSA, and a plasmid backbone and fragment (1:1 molar ratio, 10 μL total) in a total volume of 15 μL. PCR ligation conditions were: 37°C for 3 minutes, 22°C for 4 minutes (40 cycles); 22°C for 30 minutes; 80°C for 5 minutes; and 4°C for 10 minutes. The ligation system was then introduced into E. coli DH5α competent cells using the conventional E. coli heat shock transformation method to obtain a recombinant plasmid library.

[0020] Example 2 Screening and Identification of Aspartate Kinase LysC Variant Library

[0021] The present invention establishes a screening method for aspartate kinase LysC mutants using a homoserine biosensor. The homoserine biosensor, PhomBio, has been disclosed in the applicant's previously mentioned patent application (Application No. 202110059860.2). It is constructed based on the Corynebacterium glutamicum transcriptional regulator NCgl0581. The genomic region containing the NCgl0581 open reading frame and the NCgl0580 promoter is cloned in front of GFP, so that GFP expression is controlled by the NCgl0580 promoter. This biosensor can link L-homoserine concentration with fluorescence signal intensity, allowing visualization of L-homoserine concentration levels and sensitive detection of the target product, L-homoserine.

[0022] Homoserine biosensor can respond to L-homoserine. As the intracellular L-homoserine concentration increases, the fluorescence intensity also increases. Therefore, the present invention establishes a high-throughput screening method based on the L-homoserine biosensor. The recombinant plasmid mutation library and homoserine biosensor plasmid in Example 1 are introduced into Escherichia coli in which the homoserine degradation pathway has been knocked out, and beneficial mutants with high fluorescence intensity are obtained by flow cytometry screening. For mutants with improved aspartate kinase enzyme activity, the concentration of L-homoserine produced in the cell will increase accordingly, and the fluorescence intensity will also increase. The Escherichia coli in which the homoserine degradation pathway has been knocked out refers to E. coli Knockout in MG1655 metA and thrB , the strain genotype is: ∆metA ∆thrB .

[0023] The specific screening steps are as follows: first, transfer the homoserine biosensor plasmid into E. coli MG1655 ∆metA∆thrB The strain was then made competent and transformed into the recombinant plasmid mutation library described in Example 1. The strain was then plated onto selective antibiotic agar plates containing chloramphenicol and kanamycin. Colonies on the plates were washed with sterile PBS and inoculated into shake flasks containing fermentation medium. Initial OD = 0.5 was achieved, and fermentation was carried out overnight at 30°C and 200 rpm. One ml of the fermentation broth was centrifuged, washed three times with PBS, and diluted to an OD of 0.1. The broth was then sorted by flow cytometry. Mutants with higher fluorescence intensity than the wild-type control were collected and plated on plates and incubated overnight at 37°C. Use a sterilized toothpick to pick a single colony to a 96-well plate containing 200ul LB medium, culture at 37℃ for about 12h, transfer to a 96-deep-well plate containing 600μL LB medium to OD=0.6, add 0.4mM IPTG, induce overnight at 16℃, 800rpm, collect the bacteria by centrifugation, add 200μL lysis buffer containing 3mg / ml lysozyme to resuspend, react at 37℃ for 2h, centrifuge and take the supernatant to measure enzyme activity; the mutants with improved enzyme activity obtained in the initial screening were transferred to a test tube containing 5ml LB medium, cultured at 37℃ to OD=0.6, and added 0.4mM IPTG was used for overnight induction at 16°C, the cells were collected by centrifugation, disrupted by ultrasound, and the supernatant was collected by centrifugation to determine the enzyme activity. The mutation sites were determined by Sanger sequencing. After three rounds of screening, six different beneficial LysC mutants were finally obtained, namely V299A, N317S, N485S, Q107R / N317S, T114S / Q510R, and T155A / K390E.

[0024] The LB medium components include: 1% yeast extract, 2% tryptone, and 1% NaCl. The fermentation medium components include: 50 g / L glucose, 2 g / L yeast extract, 2 g / L MgSO4·7H2O, 4 g / L KH2PO4, 14 g / L (NH4)2SO4, 0.5 g / L methionine, 0.5 g / L threonine, and 20 mL of an inorganic salt ion stock solution, wherein the inorganic salt ion stock solution includes 10 g / L FeSO4·7H2O and 0.5 g / L MnSO4·4H2O.

