Glyceraldehyde-3-phosphate dehydrogenase mutant and its application in increasing the production of O-succinyl-L-homoserine

Abstract

The present invention discloses a glyceraldehyde 3-phosphate dehydrogenase mutant and its application in increasing the yield of O-succinyl-L-homoserine, wherein the mutant is obtained by single mutation of the arginine at position 233 of the amino acid sequence shown in SEQ ID NO.2. The present invention uses recombinant Escherichia coli producing O-succinyl-L-homoserine as the target strain, introduces exogenous glyceraldehyde 3-phosphate dehydrogenase gapC and its mutant to provide more NADPH supply for the metabolic pathway to increase the yield of OSH. The wild-type gapC provided by the present invention is expressed in recombinant Escherichia coli and can increase the yield of OSH in a 5L fermentor from the original 87g / L to 102g / L, and after the gapC mutant is expressed in recombinant Escherichia coli, the yield of OSH in a 5L fermentor increases to 135g / L, and the supply of NADPH is increased by 50% over the original strain.

Description

(1) Technical field

[0001] The present invention belongs to the fields of synthetic biology and microbial fermentation technology, and particularly relates to a method for increasing the yield of O-succinyl-L-homoserine, and a glyceraldehyde-3-phosphate dehydrogenase mutant derived from Clostridium acetobutylicum for increasing the yield. (2) Background technology

[0002] O-Succinyl-L-homoserine (OSH) is a homoserine derivative. As an important platform compound, it is widely used in the synthesis of substances such as homoserine lactone, γ-butyrolactone, and L-methionine. Its green and efficient synthesis is attracting increasing attention. Currently, O-Succinyl-L-homoserine is mainly synthesized by small-scale chemical synthesis, but it has problems such as expensive raw materials, harsh reaction conditions, complex extraction process, and environmental pollution. In contrast, constructing an efficient cell factory and achieving production through microbial fermentation has the advantages of mild reaction conditions, abundant and cheap raw materials, and environmental friendliness. With the development of technologies such as synthetic biology, microbial cell factories have been widely used in the synthesis of important compounds such as aspartic acid family amino acids and their derivatives, providing an important reference for the design and construction of cell factories for OSH.

[0003] In the early stages, our laboratory used E. coli as a base cell to increase OSH production by blocking homoserine competition and degradation pathways and overexpressing genes related to the homoserine synthesis pathway in the genome (3Biotech, 2018, 8:310, http: / / doi.org / 10.1007 / s13205-018-1332-x). However, during the fermentation process, it was found that insufficient NADPH supply became a limiting factor for further increasing OSH production.

[0004] As an OSH-producing strain, Escherichia coli has two main pathways for producing NADPH: the pentose phosphate pathway and the citric acid cycle. However, the ability of these two pathways to produce NADPH in bacteria is limited. If you want to obtain more NADPH supply, you need to introduce exogenous efficient NADPH synthase. Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the central carbon metabolism pathway, which can catalyze the conversion of NADP to NADPH. + and glyceraldehyde-3-phosphate to produce NADPH and 3-phospho-D-glyceryl phosphate. (3) Summary of the invention

[0005] The present invention aims to provide a glyceraldehyde-3-phosphate dehydrogenase mutant and its use in increasing O-succinyl-L-homoserine production. The present invention effectively increases O-succinyl-L-homoserine production by overexpressing the glyceraldehyde-3-phosphate dehydrogenase mutant. Specifically, through molecular engineering techniques, the glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum is mutated to obtain a beneficial mutant. This mutant enzyme is then heterologously expressed in an earlier constructed Escherichia coli OSH-producing bacterium, thereby increasing the supply of NADPH during OSH synthesis and thereby improving the fermentation capacity for OSH production.

