Phosphoglycerate dehydrogenase mutants and their use in the production of l-serine

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a phosphoglycolate dehydrogenase mutant and application thereof in L-serine production. The mutant contains a mutation of the 370th amino acid compared with a protein with an amino acid sequence as shown in SEQ ID NO:1. The mutant protein releases feedback inhibition, and the catalytic activity is significantly improved compared with the parent enzyme, thereby promoting the production of L-serine catalyzed by the phosphoglycolate dehydrogenase, and providing a new synthetic route and an engineering bacterium for the production of L-serine.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to phosphoglycerate dehydrogenase mutants and their application in the production of L-serine. Background Technology

[0002] L-Serine, as a basic amino acid that makes up proteins, is an important source of one-carbon units in organisms. It is also a precursor for the synthesis of glycine, cysteine, tryptophan, and thymine, and has significant application value in the pharmaceutical, food, and skincare industries.

[0003] The intracellular synthesis of L-serine involves a three-step reaction: 3-phosphoglycerate is successively reacted by phosphoglycerate dehydrogenase (PGDH). serA Gene-encoded), phosphoserine transaminase (PAST, by serC Gene-encoded) and phosphoserine phosphatase (PSP, by) serB The gene encodes (PGDH) to catalyze the production of L-serine. The first step of this reaction is the rate-limiting reaction in L-serine biosynthesis, and the enzyme PGDH catalyzing this reaction possesses NAD+. + L-serine is dependent on the activity of PDGH, which is strictly inhibited by feedback within the cell. To enhance the synthesis and metabolism of L-serine, it is necessary to first remove the feedback inhibition of the end product on the key enzyme PDGH.

[0004] However, existing phosphoglycerate dehydrogenases exhibit weak tolerance to feedback inhibition from serine, resulting in less than ideal serine biosynthesis efficiency. Therefore, developing phosphoglycerate dehydrogenases unaffected by serine feedback inhibition is crucial for efficient serine biosynthesis. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a phosphoglycerate dehydrogenase mutant and its application in the production of L-serine. This mutant protein relieves feedback inhibition and exhibits significantly enhanced catalytic activity compared to the parent enzyme, thereby promoting the production of L-serine catalyzed by phosphoglycerate dehydrogenase.

[0006] The technical solution of this invention is as follows:

[0007] The present invention provides a phosphoglycerate dehydrogenase mutant, which, compared with the protein shown in SEQ ID NO: 1, contains a mutation at amino acid position 370.

[0008] The present invention also provides the encoding gene of the above-mentioned phosphoglycerate dehydrogenase mutant.

[0009] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes.

[0010] The present invention also provides a host cell containing the above-described coding gene or recombinant vector.

[0011] The present invention also provides the application of the above-mentioned phosphoglycerate dehydrogenase mutant, which uses the mutant protein to catalyze the conversion of 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP).

[0012] The present invention also provides a method for preparing the above-mentioned phosphoglycerate dehydrogenase mutant, which involves gene recombination and expression using the coding gene of the phosphoglycerate dehydrogenase mutant or an expression vector containing the coding gene.

[0013] The present invention also provides a method for producing L-serine, the method comprising the steps of introducing the phosphoglycerate dehydrogenase mutant into a microbial expression system containing SerB and SerC, and preparing L-serine with a suitable substrate under suitable conditions.

[0014] The present invention also provides a method for improving the activity of phosphoglycerate dehydrogenase, which is achieved by introducing an amino acid substitution at position L370 in the parental phosphoglycerate dehydrogenase, the position corresponding to the amino acid sequence of phosphoglycerate dehydrogenase shown in SEQ ID NO: 1.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. This invention modifies phosphoglycerate dehydrogenase by means of directed evolution of active sites and gene mutation, and screens out highly active mutants that are free from the feedback inhibition of L-serine.