[0025] Example 3 Enzyme Activity Assay of Aspartate Kinase LysC Variants

[0026] Based on the Goldengate method, the PCR-derived lysC The encoding gene mutation fragment was connected to the pET21b plasmid backbone to obtain lysC Expression plasmid pET21b-lysC encoding different mutants of the gene mutThe plasmid was introduced into competent E. coli BL21 cells using the standard heat-shock transformation method. The induced expression cells were harvested, the culture medium removed by centrifugation, and the pellet resuspended in 10 mL of pre-chilled lysis buffer (20 mM Na₂HPO₃, 200 mM NaCl, pH 7.5). The cells were disrupted using an ultrasonic cell disruptor at 200 W power, with sonication for 10 minutes, 2 s on, 1 s off. The cells were then centrifuged at 8000 × g for 10 minutes in a high-speed refrigerated centrifuge. The supernatant was collected for subsequent protein purification and enzyme activity assays.

[0027] Because aspartate kinase catalyzes the conversion of aspartate to aspartate phosphate, which is then reduced to aspartate semialdehyde by aspartate semialdehyde dehydrogenase (ASDH), consuming NADPH. Therefore, the enzyme activity of aspartate kinase can be calculated by continuously measuring the decrease in absorbance at 340 nm in the presence of NADPH. The reaction system consists of 11.6 mM aspartate, 70 mM ATP, 5.8 mM MgAc2, 0.60 mM NADPH, 588 mM KCl, 140 mM Tris-HCl (pH 7.5), 3–5 units of aspartate semialdehyde dehydrogenase, and an appropriate amount of aspartate kinase sample. Under these conditions, one unit of enzyme activity is defined as the oxidation of 1 μmol of NADPH per minute.

[0028] By measuring the activity of aspartate kinase, Figure 2 The results showed that compared with the unmutated aspartate kinase, most aspartate kinase LysC mutants exhibited significantly enhanced enzyme activity. The enzyme activities of the V299A and Q107R / N317S mutants reached 12.49 ± 0.57 μmol / min / mg and 13.08 ± 0.13 μmol / min / mg, respectively, which were 3.6- and 3.8-fold higher than those of the wild-type control, suggesting that they have greater prospects for industrial application.

[0029] Example 4: Expressing an aspartate kinase LysC mutant to improve L-homoserine production by engineered bacteria

[0030] Based on the aspartate kinase mutant obtained in Example 3, a microbial cell factory was constructed for fermentation production of L-homoserine and downstream derivatives. The preferred plasmid in this example is the Escherichia coli-Corynebacterium glutamicum pXMJ19 shuttle inducible expression vector. This plasmid itself contains a strong promoter tac-related sequence (including the operator sequence lacO). When an inducer such as IPTG or lactose is added, the repressor protein is forced to leave the operator sequence, thereby initiating gene expression. Based on the Goldengate method, the PCR-derived lysCThe encoding gene mutation fragment was connected to the pXMJ19 plasmid backbone to obtain lysC Expression plasmid pXMJ19-lysC encoding different mutants of the gene mut The corresponding expression plasmids were transformed into chassis engineering bacteria with certain L-homoserine production capacity to test the fermentation performance. In a specific embodiment, the engineering bacteria Ec-Hom is characterized by Escherichia coli Escherichia coli Knockout of homoserine degradation pathway genes in MG1655 thrB (ProteinID: NP_414544) and metA (Protein ID: NP_418437), and overexpressed aspartate kinase / homoserine dehydrogenase genes thrA (Protein ID: NP_414543); In another specific embodiment, the engineered bacteria Cg-Hom is characterized by Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032 knocks out its own homoserine degradation pathway genes thrB (Protein ID: CAF19888), while overexpressing the homoserine dehydrogenase gene hom (Protein ID: CAF19887).

[0031] The engineered strains obtained above were inoculated into 20 mL LBHIS medium flasks and cultured for 16 h until mid-logarithmic phase. Cells were collected by centrifugation, suspended in fresh fermentation medium, and inoculated into 500 mL flasks containing 25 mL fermentation medium. The initial OD was adjusted to 600 The LBHIS medium contains 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 18.5 g / L brain heart infusion, and 91 g / L sorbitol. The fermentation medium contains 60 g / L glucose, 0.5 g / L urea, 30 g / L corn steep liquor, 20 g / L ammonium sulfate, 5 g / L KH2PO4, 5 g / L MgSO4·7H2O, 0.2 g / L FeSO4·7H2O, 0.1 g / L MnCl2·4H2O, 0.2 mg / L biotin, 10 mg / L vitamin mixture, and 10 g / L CaCO3 as a buffer for the fermentation medium. The seed culture medium includes 20 g / L glucose, 0.2 g / L urea, 15 g / L corn steep liquor, and 20 g / L ammonium sulfate.

