D-3-phosphoglycerate dehydrogenase mutant, coding gene thereof and application of D-3-phosphoglycerate dehydrogenase mutant and coding gene

Abstract

The invention relates to the field of gene engineering, and discloses a D-3-phosphoglycerate dehydrogenase mutant, a coding gene thereof and application of the D-3-phosphoglycerate dehydrogenase mutant and the coding gene. Compared with a protein with an amino acid sequence as shown in SEQ ID NO: 1, the mutant comprises mutation at the 131 site and / or the 285 site. The mutant can specifically catalyze a substrate 3-phosphoglycerate to synthesize 3-phosphate-hydroxypyruvic acid, can effectively promote synthesis of L-serine in a metabolic pathway of microorganisms, improves the yield and conversion rate of the L-serine, and reduces the yield of byproducts at the same time.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a D-3-phosphoglycerate dehydrogenase mutant and a coding gene thereof and applications thereof. Background Art

[0002] L-serine is a basic amino acid that makes up proteins. It has important applications in medicine, food, and cosmetics. In food, L-serine can mainly improve the flavor and sensory properties of food. In medicine, L-serine derivatives such as cycloserine and azoserine can be used as drugs. L-serine can also be used as a precursor to synthesize substances such as choline and purine. In cosmetics, L-serine is also an important natural moisturizing factor that can maintain moisture in the stratum corneum of the skin. L-serine is considered one of the top 30 most meaningful biochemicals due to its potential use as a biochemical basic material. At present, the market demand for L-serine is increasing, but its production technology is relatively backward.

[0003] Industrially, L-serine is mainly produced by protein hydrolysis, chemical synthesis, enzyme conversion and microbial fermentation. Protein hydrolysis and chemical synthesis have problems such as complex process, difficult separation and purification, and heavy pollution; enzyme conversion has the advantages of simple process and high product purity, but due to the expensive precursors and low conversion rate, large-scale industrial mass production is limited. At present, microbial fermentation is more conducive to large-scale industrial application due to its advantages such as green and environmental protection in the production process, greatly reduced costs, and easy reaction control. However, the metabolic network of microbial synthesis of L-serine is complex, and the key rate-limiting enzymes in the synthesis pathway have poor specificity and low conversion rate, which limit the industrialization process. Therefore, solving the specificity problem of the key enzymes in the microbial L-serine synthesis pathway and improving the conversion rate are currently urgent problems to be solved.

[0004] In Escherichia coli, glucose enters the L-serine branch pathway through 3-phosphoglycerate (3-PG) in the glycolysis (EMP) pathway; in the L-serine branch pathway, 3-PG is catalyzed by phosphoglycerate dehydrogenase (SerA) to synthesize 3-phospho-hydroxypyruvate (3-PHP), and then catalyzed by SerC to form the intermediate product 3-phosphoserine. This step requires glutamate as an amino donor, and glutamate is converted into α-ketoglutarate. Finally, 3-phosphoserine is synthesized into L-serine under the catalysis of SerB. Among them, SerA is the key rate-limiting enzyme in L-serine synthesis. However, it can not only catalyze 3-PG to form 3-PHP in the L-serine synthesis pathway, but also catalyze the formation of α-hydroxyglutarate as a byproduct using α-ketoglutarate as a substrate. This non-specific catalytic characteristic of SerA leads to low yield and conversion rate of L-serine synthesis. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of poor specificity of key enzymes in the L-serine synthesis pathway in the prior art, and to provide a D-3-phosphoglycerate dehydrogenase mutant and its encoding gene and their application. The D-3-phosphoglycerate dehydrogenase mutant has a specific catalytic effect on the substrate and can improve the yield and conversion rate of L-serine.

[0006] In order to achieve the above objectives, the first aspect of the present invention provides a D-3-phosphoglycerate dehydrogenase mutant, which comprises a mutation at the R site compared to the protein with the amino acid sequence shown in SEQ ID NO: 1; the R site is position 131 and / or position 285.

[0007] Preferably, compared with the protein having the amino acid sequence shown in SEQ ID NO: 1, the mutant comprises a mutation of asparagine at position 131 to alanine and / or a mutation of aspartic acid at position 285 to leucine.

[0008] Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:2.

[0009] The second aspect of the present invention provides a gene encoding a D-3-phosphoglycerate dehydrogenase mutant, wherein the gene has a nucleotide sequence encoding the D-3-phosphoglycerate dehydrogenase mutant as described above.

[0010] Preferably, the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2.

[0011] Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0012] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0013] Preferably, the expression vector of the recombinant vector is the ptrc99a plasmid.