[0017] 2. This invention combines an enhanced phosphoglycerate dehydrogenase with other enzymes in the L-serine synthesis pathway (e.g., SerB and SerC) for the biosynthesis of L-serine, effectively relieving the feedback inhibition of the first step of the catalytic reaction and improving the microbial fermentation level of L-serine.

[0018] 3. This invention is the first to discover the 370th active site of phosphoglycerate dehydrogenase from Escherichia coli. Mutation of the above key amino acid sites can effectively relieve L-serine feedback and improve enzyme activity, providing a key target for further enhancement of the enzyme's subsequent activity.

[0019] 4. Taking the *E. coli* expression system as an example, this invention obtained a genetically engineered bacterium containing the phosphoglycerate dehydrogenase mutant of this invention, and applied the genetically engineered bacterium to L-serine production. Compared with the unmutated engineered bacterium, the engineered bacterium containing the mutant of this invention exhibited high catalytic activity, with the highest L-serine concentration reaching 23.1 times that of the control bacterium, and the highest yield in shake-flask fermentation reaching 12.4 times that of the control bacterium. The mutant of this invention effectively improves the synthesis efficiency of L-serine, providing a new synthetic route and genetically engineered bacterium for L-serine production. Attached Figure Description

[0020] Figure 1 In Example 2, the recombinant plasmids pSTV28 and pSTV28- serA Plasmid map. Detailed Implementation

[0021] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0022] The following definitions are used in this invention:

[0023] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0024] Amino acid residues are named using the recognized IUPAC nomenclature, either in three-letter abbreviations or single-letter symbols. DNA nucleic acid sequences are named using the recognized IUPAC nomenclature.

[0025] 2. Identification of phosphoglycerate dehydrogenase mutants

[0026] The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the phosphoglycerate dehydrogenase mutant. For example, L370W indicates that the 370th amino acid residue in the sequence from the N-terminus to the C-terminus is replaced by tryptophan (W) instead of leucine (L) from the parental phosphoglycerate dehydrogenase. The numbering method of this invention is based on the sequence shown in SEQ ID NO: 1.

[0027] 3. The amino acid sequence of phosphoglycerate dehydrogenase is shown in SEQ ID NO: 1:

[0028] MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGALDDEQLKESIRDAHFIGRSRTHLTEDVINAAEKLVAIGCFCIGTNQVDLDAAAKRGIPVF NAPFSNTRSVAELVIGELLLLLRGVPEANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAESLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSD VVSLHVPENPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASKHLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEAQENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHIHENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEADEDVAEKALQAMKAIPGTIRARLLY

[0029] The present invention provides a phosphoglycerate dehydrogenase mutant, which, compared with the protein shown in SEQ ID NO: 1, contains a mutation at amino acid position 370.

[0030] Preferably, the phosphoglycerate dehydrogenase mutant, compared with the protein whose amino acid sequence is as shown in SEQ ID NO: 1, has leucine at position 370 mutated to one of the following amino acids: glycine, alanine, valine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, or histidine.

[0031] The present invention also provides the encoding gene of the above-mentioned phosphoglycerate dehydrogenase mutant.

[0032] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes, such as a recombinant vector composed of vector plasmid pSTV28 and the coding gene described in the present invention.

[0033] The present invention also provides a host cell comprising the above-described coding gene or recombinant vector. The host cell can be any host suitable for producing the phosphoglycerate dehydrogenase mutant of the present invention from the gene or vector of the present invention, such as microbial expression systems like *Escherichia coli*, *Bacillus subtilis*, *Corynebacterium glutamicum*, and *Saccharomyces cerevisiae*.

[0034] The present invention also provides the application of the above-mentioned phosphoglycerate dehydrogenase mutant, which uses the mutant protein to catalyze the conversion of 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP).

[0035] The phosphoglycerate dehydrogenase mutant of the present invention has improved enzyme activity or catalytic activity compared with its parent phosphoglycerate dehydrogenase, for example, more than 5 times in some embodiments, more than 10 times in others, and more than 20 times in still others.