[0006] The technical solution adopted in the present invention is:

[0007] The present invention provides a glyceraldehyde-3-phosphate dehydrogenase gapC mutant, which is obtained by single-mutating the arginine at position 233 of the amino acid sequence shown in SEQ ID NO.2. The glyceraldehyde-3-phosphate dehydrogenase gapC mutant is artificially designed and synthesized based on the original gapC sequence, connected to the pRSFDuet-1 plasmid, and successfully transformed into recombinant Escherichia coli for gene expression. The nucleotide sequence of gapC is mutated using an error-prone PCR method, and the obtained amplified product is purified and similarly connected to pRSFDuet-1, and heterologously expressed in recombinant Escherichia coli. By screening, mutants with significantly improved NADPH supply capacity are obtained, so that the recombinant strain can increase its synthesis yield of O-succinyl-L-homoserine.

[0008] Furthermore, preferably, the mutant is a mutant in which the arginine at position 233 of the amino acid sequence shown in SEQ ID NO.2 is mutated to alanine, and the amino acid sequence of the mutant is shown in SEQ ID NO.4.

[0009] The present invention provides a gene encoding the glyceraldehyde-3-phosphate dehydrogenase gapC mutant, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.3.

[0010] The present invention also relates to a recombinant vector containing a gene encoding a glyceraldehyde-3-phosphate dehydrogenase gapC mutant, and a recombinant bacterium constructed by the recombinant vector.

[0011] Furthermore, the recombinant bacteria containing the coding gene of the glyceraldehyde-3-phosphate dehydrogenase gapC mutant were constructed as follows: the coding gene of the glyceraldehyde-3-phosphate dehydrogenase gapC mutant with the nucleotide sequence shown in SEQ ID NO.3 was double-digested with EcoRI and BamHI and then inserted into the multiple cloning site MCS1 of the plasmid pRSFDuet-1, and metA was inserted into the recombinant bacteria. fbrThe gene was inserted into another MCS2 site of the plasmid after double digestion with KpnI and XhoI, and then transformed into E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr Competent cells were coated on LB plates containing 50 μg / ml kanamycin, cultured at 37°C overnight, and positive transformants were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin. After cultured at 37°C overnight, recombinant bacteria were obtained; the E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr After knocking out metI, metJ, metB, and thrB genes in the Escherichia coli genome, thrA fbr The promoter of metL gene was replaced by the strong Trc promoter.

[0012] The present invention provides an application of a glyceraldehyde-3-phosphate dehydrogenase gapC mutant in increasing the yield of O-succinyl-L-homoserine, wherein the application method is:

[0013] The recombinant bacteria were inoculated into a fermentation medium containing 50 μg / mL kanamycin, cultured at 37°C and 180 rpm for 3 hours, and then lactose was added to a final concentration of 0.5 mmol / L. The mixture was induced and cultured at 30°C for 48 hours. The mixture was centrifuged, and the supernatant was collected for separation and purification to obtain O-succinyl-L-homoserine. The fermentation medium composition was as follows: 20 g / L glucose, 2.5 g / L yeast powder, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.23 g / L L-threonine, 0.1 g / L L-methionine, 1 g / L succinic acid, 2 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·4H2O, 0.005 g / L MnSO4, and 0.01 g / L ZnSO4. 0.0025g / L, L-arginine 0.004g / L, L-glutamic acid 0.003g / L, the solvent is deionized water, and the pH value is natural.

[0014] Furthermore, the recombinant bacteria are cultured in a fermenter: the recombinant bacteria are inoculated into a 5-L fermenter containing 2 L of fermentation medium containing a final concentration of 50 μg / ml kanamycin, the initial rotation speed is 400 rpm, the initial ventilation flow rate is 1.0 L / min, the rotation speed and ventilation flow rate are adjusted as the bacterial concentration increases to maintain the dissolved oxygen value at 20-30% air saturation, the pH is adjusted to be stable at 6.8-7.0 with a volume concentration of 50% concentrated ammonia water and 50% phosphoric acid, and after culturing at 37°C for 8-12 hours, lactose is added to a final concentration of 0.5 mmol / L, the temperature is lowered to 30°C to induce expression, and the culture is continued for 100-120 hours; during the fermentation process, a feed medium is added to maintain a glucose concentration of 1-5 g / L; after the fermentation is completed, the fermentation broth is centrifuged, the supernatant is retained, and separation and purification are performed to obtain O-succinyl-L-homoserine;