[0014] A fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0015] Preferably, the host strain of the recombinant strain is Escherichia coli with auxiliary genes knocked out, and the auxiliary genes include sdaA gene, sdaB gene and tdcG gene.

[0016] The fifth aspect of the present invention provides the use of at least one of the above-mentioned D-3-phosphoglycerate dehydrogenase mutant, the above-mentioned gene, the above-mentioned recombinant vector and the above-mentioned recombinant strain in the production of L-serine.

[0017] A sixth aspect of the present invention provides a method for producing L-serine, the method comprising the following steps: inoculating the recombinant strain described above into a fermentation medium for fermentation culture.

[0018] Preferably, the fermentation medium contains glucose, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, threonine, glutamic acid, alanine, biotin and trace element mother solution.

[0019] Preferably, the fermentation medium contains 2-6 g / L of glucose, 1-3 g / L of potassium dihydrogen phosphate, 1-3 g / L of magnesium sulfate, 2-4 g / L of ammonium sulfate, 0.1-0.2 g / L of threonine, 0.5-1 g / L of glutamic acid, 0.03-0.1 g / L of alanine, 0.5-1.5 mL / L of trace element mother solution, and 4-8 mg / L of biotin; wherein the trace element mother solution contains 3-8 mg / L of zinc sulfate, 1-3 mg / L of manganese sulfate, 0.3-0.8 mg / L of cobalt chloride, 0.1-0.3 mg / L of copper sulfate, and 4-8 mg / L of vitamin B12. 1 3-8mg / L.

[0020] Preferably, the fermentation culture conditions include: a temperature of 35-40°C and a rotation speed of 180-220 rpm.

[0021] Through the above technical solution, the beneficial effects of the present invention are:

[0022] The D-3-phosphoglycerate dehydrogenase mutant provided by the present invention can specifically catalyze the synthesis of 3-phosphoglycerate from the substrate 3-phosphoglycerate, but has a weaker catalytic effect on the conversion of the substrate α-ketoglutarate into α-hydroxyglutarate, and can effectively promote the synthesis of L-serine in the metabolic pathway of microorganisms, increase the yield and conversion rate of L-serine, and reduce the yield of by-products such as α-hydroxyglutarate. DETAILED DESCRIPTION

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] In the first aspect, the present invention provides a D-3-phosphoglycerate dehydrogenase mutant (hereinafter referred to as SerA mutant), which comprises a mutation at the R site compared to the protein having an amino acid sequence as shown in SEQ ID NO: 1; the R site is position 131 and / or position 285.

[0025] Based on the fact that the non-specific catalytic effect of D-3-phosphoglycerate dehydrogenase (hereinafter referred to as SerA) directly affects the synthesis of L-serine, the inventors of the present invention cleverly discovered during the research and development process that, taking SerA (amino acid sequence shown in SEQ ID NO: 1, and the specific acquisition method can refer to the document The Mechanism of Velocity Modulated Allosteric Regulation in D-3-Phosphoglycerate Dehydrogenase, J. Biol. Chem. 271, 23235-23238) which has released the feedback inhibition of L-serine as wild-type SerA, after mutation occurs at specific sites (position 131 and / or position 285), the obtained mutant can effectively enhance the specific catalytic effect on the substrate 3-phosphoglycerate while maintaining the original enzyme activity, thereby promoting the metabolic flow of L-serine synthesis in the metabolic process of microorganisms, and effectively improving the yield and conversion rate of L-serine synthesized by microbial culture.

[0026] According to the present invention, preferably, the mutant comprises a mutation of asparagine at position 131 to alanine and / or a mutation of aspartic acid at position 285 to leucine compared to the protein having an amino acid sequence as shown in SEQ ID NO: 1. The inventors have found that under this preferred embodiment, it is beneficial to further enhance the specific catalytic effect of the mutant on 3-phosphoglycerate and increase the yield of L-serine.

[0027] In the present invention, the asparagine at position 131 of the protein shown in the amino acid sequence SEQ ID NO: 1 is mutated to alanine and the aspartic acid at position 285 is mutated to leucine to form a mutant serA (N131A) (D285L) with an amino acid sequence such as SEQ ID NO: 2; the asparagine at position 131 of the protein shown in the amino acid sequence SEQ ID NO: 1 is mutated to alanine to form a mutant serA (N131A) with an amino acid sequence such as SEQ ID NO: 4; the aspartic acid at position 285 of the protein shown in the amino acid sequence SEQ ID NO: 1 is mutated to leucine to form a mutant serA (D285L) with an amino acid sequence such as SEQ ID NO: 5. Preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 2.