[0036] The present invention also provides a method for preparing the above-mentioned phosphoglycerate dehydrogenase mutant, which involves gene recombination and expression using the coding gene of the phosphoglycerate dehydrogenase mutant described in this invention or an expression vector containing the coding gene. Gene recombination methods and expression hosts known to those skilled in the art can be used, and suitable culture media and culture conditions for host expression can be selected. The method may further include a step of recovering the phosphoglycerate dehydrogenase mutant, which may involve isolating or purifying the phosphoglycerate dehydrogenase mutant from the host culture or expression product, and can be performed using any method known to those skilled in the art.

[0037] The present invention also provides a method for producing L-serine, the method comprising the steps of introducing the phosphoglycerate dehydrogenase mutant into a microbial expression system containing SerB and SerC (e.g., an Escherichia coli expression system), and preparing L-serine under suitable conditions with a suitable substrate (e.g., glucose).

[0038] In the above method for producing L-serine, the preferred culture medium composition is as follows:

[0039] The culture medium contains 10-30 g / L glucose, 1-5 g / L peptone, 1-6 g / L yeast extract, 1-3 g / L sodium citrate, 1-3 g / L potassium salt, 1-3 g / L magnesium salt, 15-20 mg / L iron salt, 15-20 mg / L manganese salt, 0.5-2 g / L methionine, 0.5-2 g / L glycine, and 1-3 mg / L each of vitamin B1, vitamin B3, vitamin B5, vitamin B12, and vitamin H, with the remainder being water; the pH of the culture medium is 7.0-7.2.

[0040] The present invention also provides a method for improving the activity of phosphoglycerate dehydrogenase, which is achieved by introducing an amino acid substitution at position L370 in the parental phosphoglycerate dehydrogenase, the position corresponding to the amino acid sequence of phosphoglycerate dehydrogenase shown in SEQ ID NO: 1.

[0041] In the above method, preferably, the amino acid substitutions of the phosphoglycerate dehydrogenase mutant compared to the protein with the amino acid sequence shown in SEQ ID NO: 1 are selected from one of the following: L370A, L370W, L370T, L370E, L370D, L370S, L370F, L370M, L370K, L370N, L370I, L370R, L370Y, L370G, L370H, L370P, L370V, L370Q, or L370C.

[0042] The present invention will be described in more detail below through specific embodiments. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] Example 1:

[0044] This embodiment provides a method for constructing a chassis strain, the method comprising the following steps:

[0045] 1. Gene editing operations

[0046] (1) Construction of pGRB plasmid: The purpose of constructing pGRB plasmid is to transcribe the corresponding gRNA, thereby forming a complex with the Cas9 protein, and to achieve double-strand breaks in the target DNA by recognizing the target gene site through base pairing and PAM. The pGRB plasmid is constructed by recombination of a DNA fragment containing the target sequence with a linearized vector fragment, as described in step af below:

[0047] a. Target sequence design: The target sequence (PAM: 5'-NGG-3') was designed using CRISPR RGEN Tools;

[0048] b. Preparation of DNA fragments containing the target sequence: Design primers: 5'-linearized vector terminal sequence (15 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (15 bp)-3' and its reverse complementary primers, and prepare DNA fragments containing the target sequence by annealing single-stranded DNA.

[0049] c. Preparation of linear vectors: The vectors were linearized using reverse PCR amplification.

[0050] d. Recombination reaction;

[0051] e. Plasmid transformation: Take 10 μL of reaction solution and add it to 100 mL of DH5α-transformed competent cells. Gently mix and incubate on ice for 20 min. Heat shock at 42℃ for 45-90 s, then immediately incubate on ice for 2-3 min. Add 900 μL of SOC and incubate at 37℃ for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, and resuspend the bacterial cells in about 200 μL. Spread the resuspended cells onto a plate containing 100 mg / L ampicillin. Invert the plate and incubate overnight at 37℃. After single cells grow on the plate, identify them by colony PCR and select positive recombinants.