[0015] The fermentation medium is composed of: 20 g / L glucose, 2.5 g / L yeast powder, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.23 g / L L-threonine, 0.1 g / L L-methionine, 1 g / L succinic acid, 2 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·4H2O, 0.005 g / L MnSO4, 0.0025 g / L ZnSO4, 0.004 g / L L-arginine, 0.003 g / L L-glutamic acid, 10 g / L CaCO3 (sterilized separately), deionized water as the solvent, and natural pH value;

[0016] The feed medium is composed of: 500 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 1 g / L L-threonine, 0.05 g / L L-methionine, 0.5 g / L MgSO4·7H2O, 0.005 g / L MnSO4, 0.01 g / L FeSO4·4H2O, 0.0025 g / L ZnSO4, 0.004 g / L L-arginine, and 0.003 g / L L-glutamic acid, and the solvent is natural water.

[0017] Furthermore, before the recombinant bacteria are inoculated into the fermentation tank, slant activation and seed expansion culture are first performed, and the seed solution is inoculated into the fermentation tank culture medium at an inoculum concentration of 5-10% by volume. The seed solution is prepared as follows:

[0018] (1) Plate culture: Use an inoculating loop to pick the recombinant bacteria from the glycerol tube, streak inoculate on a LB solid plate with a final concentration of 50 μg / ml kanamycin resistance, seal with sealing film, and place in a 37°C constant temperature incubator for 12-16 h (until a single colony of appropriate size grows); the final concentration of the LB solid plate medium is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, the solvent is deionized water, pH 7.0;

[0019] (2) Slant liquid culture: Use an inoculation loop to pick a single colony from the plate in step (1) and inoculate it into a test tube (10 ml) containing 10 ml of LB liquid medium containing a final concentration of 50 μg / ml kanamycin, and place it in a constant temperature shaking incubator at 37°C and 150 rpm for 8-12 hours for activation; the final concentration of the LB liquid medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7.0;

[0020] (3) Seed culture: The bacterial solution in the test tube activated in step (2) was inoculated into a 500-ml shake flask containing 100 ml of LB liquid medium containing a final concentration of 50 μg / ml kanamycin at an inoculum size of 2% (v / v), and the culture was placed in a 37°C constant temperature shaker for 8-12 h to obtain a seed solution.

[0021] The present invention can be constructed by connecting the nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase and its mutants of the present invention to various vectors by conventional methods in the art. The recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and / or eukaryotic cells. The vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., preferably pRSFDuet-1. Preferably, the recombinant expression vector of the present invention can be obtained by the following method: the obtained wild-type gapC or mutant gapC M The gene product was inserted into the EcoRI and BamHI sites of the vector pRSFDuet-1 to construct the recombinant expression plasmids pRSFDuet-1-gapC and pRSFDuet-1-gapC of the present invention. M .

[0022] Introducing the gapC encoding the present invention and its mutant gapC MThere is no restriction on the host cell of the DNA, as long as a recombinant expression system has been established for it, the recombinant expression vector can stably replicate itself and the gapC and mutant genes of the present invention carried therein can be effectively expressed. For example, Escherichia coli, Corynebacterium glutamicum, yeast, actinomycetes, Aspergillus, as well as animal cells and higher plant cells. The present invention preferably uses Escherichia coli, more preferably Escherichia coli (E. coli-ΔBBIJ) constructed in the early laboratory to knock out metI, metJ, metB, and thrB genes as the chassis (3Biotech, 2018, 8: 310, http: / / doi.org / 10.1007 / s13205-018-1332-x), and the thrA gene on the genome is removed. fbr The promoter of metL gene was replaced with Trc strong promoter (E.coli-ΔBBIJ-TrcmetL-TrcthrA fbr ), will also carry metA fbr and gapC or metA fbr and gapC M The recombinant plasmid pRSFDuet-1-metA fbr -gapC or pRSFDuet-1-metA fbr -gapC M Transformed into E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr The engineered bacteria are obtained.