[0028] According to the present invention, there is no particular limitation on the method for obtaining the SerA mutant, as long as a SerA mutant having an amino acid sequence with the above characteristics can be obtained. For example, the SerA mutant can be obtained by artificial synthesis, or its encoding gene can be obtained through the amino acid sequence of the mutant, and then obtained by corresponding biological expression.

[0029] The second aspect of the present invention provides a gene encoding a D-3-phosphoglycerate dehydrogenase mutant, wherein the gene has a nucleotide sequence encoding the D-3-phosphoglycerate dehydrogenase mutant as described above.

[0030] In the present invention, any gene capable of encoding and expressing the above-mentioned SerA mutant belongs to the protection scope of the present invention. It is well known to those skilled in the art that genetic codons are degenerate. Therefore, when the amino acid sequence of the above-mentioned SerA mutant is known, those skilled in the art can obtain genes with different nucleotide sequences and capable of encoding the above-mentioned SerA mutant according to conventional technical means. For example, according to the coding gene of wild-type SerA (for example, the coding gene with a nucleotide sequence as shown in SEQ ID NO: 8), the codon encoding the amino acid at the mutation site can be adjusted accordingly according to the change of the amino acid at the mutation site. For another example, according to the characteristics of the expression host, the coding gene can also be optimized to increase the expression amount and / or expression efficiency of the SerA mutant.

[0031] According to the present invention, preferably, the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5. More preferably, the gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2.

[0032] In the present invention, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 may be shown as SEQ ID NO: 3, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 may be shown as SEQ ID NO: 6, and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5 may be shown as SEQ ID NO: 7. Further preferably, the nucleotide sequence of the gene is shown in SEQ ID NO: 3. The inventors have found that under this preferred embodiment, it is beneficial to further improve the synthesis conversion rate of L-serine.

[0033] The nucleotide sequences provided by the present invention can usually be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis. Once the relevant nucleotide sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombination. The obtained nucleotide sequence is usually cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequence is isolated from the propagated host cells by conventional methods.

[0034] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0035] The third aspect of the present invention provides a recombinant vector, which contains the gene as described above.

[0036] In the present invention, the expression vector used in the recombinant vector can be selected from various vectors known in the art, such as various commercially available plasmids, cosmids, phages and retroviruses, etc. The preferred expression vector of the present invention is the ptrc99a plasmid. The construction method of the recombinant vector can adopt various methods commonly used in the art, such as connecting the target gene to the expression vector after enzyme digestion.

[0037] A fourth aspect of the present invention provides a recombinant strain, which contains the gene or the recombinant vector as described above.

[0038] According to the present invention, the method for constructing the recombinant strain can be accomplished by conventional technical means in the art, for example, by introducing a vector inserted with a target gene into the corresponding strain.

[0039] In the present invention, any host cell such as an engineered bacterium that can be used to express exogenous genes in the art can be used to construct the recombinant strain provided by the present invention, as long as it can express the SerA mutant described in the present invention. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, such as Escherichia coli, Bacillus subtilis, etc.

[0040] According to the present invention, preferably, the host strain of the recombinant strain is Escherichia coli. There is no special requirement for the type of Escherichia coli used to construct the recombinant strain, and it can be various Escherichia coli commonly used in the art that can express the target gene. Further preferably, the host strain of the recombinant strain is Escherichia coli with auxiliary genes knocked out, and the knockout of the auxiliary genes is to knock out the catabolic pathway of serine to pyruvate in Escherichia coli, and the auxiliary genes include sdaA gene, sdaB gene and tdcG gene. The inventors found that under this preferred embodiment, it is beneficial to further improve the yield and conversion rate of L-serine.

[0041] Wherein, the starting strain of Escherichia coli can be a commercially available strain, such as Escherichia coli W3110; the sdaA gene is a gene encoding L-serine dehydratase, the sdaB gene is a gene encoding an L-serine dehydratase isozyme, and the tdcG gene is a gene encoding L-serine dehydrogenase. Exemplarily, the nucleotide sequence of the sdaA gene is shown in SEQ ID NO: 9, the nucleotide sequence of the sdaB gene is shown in SEQ ID NO: 10, and the nucleotide sequence of the tdcG gene is shown in SEQ ID NO: 11. The Escherichia coli with the auxiliary gene knocked out can be obtained by knocking out the sdaA gene, the sdaB gene, and the tdcG gene in the starting strain of Escherichia coli using a gene knockout method commonly used in the art, for example, including a λRed recombination system, a CRISPR-Cas9 recombination system, and RNAi, etc.