[0052] f. Cloning identification: PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight. Plasmids were then extracted and identified by enzyme digestion.

[0053] (2) Preparation of recombinant DNA fragments: Primer design software Primer5 was used to... yjgX and serB Using the upstream and downstream sequences of the gene as templates, primers for upstream and downstream homologous arms were designed. The upstream and downstream homologous arms and the target gene fragment were amplified by PCR, and then recombinant fragments were prepared by overlapping PCR.

[0054] (3) Transformation of plasmids and recombinant DNA fragments

[0055] a. Transformation of pREDCas9: The pREDCas9 plasmid was electroporated into the electrocompetent cells of the starting strain. After cell resuscitation and culture, the cells were plated on LB agar plates containing zizomycin and incubated overnight at 32°C. Single colonies growing on the resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0056] b. Preparation of electroconversion competent cells of the target strain containing pREDCas9: cultured at 32℃ until OD 600 When the concentration reaches 0.1–0.2, add 0.1 M IPTG (to bring the final concentration to 0.1 mM) and continue culturing until OD reaches 0.2. 600 Competent cells were prepared when the pH was 0.6–0.7. The purpose of adding IPTG was to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process for competent cells followed standard operating procedures.

[0057] c. Transformation of pGRB and recombinant DNA fragment: pGRB and donor DNA fragment were simultaneously electroporated into electrocompetent cells containing pREDCas9. The revived cells after electroporation were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Colony PCR was performed using specially designed identification primers to verify the colonies, screen for positive recombinants, and maintain the cells.

[0058] (4) Plasmid elimination

[0059] a. Elimination of pGRB: Positive recombinants were incubated overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread onto LB plates containing zirconia-resistant bacteria and incubated overnight at 32°C. Single colonies that did not grow on ampicillin-resistant plates but grew on zirconia-resistant plates were selected and preserved.

[0060] b. Elimination of pREDCas9 plasmid: The positive recombinant was transferred to antibiotic-free LB liquid medium and incubated overnight at 42°C. After appropriate dilution, it was plated onto antibiotic-free LB plates and incubated overnight at 37°C. Single colonies that did not grow on bilirubin-resistant plates and grew on antibiotic-free plates were selected for preservation.

[0061] 2 Chassis strains E. coli Construction of SER01: serB Genetic yjgX Site integration

[0062] yjgX ::P trc -serB Reconstructed fragments: First, based on serB Designing upstream and downstream primers for gene (Gene ID: 948913) serB -F、 serB -R get serB Gene fragments, and then based on E. coli In the MG1655 genome yjgX Based on the nucleotide sequence of the gene, primers for amplifying the upstream homologous arm were designed. yjgX -UP-F、 yjgX Primers required for UP-R and downstream homologous arm amplification yjgX -DN-F、 yjgX -DN-R. The upstream and downstream homologous arms are connected by P. trc The promoter sequence is used as the overlapping region, and the above primers are used to obtain the overlapping PCR. yjgX - serB Recombined fragments.

[0063] pGRB-serB: According to E. coli In the MG1655 genome serB The sequence was used to design and construct the plasmid pGRB- using the online tool CRISPR RGENTools. serB Required primers serB -F and serB -R, pGRB- was constructed and obtained via circular PCR. serB Plasmid.

[0064] Reconstructed fragments yjgX ::P trc -serB With plasmid pGRB- serB Common electroconversion to a plasmid containing pREDCas9 E. coli Electrotransfer of MG1655 strain to competent cells using primers yjgX -UP-F、 yjgX -DN-R was used as a primer for colony PCR identification to obtain the strain. E. coli SER01.