[0023] The present invention induces the culture of recombinant Escherichia coli containing gapC and its mutant sequences to achieve normal expression of related enzymes in the metabolic process and improve the efficiency of O-succinyl-L-homoserine synthesis. The seed culture medium can be a culture medium in the art that can grow transformants and produce gapC of the present invention, preferably LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent is deionized water, pH 7.2.

[0024] Compared with the existing technology, the beneficial effects of the present invention are mainly reflected in the following: the present invention uses recombinant Escherichia coli that produces O-succinyl-L-homoserine as the target strain, introduces exogenous glyceraldehyde-3-phosphate dehydrogenase gapC and its mutants into the metabolic pathway to provide more NADPH supply, thereby increasing OSH production. The wild-type gapC provided by the present invention, when expressed in recombinant E. coli, can increase OSH production in a 5L fermentor from the original 87g / L to 102g / L. After expressing the gapC mutant in the recombinant E. coli, OSH production in a 5L fermentor increased to 135g / L, and the NADPH supply was increased by 50% compared to the original strain. (IV) Description of the accompanying drawings

[0025] Figure 1 .NADPH standard curve. (V) Specific implementation methods

[0026] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0027] The LB solid culture medium of the present invention has a final concentration of 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder, and the solvent is deionized water with a pH of 7.0.

[0028] The LB liquid culture medium of the present invention has a final concentration of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and the solvent is deionized water with a pH of 7.0.

[0029] The wild-type strain E. coli-ΔBBIJ was constructed according to 3Biotech, 2018, 8:310, http: / / doi.org / 10.1007 / s13205-018-1332-x. On this basis, the thrA fbr The promoter of metL gene was replaced with Trc strong promoter to obtain E.coli-ΔBBIJ-TrcmetL-TrcthrA fbr The recombinant bacteria were further constructed, and the target gene was carried by the pRSFDuet-1 plasmid to construct an engineered bacteria with improved OSH production.

[0030] Example 1: Construction of gapC recombinant bacteria

[0031] The glyceraldehyde-3-phosphate dehydrogenase (gapC) gene (NC-017295.1) from Clostridium acetobutylicum was codon-optimized (Escherichia coli preference) and fully synthesized at Beijing Qingke Biotechnology Co., Ltd. (nucleotide sequence shown in SEQ ID NO.1, amino acid sequence shown in SEQ ID NO.2). GapC was then inserted into the MCS1 site of pRSFDuet-1 via EcoRI and BamHI digestion and ligation. The metA gene had been previously inserted into the MCS2 site of the plasmid. fbr Gene, the recombinant plasmid pRSFDuet-1-metA carrying the two genes fbr -gapC was transformed into E. coli DH5 competent cells, coated on LB plates containing kanamycin (50 μg / ml), cultured at 37°C overnight, and positive transformants were picked and identified and sequenced.

[0032] SEQ ID NO: 1:

[0033]

[0034] SEQ ID NO: 2:

[0035] MAKIAINGFGRIGRLALRRILEVPGLEVVAINDLTDAKMLAHLFKYDSSQGRFNGEIEVKEGAFVVNGKEVKVFAEADPEKLPWGDLGIDVVLECTGFFTKKEKAEAHVRAGAKKVVISAPAGNDLKTIVFNVNNEDLDGTETVISGASCTTNCLAPMAKVLNDKFG IEKGFMTTIHAFTNDQNTLDGPHRKGDLRRARAAAVSIIPNSTGAAKAISQVIPDLAGKLDGNAQRVPVPTGSITELVSVLKKKVTVEEINAAMKEAADESFGYTEDPIVSADVVGINYGSLFDATLTKIVDVNGSQLVKTAAWYDNEMSYTSQLVRTLAYFAKIAK.