[0042] The D-3-phosphoglycerate dehydrogenase mutant provided by the present invention can specifically catalyze the synthesis of 3-phosphoglycerate from the substrate 3-phosphoglycerate, but has a weak catalytic effect on the conversion of the substrate α-ketoglutarate into α-hydroxyglutarate, and can effectively promote the synthesis of L-serine in the metabolic pathway of microorganisms. In a fifth aspect, the present invention provides the use of at least one of the D-3-phosphoglycerate dehydrogenase mutant, the gene, the recombinant vector, and the recombinant strain described above in the production of L-serine.

[0043] A sixth aspect of the present invention provides a method for producing L-serine, the method comprising the following steps: inoculating the recombinant strain described above into a fermentation medium for fermentation culture.

[0044] According to the present invention, the fermentation culture conditions can adopt conventional culture conditions, as long as the recombinant strain is proliferated in large quantities through the fermentation culture process. Preferably, the fermentation culture conditions include: a temperature of 35-40°C and a rotation speed of 180-220rpm. Exemplarily, the recombinant strain and / or the culture product of the recombinant strain is inoculated into the fermentation medium, and cultured at a temperature of 35-40°C and a rotation speed of 180-220rpm until the OD600 The induction agent is 0.6-0.7, and then isopropyl-β-D-thiogalactoside (IPTG) is added to continue inducing expression for 20-30 hours at a temperature of 35-40°C and a rotation speed of 180-220rpm. During fermentation and culture, the above gene can be translated into the corresponding protein, and the corresponding protein can play its role to promote the synthesis of L-serine. The final concentration of the inducer can be as long as it reaches the level that prompts the recombinant strain to express and produce the target protein (i.e., the D-3-phosphoglycerate dehydrogenase mutant provided by the present invention), for example, it can be 0.1-0.2mM.

[0045] In the present invention, the recombinant strain can be first subjected to seed culture before fermentation culture to enhance the growth activity of the recombinant strain. Specifically, a single colony of the recombinant strain can be inoculated into a seed culture medium containing an antibiotic as a screening marker for seed culture to obtain a seed solution, and then the seed solution can be inoculated into a fermentation culture medium for the fermentation culture. Wherein, the single colony of the recombinant strain can be selected from the freshly prepared recombinant strain or the recombinant strain frozen at low temperature (for example, a recombinant strain frozen in a glycerol cryopreservation tube in a -80°C refrigerator); the screening marker antibiotic can be kanamycin (the concentration in the culture medium is 50 μg / μL), the seed culture medium can use a conventional culture medium, such as LB culture medium, and the conditions of the seed culture include: a temperature of 35-40°C, a rotation speed of 180-220rpm, and a time of 8-15h. Accordingly, the inoculation amount of the seed solution in the fermentation culture medium is 0.5-1.5% by volume.

[0046] According to the present invention, preferably, the fermentation medium contains glucose, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, threonine, glutamic acid, alanine, biotin and trace element mother solution. The inventors found that under this preferred embodiment, it is conducive to promoting the growth of the recombinant strain and improving the efficiency of synthesizing L-serine. Wherein, the trace element mother solution can adopt a solution containing trace elements required for microbial growth conventional in the art, for example, the trace element mother solution contains zinc, manganese, cobalt, copper, etc.

[0047] According to the present invention, preferably, the fermentation medium contains 2-6 g / L of glucose, 1-3 g / L of potassium dihydrogen phosphate, 1-3 g / L of magnesium sulfate, 2-4 g / L of ammonium sulfate, 0.1-0.2 g / L of threonine, 0.5-1 g / L of glutamic acid, 0.03-0.1 g / L of alanine, 0.5-1.5 mL / L of trace element mother solution and 4-8 mg / L of biotin; wherein the trace element mother solution contains 3-8 mg / L of zinc sulfate, 1-3 mg / L of manganese sulfate, 0.3-0.8 mg / L of cobalt chloride, 0.1-0.3 mg / L of copper sulfate, and 4-8 mg / L of vitamin B12. 13-8 mg / L. The inventors found that under this preferred embodiment, it is beneficial to further increase the yield of L-serine and reduce the synthesis of by-products.

[0048] The present invention will be described in detail below through examples.

[0049] In the following examples, the experimental methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0050] In the following examples, 2×Phanta Mix high-fidelity enzyme was purchased from Nanjing Novozymes Biotech Co., Ltd. with the catalog number P525-01; 2×rapid taq enzyme was purchased from Nanjing Novozymes Biotech Co., Ltd. with the catalog number P222-01; Escherichia coli DH5α competent cells were purchased from Nanjing Novozymes Biotech Co., Ltd. with the catalog number C502-03; DpnI digestion enzyme was purchased from Thermo Fisher Scientific, Germany with the catalog number E101; and one-step cloning kit ClonExpress IIOne StepCloning Kit was purchased from Nanjing Novozymes Biotech Co., Ltd. with the catalog number C112-01.