[0065] 3 Chassis strains E. coli SER02 construction: serC Genetic yghE Site integration

[0066] yghE::P trc - serC Reconstructed fragments: First, based on serC Designing upstream and downstream primers based on gene (Gene ID: 945527) sequence serC -F、 serC -R get serC Gene fragments, and then based on E. coli In the MG1655 genome yghE Based on the nucleotide sequence of the gene, primers for amplifying the upstream homologous arm were designed. yghE -UP-F、 yghE Primers required for UP-R and downstream homologous arm amplification yghE -DN-F、 yghE -DN-R. The upstream and downstream homologous arms are connected by P. trc The promoter sequence is used as the overlapping region, and the above primers are used to obtain the overlapping PCR. yghE - serC Recombined fragments.

[0067] pGRB- serC :according to E. coli In the MG1655 genome serC The sequence was used to design and construct the plasmid pGRB- using the online tool CRISPR RGENTools. serC Required primers serC -F and serC -R, pGRB- was constructed and obtained via circular PCR. serC Plasmid.

[0068] The recombined fragment yghE::P trc - serC With plasmid pGRB- serC Common electroconversion to a plasmid containing pREDCas9E. coli Electrotransfer of SER01 strain to competent cells using primers yghE -UP-F、 yghE -DN-R was used as a primer for colony PCR identification to obtain the strain. E. coli SER02.

[0069] Example 2:

[0070] This embodiment provides serA- The method for constructing the L370 saturated mutant and its mutant strain includes the following steps:

[0071] 1. Recombinant expression vector pSTV28- serA Construction

[0072] The recombinant expression vector uses pSTV28 plasmid as its backbone. Figure 1 ), inserted with potentially high activity serA Mutant protein. First, using the pSTV28 plasmid as a template, a linearized vector was obtained via reverse PCR using primers pSTV28-F and pSTV28-R. This vector was then used with primers... serA -F、 serA -R serA Gene (Gene ID: 945258) fragment via ClonExpress ® II OneStep Cloning Kit series of homologous recombinases are used for ligation.

[0073] The PCR reaction system consisted of: a total volume of 50 μL, 19 μL of sterile water, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of template, and 25 μL of Primestar DNA Polymerase. The PCR program was: 98℃ for 30 s, 98℃ for 10 s, 57℃ for 30 s, 72℃ for 5 s / kb, 72℃ for 10 min, 16℃ ± ∞, with 30 cycles. The homologous recombination reaction conditions were: 37℃ for 10 min. The reaction system consisted of: a total volume of 10 μL, 2 μL of 5×CE II Buffer, 1 μL each of the fragment and the linearized cloning vector, and Exnase. ® II1 μL, ddH2O 5 μL.

[0074] The recombinant vector pSTV28- serA The ligation system was transferred into Escherichia coli via CaCl2 chemical conversion. E.coliE.coliAfter DH5α, the bacteria were spread onto LB agar plates containing 100 mg / L chloramphenicol and incubated overnight at 37°C. Once single colonies appeared on the plates, colony PCR was performed using primers JD-pSTV28-F and JD-pSTV28-R to identify the recombinant strain. E.coli DH5α-pSTV28- serA .

[0075] 2 serA- Construction of L370 saturated mutant strain

[0076] Based on the codon preference of E. coli, the recombinant expression vector was used as the backbone to verify the potential highly active SerA mutant protein. Two complementary primers were designed to mutate leucine at position 370 to the other 19 amino acids (taking L370A as an example, and the same applies to the others: L370A-F: 5'-GCGCAATATGCACAAACTTCCGCCCAGATGGG-3'; L370A-R: 5'-GGAAGTTTGTGCATATTGCGCGGCGATGTT-3') to construct a single-point mutant.