[0036] The verified positive monoclonal clone was inoculated into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin and cultured at 37°C overnight. After the plasmid was extracted and verified, the recombinant expression vector pRSFDuet-1-metA was cloned into the fbr -gapC was transformed into E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr Recombinant bacteria were obtained and cultured overnight at 37°C on LB plates containing kanamycin (50 μg / ml). A single colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. After overnight culture at 37°C, wet cells were collected by centrifugation at 8000 rpm for 10 min at 4°C and stored in glycerol at -80°C for later use.

[0037] Example 2: Construction of gapC mutant library

[0038] The recombinant expression vector pRSFDuet-1-metA obtained in Example 1 fbr -gapC was used as a template for error-prone PCR amplification. The PCR products were analyzed by 1% agarose gel electrophoresis and recovered by gel excision. They were then double-digested with EcoRI and BamHI and compared with pRSFDuet-1-metA which had also been digested with EcoRI and BamHI. fbr The ligation solution was transformed into E. coli W3110 competent cells by electroporation, and spread on LB plates containing kanamycin (50 μg / ml). The cells were cultured at 37° C. overnight to obtain a culture plate containing the gapC mutant library.

[0039] Amplification primers (5′-CAATTGGATATCGGCCGGCCATGGCAAAAATCGCGATTAA-3′) and (5′-AGGGTACCGACGTCAGCGATTTATTTTGCAATTTTTGCAA-3′).

[0040] Amplification system: 50 μl reaction system: 10xTaq polymerase buffer: 5 μl; Mg 2+ (25mM): 5μl; Mm 2+ (25mM): 3μl; 10mM dNTP mixture (2.5mM each of dATP, dCTP, dGTP and dTTP) 4μl; 1μl each of 50μM upstream primer and downstream primer, DNA template: 1μl; Taq DNA polymerase: 10U; make up the system with double-distilled water.

[0041] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 1 min, followed by 30 cycles of temperature cycling at 95°C for 10 s, 56°C for 90 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The termination temperature was 4°C.

[0042] Example 3. Screening of gapC-positive mutants and obtaining recombinant bacteria containing the mutants

[0043] A single clone was picked from the mutant library constructed in Example 2 and inoculated into a well of a 96-well plate with 1 mL of LB liquid medium containing 50 μg / mL kanamycin. After culturing at 37°C and 180 rpm for 8-12 h, 100 μL of the culture solution was inoculated into a well of a new 96-well plate with 1 mL of fermentation medium containing 50 μg / mL kanamycin. After culturing at 37°C and 180 rpm for 3 h, lactose was added to a final concentration of 0.5 mmol / L and induced at 30°C for 48 h. The bacterial solution was collected and centrifuged. The wet bacteria were detected for NADPH content in the cells using the NADP+ / NADPH Assay Kit (WST-8 method) of Biyuntian. The results of some mutants are shown in Table 1. Positive clones with improved NADPH synthesis capacity were selected and sent to a sequencing company for sequencing. After sequencing analysis, the positive mutants were determined.

[0044] The sequencing-verified gapC M The recombinant plasmid pRSFDuet-1-metA fbr -gapC M , transformed into E.coli-ΔBBIJ-TrcmetL-TrcthrA according to the method of Example 1 fbrThe recombinant E. coli-ΔBBIJ-TrcmetL-TrcthrA was obtained. fbr -pRSFDuet-1-metA fbr -gapC M , E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr -pRSFDuet-1-metA fbr Recorded as E. coli M On this basis, the recombinant strain containing wild-type gapC was recorded as E. coli M -gapC, and the recombinant bacteria containing different gapC mutants were recorded as E. coli M -gapC M (Where M represents a specific mutant, such as E. coli M -gapC R233A ).

[0045] The best mutant was the single mutant R233A (ie, the arginine at position 233 of the amino acid sequence of SEQ ID NO.2 was mutated to alanine), the amino acid sequence of which was shown in SEQ ID NO.4, and the nucleotide sequence of which was shown in SEQ ID NO.3.