[0051] In the following examples, the HPLC detection method of L-serine is the HPLC pre-column derivatization method, and the specific process is: after the fermentation broth is diluted 10 times, it is centrifuged at 12000rpm for 10min, and the supernatant is filtered with a filter membrane and then tested by HPLC; before entering the chromatographic column, the supernatant is mixed with an OPA derivatizer to react to generate a fluorescent product, which is detected by a UV detector; the chromatographic column is EC C18, and the preparation process of the mobile phase A is as follows: 31.2g of sodium dihydrogen phosphate dihydrate is weighed, ultrapure water is added to nearly 5L, and then the pH of the solution is adjusted to 7.80 with sodium hydroxide, and then the volume is fixed to 5L, and 0.22μm water membrane is filtered; the preparation process of mobile phase B is as follows: 900mL of methanol, 900mL of acetonitrile, and 200mL of ultrapure water are measured, mixed, and filtered with a 0.22μm water membrane; the chromatographic column temperature is 40°C, and the detection wavelength is 338nm;

[0052] Preparation of OPA derivative: Weigh 1.372 g of o-phthalaldehyde (OPA) and 0.5888 g of N-acetyl-L-cysteine ​​in a 100 mL volumetric flask, add 20 mL of anhydrous ethanol, dissolve by ultrasonication, and then dilute to 100 mL with 0.05 M sodium borate buffer (0.05 M, pH adjusted to 9.5 with sodium hydroxide), and keep away from light for later use.

[0053] The elution conditions of HPLC are shown in Table 1.

[0054] Table 1

[0055] Time min A％ B％ Flow rate ml / min Maximum pressure limit (bar) 0 100 0 1.00 400.00 1 100 0 1.00 400.00 13 43 57 1.00 400.00 16 0 100 1.00 400.00 19 100 0 1.00 400.00

[0056] In the following examples, the components of the fermentation medium are: 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 4 g / L glucose, 0.12 g / L threonine, 0.7 g / L glutamic acid, 0.05 g / L alanine, 3 g / L ammonium sulfate, 1 mL / L 1X trace element mother solution (formula see Table 2), 15 mL / L biotin mother solution (biotin concentration is 0.4 g / L);

[0057] Table 2

[0058] Reagents Final concentration (g / L) Mother liquor (1000×)(g / L) Zinc sulfate heptahydrate 0.005 5 Manganese sulfate monohydrate 0.002 2 Cobalt chloride hexahydrate 0.0005 0.5 Copper sulfate pentahydrate 0.0002 0.2 <![CDATA[Vitamin B 1 > 0.005 5

[0059] Example 1

[0060] 1. Construction of engineered strains Ls001, Ls002 and Ls003

[0061] (1) PCR amplification was performed using primer pair ΔsdaA-UF (nucleotide sequence shown in SEQ ID NO: 20) and ΔsdaA-UR (nucleotide sequence shown in SEQ ID NO: 21) to obtain the upstream homology arm ΔsdaA-UP fragment; PCR amplification was performed using primer pair ΔsdaA-DF (nucleotide sequence shown in SEQ ID NO: 22) and ΔsdaA-DR (nucleotide sequence shown in SEQ ID NO: 23) to obtain the downstream homology arm ΔsdaA-DOWN fragment; overlapping PCR was used to connect UP and DOWN to obtain the homologous recombinant fragment UP-DOWN (ΔsdaA-donor fragment); primer pair ΔsdaA-sgF (nucleotide sequence shown in SEQ ID NO: 24) and ΔsdaA-sgR (nucleotide sequence shown in SEQ ID NO: 25) to obtain the downstream homology arm ΔsdaA-DOWN fragment; NO: 25) was used to amplify the pTarget-ΔsdaA-sgRNA fragment by PCR, which was transformed into Escherichia coli DH5α competent cells, cultured overnight, verified by colony PCR, and sent for sequencing to obtain the pTarget-ΔsdaA-sgRNA targeting plasmid.

[0062] The PCR amplification reaction system (50 μL) in this process is: ddH 2 O 20μL, upstream primer (10mM) 2μL, downstream primer (10mM) 2μL, corresponding genomic DNA template 1μL, 2×Phanta Mix high-fidelity enzyme 25μL; PCR reaction program is: step 1: 95℃, 3min; step 2: 95℃, 15s, 60℃, 10s; step 3: 72℃, 1min; this step is repeated 30 cycles, 72℃, 5min; step 4: store at 4℃.