[0032] The engineered strains obtained above were inoculated into 250 mL Erlenmeyer flasks containing 20-25 mL seed culture medium and cultured for 12-16 hours until the mid-logarithmic phase. The cells were then collected by centrifugation, suspended in fresh fermentation medium, and inoculated into 250 mL Erlenmeyer flasks containing 50 mL fermentation medium. The initial OD 600 The pH value was 0.8-1.2, and 5-15 g / L CaCO₃ was added. Fermentation conditions were 30°C, 150-200 rpm, and constant shaking. The fermentation medium consisted of: 50-60 g / L glucose, 0.1-0.2 g / L urea, 5-10 g / L corn steep liquor, 15-20 g / L ammonium sulfate, 5-6 g / L KH₂PO₄, 2-5 g / L MgSO₄·7H₂O, 0.2-0.5 g / L FeSO₄·7H₂O, 0.2-0.4 g / L MnCl₂·4H₂O, 0.2-0.4 mg / L biotin, 1-2 mg / L vitamin B1, 1-2 mg / L vitamin B6, and 5-15 g / L CaCO₃ as a buffer for the fermentation medium. The seed culture medium includes 10-20 g / L glucose, 0.1-0.2 g / L urea, 15-20 g / L corn steep liquor, 15-20 g / L ammonium sulfate, and IPTG at a concentration of 0.2 mM. The results are shown in Table 1:

[0033] Table 1 Effect of expression of aspartate kinase LysC mutant on fermentation production of L-homoserine by chassis engineering bacteria

[0034]

[0035] After 72 hours of fermentation, overexpression of the LysC mutant aspartate kinase in the Ec-Hom engineered strain of Escherichia coli increased L-homoserine production by 80%-285% compared to the control starting strain. Overexpression of the LysC mutant in the Cg-Hom engineered strain of Corynebacterium glutamicum increased L-homoserine production by 5%-49%. Compared to unmutated aspartate kinase, expression of the LysC mutant in the chassis engineered strain significantly enhanced the strain's ability to produce L-homoserine.

Claims

1. A LysC mutant of aspartate kinase derived from Saccharomyces cerevisiae, characterized in that: With respect to the amino acid sequence shown in SEQ ID No. 2, There is only a combined mutation of glutamine Q to arginine R at position 107 and asparagine N to serine S at position 317; There is only a combined mutation of Threonine T to Serine S at position 114 and Glutamine Q to Arginine R at position 510; or There was only a combined mutation of threonine T to alanine A at position 155 and lysine K to glutamate E at position 390.

2. The gene encoding the aspartate kinase LysC mutant according to claim 1.

3. The coding gene according to claim 2, wherein The nucleotide sequence is obtained by performing mutation on the basis of the nucleotide sequence shown in SEQ ID No.

1.

4. An expression vector containing the coding gene according to claim 2 or 3.

5. A host cell containing the encoding gene according to claim 2 or 3, characterized in that It is Escherichia coli, Corynebacterium glutamicum or Saccharomyces cerevisiae.

6. Use of the aspartate kinase mutant or the encoding gene thereof according to claim 1 in the preparation of L-homoserine.

7. A method for preparing L-homoserine, characterized in that: The recombinant microorganism containing the coding gene according to claim 2 or 3 is fermented to produce the L-homoserine; the microorganism is Escherichia coli, Corynebacterium glutamicum or Saccharomyces cerevisiae having the ability to produce L-homoserine.

8. The method according to claim 7, wherein The homoserine degradation pathway gene of the Escherichia coli is knocked out thrB and metA genes, and simultaneously overexpressed the fusion aspartate kinase / homoserine dehydrogenase gene thrA .

9. The method according to claim 7, wherein The homoserine degradation pathway gene of Corynebacterium glutamicum is knocked out thrB , while overexpressing the homoserine dehydrogenase gene hom .

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