[0046] Fermentation medium composition: glucose 20 g / L, yeast powder 2.5 g / L, (NH4)2SO4 16 g / L, KH2PO4 2 g / L, L-threonine 0.23 g / L, L-methionine 0.1 g / L, succinic acid 1 g / L, citric acid 2 g / L, MgSO4·7H2O0.5 g / L, FeSO4·4H2O 0.01 g / L, MnSO4 0.005 g / L, ZnSO4 0.0025 g / L, L-arginine 0.004 g / L, L-glutamic acid 0.003 g / L, solvent is deionized water, pH value is natural.

[0047] Table 1 NADPH content in cells of different strains

[0048]

[0049] The NADP+ / NADPH Assay Kit for detecting NADPH content in cells is a WST-8-based colorimetric assay that detects the amount, ratio, and total amount of NADP+ (oxidized coenzyme II) and NADPH (reduced coenzyme II) in cells, tissues, or other samples. The NADP+ / NADPH Assay Kit includes G6PDH, a colorimetric solution, NADPH, a NADP+ / NADPH extract, and a reaction extract. Specific instructions are as follows:

[0050] (1) Sample preparation:

[0051] Centrifuge the fermentation culture in the 96-well plate at 600g for 5 minutes. Aspirate the culture medium and add 200μl of ice-cold NADP+ / NADPH extraction solution using a pipette. Gently pipette to promote cell lysis. Lysis can be performed at room temperature or on ice. Subsequently, centrifuge at 12,000g at 4°C for 5-10 minutes. Collect the supernatant for use as the sample to be tested.

[0052] (2) Preparation of the kit:

[0053] a. Preparation of NADPH Standard: Dissolve the 5 mg of NADPH provided in this kit in 6 ml of ultrapure water to obtain a 1 mM NADPH standard. Aliquot and store the 1 mM NADPH standard at -80°C in the dark.

[0054] b. NADPH standard curve setup: Dilute a 1 mM NADPH standard with the NADP+ / NADPH extract to an appropriate concentration gradient. For example, for an initial assay, dilute to 0, 0.25, 0.5, 1, 2, 4, 6, and 8 μM. Add 50 μl of standard to each well of a 96-well plate, equivalent to 0, 12.5, 25, 50, 100, 200, 300, and 400 pmol NADPH per well. If necessary, adjust the standard concentration range in subsequent experiments based on the NADPH content in the samples. The 0 μM concentration point is a blank control containing only the NADP+ / NADPH extract. Note: Because NADPH is very unstable, use it as soon as possible after preparation.

[0055] c. Preparation of G6PDH working solution: Dilute G6PDH 50-fold with reaction buffer. For example, add 2 μl of G6PDH to 98 μl of reaction buffer to obtain 100 μl of G6PDH working solution. Each standard or sample requires 100 μl of G6PDH working solution. Prepare an appropriate amount of G6PDH working solution based on the number of standards and samples to be tested, and ensure it is used immediately.

[0056] (3) Sample determination:

[0057] a. Determination of NADPH content in the sample: Pipette 100-200μl of the sample to be tested into a centrifuge tube and heat in a 60°C water bath or PCR instrument for 30 minutes to decompose NADP+. If insoluble material is produced after heating, centrifuge at 10,000g for 5 minutes at room temperature or 4°C. Pipette 50μl of the supernatant as the sample to be tested into a 96-well plate. To reduce experimental error, it is recommended to set up duplicate wells for the sample. If the NADP+ or NADPH content in the sample is found to be too high and exceeds the range of the standard curve, it is necessary to appropriately dilute the sample with NADP+ / NADPH extraction solution before testing. If the content is too low, it is necessary to increase the amount of cell sample.

[0058] Use a 96-well plate to set up blank control wells, standard wells, and sample wells, referring to Table 2. Add G6PDH working solution and mix thoroughly.

[0059] Table 2 NADPH content determination settings

[0060]

[0061] b. Incubate at 37°C in the dark for 10 minutes. Note: This incubation step converts NADP+ in the sample to NADPH. Be gentle when adding the G6PDH working solution to avoid bubbles. If bubbles are present, puncture them with a small pipette tip or needle.