[0063] The overlapping PCR reaction system (50 μL) in this process was as follows: 2×Phanta Mix high-fidelity enzyme 25 μL, ΔsdaA-UP and ΔsdaA-DOWN fragments 1 μL each, ΔsdaA-UF (10 mM) and ΔsdaA-DR (10 mM) 2 μL each, ddH 2 O 19μL; overlapping PCR reaction program is: step 1: 95℃, 3min; step 2: 95℃, 15s, 60℃, 10s; step 3: 72℃, 2min; this step is repeated 30 cycles, 72℃, 5min; step 4: store at 4℃.

[0064] (2) The pTarget-ΔsdaA-sgRNA targeting plasmid and ΔsdaA-donor fragment constructed above were electroporated into wild-type Escherichia coli (purchased from Shanghai Collection Biotechnology Center, numbered W3110) for gene editing. Single colony PCR verification was performed using ΔsdaA-UF / ΔsdaA-DR verification primers. The correctly verified strain was sent for sequencing. The strain with the correct sequencing was the strain with successful gene editing and was named the engineered strain Ls001.

[0065] (3) The ΔsadB-UP fragment, ΔsadB-DOWN fragment, ΔsadB-donor fragment, and pTarget-ΔsadB-sgRNA targeting plasmid were obtained by the same method as step (1), and then the pTarget-ΔsadB-sgRNA targeting plasmid and ΔsdaB-donor fragment were electroporated into the engineering strain Ls001 by the same method as step (2) to obtain the engineering strain Ls002; the ΔtdcG-UP fragment, ΔtdcG-DOWN fragment, ΔtdcG-donor fragment, and pTarget-ΔtdcG-sgRNA targeting plasmid were obtained by the same method as step (1), and then the pTarget-ΔtdcG-sgRNA targeting plasmid and ΔtdcG-donor fragment were electroporated into the engineering strain Ls002 by the same method as step (2) to obtain the engineering strain Ls003; the nucleotide sequences of the primer pairs used are shown in Table 3.

[0066] Example 2

[0067] 1. Construction of a recombinant vector ptrc99a-serA(WT) expressing wild-type SerA: PCR amplification was performed using primers ptrc99a-CF (nucleotide sequence as shown in SEQ ID NO: 12) and ptrc99a-CR (nucleotide sequence as shown in SEQ ID NO: 13) to obtain an expression vector ptrc99a plasmid, and primers serA(WT)-F (nucleotide sequence as shown in SEQ ID NO: 14) and serA(WT)-R (nucleotide sequence as shown in SEQ ID NO: 15) to obtain an expression vector ptrc99a plasmid. NO: 15) was amplified by PCR to obtain the serA (WT) gene fragment; the serA (WT) gene fragment was connected to the expression vector ptrc99a plasmid using a one-step cloning kit to obtain a connected mixture; 10 μL of the above-mentioned mixture was transferred into the competent cells of Escherichia coli DH5a, cultured overnight, verified by colony PCR, and sent for sequencing to obtain a recombinant vector ptrc99a-serA (WT) expressing wild-type SerA; wherein, based on the feedback inhibition of L-serine on SerA, a large number of studies have been conducted to optimize the amino acid sequence of SerA to eliminate the feedback inhibition of L-serine. The SerA (WT) used in this embodiment is a SerA variant that has been freed from the feedback inhibition of L-serine, and the amino acid sequence is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 8. For details, please refer to the document "Al-Rabiee, R., Zhang, Y., and Grant, GA (1996) The Mechanism of Velocity Modulated Allosteric Regulation in D-3-PhosphoglycerateDehydrogenase.J.Biol.Chem.271,23235-23238";

[0068] The PCR reaction system (50 μL) in this process is: ddH 2 O 20μL, upstream primer (10mM) 2μL, downstream primer (10mM) 2μL, corresponding DNA template 1μL, 2×Phanta Mix high-fidelity enzyme 25μL; PCR reaction program is: step 1: 95℃, 3min; step 2: 95℃, 15s, 60℃, 10s; step 3: 72℃, 3min; this step is repeated for 30 cycles, 72℃, 5min; step 4: store at 4℃.

[0069] 2. Construction of a recombinant vector ptrc99a-serA(N131) expressing a mixed mutant serA(N131): using serA(N131)-F (nucleotide sequence as shown in SEQ ID NO: 16) and serA(N)-R primers (nucleotide sequence as shown in SEQ ID NO: 17), and using the ptrc99a-serA(WT) wild-type expression plasmid as a template, PCR was performed to randomly mutate the amino acid at position 131 of SerA to obtain a mixed mutant fragment of ptrc99a-serA(N131) using ptrc99a as an expression vector;

[0070] The PCR reaction system (50 μL) in this process is: ddH 2 O 20μL, upstream primer (10mM) 2μL, downstream primer (10mM) 2μL, plasmid template 1μL, 2×Phanta Mix high-fidelity enzyme 25μL; PCR reaction program is: step 1: 95℃, 3min; step 2: 95℃, 15s, 60℃, 10s; step 3: 72℃, 3min; this step is repeated for 30 cycles, 72℃, 5min; step 4: store at 4℃.