[0077] Self-recombining strains using the OMEGA DNA extraction kit E.coli DH5α-pSTV28- serA Extracting plasmid pSTV28- serA Using this as a template, a saturation mutation was performed on leucine at position 370 of the amino acid sequence of phosphoglycerate dehydrogenase via whole-plasmid PCR to obtain a recombinant expression vector containing the phosphoglycerate dehydrogenase mutant. The PCR product was verified by agarose gel electrophoresis and then subjected to restriction endonuclease assays. Q.Cut Dpn I was digested at 37℃ for 1 h; the digestion system consisted of: a total volume of 50 μL, 43 μL of PCR product, 5 μL of Cutsmart Buffer, and restriction endonuclease. Q.Cut Dpn I 2 μL; after purification and recovery, the digestion product was transformed into a strain capable of serine synthesis via chemical transformation. E.coli In SER02, after single colonies grew on the plate, colony PCR was performed to identify recombinant strains containing the nucleotide sequence encoding the phosphoglycerate dehydrogenase mutant. The primers used in the strain construction process are shown in Table 1.

[0078] Table 1 Primers used in strain construction

[0079]

[0080] Example 3:

[0081] This embodiment provides a method for producing L-serine by fermentation using a phosphoglycerate dehydrogenase mutant strain, the method comprising at least one of the following steps:

[0082] The plate culture method is as follows: the recombinant strain stored at -80℃ is streaked and inoculated onto activation plates, and cultured at 37℃ for 12 h to obtain slant seed culture; wherein, the preferred solid culture medium composition is: 5 g / L peptone, 5 g / L yeast extract, 2.5 g / L NaCl, 10 g / L agar, and the remainder is water, with a pH of 7.0-7.2.

[0083] The in vitro culture method is as follows: use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a test tube containing 5 mL of seed culture medium. Culture at 37℃ and 200 rpm for 12 h to obtain primary seed solution.

[0084] 24-well plate seed culture: The primary seed culture was inoculated into 24-well plates (250 mL shake flasks) containing a final volume of 5 mL (50 mL) at an inoculation rate of 2% (v / v) of the seed culture medium. The plates were then shaken and cultured at 37℃ and 200 rpm for 12 h to obtain the secondary seed culture. The preferred seed culture medium composition was as follows: glucose 30 g / L, peptone 5 g / L, yeast extract 6 g / L, potassium salt 3 g / L, magnesium salt 3 g / L, iron salt 20 mg / L, manganese salt 20 mg / L, methionine 1 g / L, glycine 1 g / L, vitamin B1, vitamin B3, vitamin B5, vitamin B12, and vitamin H 3 mg / L each, with the remainder being water, and the pH being 7.0-7.2.

[0085] Fermentation culture: The secondary seed culture was inoculated into a 24-well plate (250 mL shake flask) containing a final volume of 5 mL (50 mL) at an inoculation rate of 2% (v / v) of the seed culture liquid. The culture was shaken and cultured for 12 h at 37℃ and 200 rpm to obtain the fermentation broth. The preferred fermentation medium composition was as follows: glucose 20 g / L, peptone 3 g / L, yeast extract 3 g / L, sodium citrate 3 g / L, potassium salt 3 g / L, magnesium salt 3 g / L, iron salt 20 mg / L, manganese salt 20 mg / L, methionine 2 g / L, glycine 2 g / L, vitamin B1, vitamin B3, vitamin B5, vitamin B12, and vitamin H 3 mg / L each, with the remainder being water. The pH of the medium was 7.0-7.2.

[0086] The detection method for serine in fermentation broth is as follows: Take the fermentation broth, mix thoroughly, dilute with deionized water by a certain factor, centrifuge at 13000 r / min, and collect the supernatant for later use. Sample derivatization procedure: Take 200-300 μL of 50 mM NaHCO3 solution, 200-400 μL of 1% 2,4-dinitrofluorophenylacetonitrile solution, and add 10-20 μL of the diluted fermentation sample. Mix well and place in a 65℃ water bath in the dark for 45-60 min. After cooling to room temperature, add 600-700 μL of 50 mM KH2PO4 solution, filter through a 0.22 μm microporous membrane, and then analyze using a microfiltration instrument. An Agilent ZORBAX Eclipse AAA (4.6 mm × 250 mm, 5-Micron) liquid chromatography column was used with binary high-pressure gradient elution and UV detection. The mobile phase consisted of 50% acetonitrile solution and 50 mM sodium acetate aqueous solution (containing 10 mL N,N-dimethylformamide per liter). The flow rate was 1 mL / min, the column temperature was 33 °C, the UV detection wavelength was 360 nm, and the retention time was 30 min.