[0071] 3. Construction of a recombinant vector ptrc99a-serA(D285) expressing a mixed mutant serA(D285): Using serA(D285)-F (nucleotide sequence as shown in SEQ ID NO: 18) and serA(D)-R primers (nucleotide sequence as shown in SEQ ID NO: 19), and the ptrc99a-serA(WT) wild-type expression plasmid as a template, PCR was performed to randomly mutate the amino acid at position 285 of SerA to obtain a mixed mutant fragment of ptrc99a-serA(D285) using ptrc99a as an expression vector; in this process, the PCR reaction system and reaction procedure were the same as the PCR conditions of ptrc99a-serA(N131).

[0072] Table 3

[0073]

[0074]

[0075] 4. Construction of recombinant strains

[0076] The recombinant vector ptrc99a-serA (WT) obtained in step 1 was electroporated into the engineered strain Ls003 obtained in Example 1 to obtain the recombinant strain Ls003 / 0;

[0077] The ptrc99a-serA(N131) mixed mutant fragment obtained in step 2 was electroporated into the engineered strain Ls003 obtained in Example 1, and spread on a LB+Kan resistance plate. The monoclonal colonies obtained on the plate were mixed SerA(N131) mutant transformants. 32 monoclonal strains were randomly selected and named as recombinant strains Ls003 / 1-Ls003 / 32 in sequence.

[0078] The ptrc99a-serA (D285) mixed mutant fragment obtained in step 3 was electroporated into the engineered strain Ls003 obtained in Example 1 and spread on an LB+Kan resistance plate. The monoclonal colonies obtained on the plate were mixed SerA (D285) mutant transformants. 32 monoclonal strains were randomly selected and named as recombinant strains Ls003 / 33-Ls003 / 64.

[0079] Example 3

[0080] The recombinant strains Ls003 / 1-Ls003 / 64 and the control strain Ls003 / 0 obtained in Example 2 were inoculated into LB liquid culture medium test tubes supplemented with 50 μg / μL kanamycin, respectively, and cultured overnight (about 12 h) at 37°C and 200 rpm. The culture solution was transferred to 50 mL / 250 mL fermentation medium at an inoculum volume of 1% by volume, and cultured at 37°C and 200 rpm until OD 600 =0.6-0.8, 0.1 mM IPTG was added respectively, and the culture was continued at 37°C and 200 rpm for 24 h. Then 1 mL of the fermentation broth was taken, and the contents of L-serine and α-hydroxyglutaric acid in the fermentation broth were detected by HPLC, and the conversion rate of L-serine was calculated. The results are shown in Table 4; the calculation formula of the conversion rate of L-serine is shown in Formula (I);

[0081] L-serine conversion rate = [L-serine concentration / (initial sugar concentration-residual sugar concentration)]×100% (I).

[0082] Table 4

[0083]

[0084]

[0085]

[0086] The results in Table 4 show that the L-serine yield of the strain numbered Ls003 / 1 in the 131st random mutation of SerA is relatively high, reaching 1.4 g / L, and the conversion rate is 35%, and the corresponding α-hydroxyglutaric acid yield is 0.096 g / L. Compared with the control strain Ls003 / 0, its L-serine yield is increased by nearly 43%, and the yield of α-hydroxyglutaric acid is reduced by nearly 65%. The L-serine yield of the strain numbered Ls003 / 52 in the 285th random mutation of SerA is relatively high, reaching 1.43 g / L, and the conversion rate is 35.8%, and the corresponding α-hydroxyglutaric acid yield is 0.15 g / L. Compared with the control strain Ls003 / 0, the L-serine yield is increased by nearly 46%, and the yield of α-hydroxyglutaric acid is reduced by nearly 46%. Ls003 / 1 and Ls003 / 52 were sent for sequencing, and the SerA mutant in the recombinant strain Ls003 / 52 was SerA(N131A), the amino acid sequence of SerA(N131A) was shown in SEQ ID NO:4, and the nucleotide sequence was shown in SEQ ID NO:6. The SerA mutant in the recombinant strain Ls003 / 1 was SerA(D285L), the amino acid sequence of SerA(D285L) was shown in SEQ ID NO:5, and the nucleotide sequence was shown in SEQ ID NO:7.

[0087] Example 4

[0088] 1. Construction of combined mutant expression strains

[0089] Using the DNA of the recombinant strain Ls003 / 1 as a template, the primer pair serA(D285L)-F (nucleotide sequence as shown in SEQ ID NO: 38) and serA(D285L)-R (nucleotide sequence as shown in SEQ ID NO: 39) were used to perform PCR amplification of Ls003 / 52 (carrying the ptrc99a-SerA(N131A) plasmid) to obtain the ptrc99a-SerA(N131A)(D285L) fragment, which was transformed into Escherichia coli DH5α competent cells, cultured and expressed in the ptrc99a-SerA(N131A)(D285L) plasmid (the amino acid sequence of SerA(N131A)(D285L) is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 3), and then the plasmid was electroporated into Ls003 using the method of step 4 in Example 2 to obtain the recombinant strain Ls003 / 65.

[0090] The PCR reaction system (50 μL) in this process is: ddH 2O 20μL, upstream primer (10mM) 2μL, downstream primer (10mM) 2μL, colony template 1μL, 2×Phanta Mix high-fidelity enzyme 25μL; PCR reaction program is: step 1: 95℃, 3min; step 2: 95℃, 15s, 60℃, 10s; step 3: 72℃, 5min; this step is repeated for 30 cycles, 72℃, 5min; step 4: store at 4℃.

[0091] 2. The recombinant strain Ls003 / 65 was fermented by the culture method in Example 3 to obtain a fermentation broth. The fermentation results showed that the L-serine production of the recombinant strain Ls003 / 65 reached 1.48 g / L, and the conversion rate was 39%, and the corresponding α-hydroxyglutaric acid production was 0.12 g / L. Compared with the control strain Ls003 / 0, the L-serine production and conversion rate were increased by about 51%, and the α-hydroxyglutaric acid production was reduced by about 55%.

[0092] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A D-3-phosphoglycerate dehydrogenase mutant, It is characterized in that Compared with the protein with the amino acid sequence shown in SEQ ID NO: 1, the mutant comprises a mutation at the R site; the R site is the 131st and / or 285th position.

2. The D-3-phosphoglycerate dehydrogenase mutant according to claim 1, It is characterized in that Compared with the protein with the amino acid sequence shown in SEQ ID NO: 1, the mutant comprises a mutation of asparagine at position 131 to alanine and / or a mutation of aspartic acid at position 285 to leucine; Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:

2.

3. A gene encoding a mutant of D-3-phosphoglycerate dehydrogenase, It is characterized in that The gene has a nucleotide sequence encoding the D-3-phosphoglycerate dehydrogenase mutant according to claim 1 or 2.

4. The gene according to claim 3, It is characterized in that The gene has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2; Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO:

3.

5. A recombinant vector, It is characterized in that The recombinant vector contains the gene according to claim 3 or 4.

6. The recombinant vector according to claim 5, It is characterized in that The expression vector of the recombinant vector is the ptrc99a plasmid.

7. A recombinant strain, It is characterized in that The recombinant strain contains the gene according to claim 3 or 4 or the recombinant vector according to claim 5 or 6.

8. The recombinant strain according to claim 7, It is characterized in that The host strain of the recombinant strain is Escherichia coli with auxiliary genes knocked out, and the auxiliary genes include sdaA gene, sdaB gene and tdcG gene.

9. Use of at least one of the D-3-phosphoglycerate dehydrogenase mutant according to claim 1 or 2, the gene according to claim 3 or 4, the recombinant vector according to claim 5 or 6, and the recombinant strain according to claim 7 or 8 in producing L-serine.

10. A method for producing L-serine, It is characterized in that The method comprises the following steps: inoculating the recombinant strain according to claim 7 or 8 into a fermentation medium for fermentation culture; Preferably, the fermentation medium contains glucose, potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate, threonine, glutamic acid, alanine, biotin and trace element mother solution; Preferably, the fermentation medium contains 2-6 g / L of glucose, 1-3 g / L of potassium dihydrogen phosphate, 1-3 g / L of magnesium sulfate, 2-4 g / L of ammonium sulfate, 0.1-0.2 g / L of threonine, 0.5-1 g / L of glutamic acid, 0.03-0.1 g / L of alanine, 0.5-1.5 mL / L of trace element mother solution, and 4-8 mg / L of biotin; The trace element mother solution contains 3-8 mg / L zinc sulfate, 1-3 mg / L manganese sulfate, 0.3-0.8 mg / L cobalt chloride, 0.1-0.3 mg / L copper sulfate, and vitamin B 1 3-8mg / L; Preferably, the fermentation culture conditions include: a temperature of 35-40°C and a rotation speed of 180-220 rpm.

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