[0087] Example 4:

[0088] This example illustrates the differences between phosphoglycerate dehydrogenase mutant strains and control strains. E.coli SER02-pSTV28- serA Differences in L-serine production performance.

[0089] The mutant strain and control strain constructed in Example 2 were cultured using the method described in Example 3. E.coli SER02-pSTV28- serA Fermentation was performed in 24-well plates. The concentration of L-serine in the fermentation supernatant was measured, and the results are shown in Table 2.

[0090] Table 2. Results of plate fermentation of SerA protease L370 site saturated mutant strains.

[0091]

[0092] Table 2 shows that the L-serine concentration in the mutant strain was significantly increased compared to the control strain, with the highest being [missing information]. E.coli SER02-pSTV28- serA The L-serine concentration in L370W increased from 0.06 g / L to 1.46 g / L, which is higher than that of the control strain. E.coli SER02-pSTV28- serA The result was 23.1 times higher than that of the strain, indicating that the phosphoglycerate dehydrogenase mutant containing the 370th amino acid mutation could significantly improve the catalytic activity of the enzyme and the L-serine production performance of the strain.

[0093] Furthermore, the strain was cultured in shake flasks using the culture method described in Example 3, and the concentration of L-serine in the fermentation supernatant was measured. The results are shown in Table 3.

[0094] Table 3 Results of shake-flask fermentation of mutant strains

[0095]

[0096] Table 3 shows that the mutation did not significantly affect the growth of the engineered strain; at the same time, it promoted the growth of the mutant strain. E.coli SER02-pSTV28- serA The L-serine concentration in L370W increased from 0.13 g / L to 1.61 g / L, which is higher than that of the control strain. E.coli SER02-pSTV28- serA It is 12.4 times that of the strain, significantly improving the L-serine production performance of the strain.

[0097] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principle of the present invention. The scope of the present invention is defined by the claims and their equivalents as contained in the description.

Claims

1. A phosphoglycerate dehydrogenase mutant, wherein the leucine at position 370 is substituted with tryptophan, as compared to the protein having the amino acid sequence set forth in SEQ ID NO:

1.

2. A gene encoding the phosphoglycerate dehydrogenase mutant of claim 1.

3. A recombinant vector, characterized in that, comprising the gene encoding of claim 2.

4. A host cell, characterized in that, comprising the gene encoding of claim 2 or the recombinant vector of claim 3.

5. Use of the phosphoglycolate dehydrogenase mutant according to claim 1, characterized in that, the mutant protein is used to catalyze the conversion of 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP).

6. A method of producing the phosphoglycerate dehydrogenase mutant of claim 1, by genetic recombination and expression using a gene encoding the phosphoglycerate dehydrogenase mutant or an expression vector comprising the gene.

7. A method of producing L-serine, comprising the step of introducing the phosphoglycerate dehydrogenase mutant of claim 1 into a microbial expression system containing SerB and SerC, and producing L-serine from a suitable substrate under suitable conditions.

8. The method of claim 7, wherein, the microbial expression system is Escherichia coli.

9. A method of increasing the enzymatic activity of phosphoglycerate dehydrogenase, by introducing a L370W substitution in a parent phosphoglycerate dehydrogenase, wherein the amino acid position corresponds to the amino acid sequence of the parent phosphoglycerate dehydrogenase set forth in SEQ ID NO: 1.

Images