[0062] c. Mix the colorimetric solution thoroughly, then add 10 μl of colorimetric solution to each well. Mix thoroughly, and incubate at 37°C in the dark for 10-20 minutes. Orange-yellow formazan will form. Measure the absorbance at 450 nm. If the color is light, extend the incubation time to 30-60 minutes. The color will gradually deepen with extended incubation time.

[0063] (4) Calculation of NADPH content in the sample:

[0064] a. Calculate the average absorbance of each point in the standard group and subtract the absorbance of the blank control group to obtain the absorbance of each standard.

[0065] b. Draw a standard curve with NADPH concentration as the horizontal axis and absorbance as the vertical axis. The standard curve of NADPH standard is shown in Figure 1 , curve equation Y=0.44786x+0.0029, R 2 = 0.99674. If the incubation time is too long, the color development of the high-concentration standard will reach a plateau. In this case, it is advisable to select a standard that has not reached the plateau to draw the standard curve, or select the absorbance data of the standard with a shorter incubation time to draw the standard curve.

[0066] Figure 1This test kit demonstrates excellent NADPH detection without interference from NADH. Actual readings may vary under different assay conditions due to differences in standard preparation, instrumentation, and other factors. The data in the figure is for reference only.

[0067] c. After heating at 60°C, the NADPH concentration in the cell sample was calculated based on the standard curve.

[0068] Example 4. Fermentation of Starting Strains, Recombinant Bacteria Containing gapC, and Recombinant Bacteria Containing a GapC Mutant

[0069] In order to test the effect of introducing exogenous glyceraldehyde-3-phosphate dehydrogenase gapC and its mutants to provide more NADPH supply to the metabolic pathway and improve the production of OSH, the starting strain E. coli M and E. coli M -gapC, E. coli M -gapC R233A Fermentation culture is carried out, and the specific fermentation culture steps are as follows:

[0070] (1) Plate culture: Use an inoculating loop to pick a small amount of the above-mentioned wild-type strain, gapC recombinant bacteria, and gapC mutant recombinant bacteria from the glycerol tube, streak inoculate on LB solid plates with a final concentration of 50 μg / ml kanamycin resistance, seal with sealing film, and place in a 37°C constant temperature incubator for 12-16 hours (until single colonies of appropriate size grow). The final concentration of LB solid plate medium is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, solvent is deionized water, pH 7.0;

[0071] (2) Slant liquid culture: Use an inoculation loop to pick a single colony from the plate in step (1) and inoculate it into a test tube (10 ml) containing 10 ml of LB liquid medium containing a final concentration of 50 μg / ml kanamycin, and culture it in a constant temperature shaking incubator at 37°C and 150 rpm for 8-12 h for activation; the final concentration of the LB liquid medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7.0;

[0072] (3) Seed culture: The bacterial solution in the test tube activated in step (2) was inoculated into a 500-ml shake flask containing 100 ml of LB liquid medium containing a final concentration of 50 μg / ml kanamycin at an inoculum size of 2% (v / v), and the culture was placed in a 37°C constant temperature shaker for 8-12 h to obtain a seed solution.

[0073] (4) Fermentation culture: The seed liquid was inoculated at a volume concentration of 10% into a 5-L fermentation tank containing 2 L of fermentation medium containing a final concentration of 50 μg / ml kanamycin. The initial rotation speed was 400 rpm and the initial ventilation flow rate was 1.0 L / min. As the bacterial concentration increased, the rotation speed and ventilation flow rate were adjusted to maintain the dissolved oxygen value at 20-30% air saturation. The pH was adjusted to 6.8-7.0 with a volume concentration of 50% concentrated ammonia water and 50% phosphoric acid. After culturing at 37°C for 8-12 h, a final concentration of 0.5 mmol / L lactose was added and the temperature was lowered to 30°C to induce expression. The culture was continued for 100-120 h. During the fermentation process, the feed medium was added to maintain the glucose concentration at 1-5 g / L. After the fermentation was completed, the supernatant was collected by centrifugation and the OSH content in the fermentation broth was detected by amino acid analyzer.

[0074] The fermentation medium is composed of 20 g / L glucose, 2.5 g / L yeast powder, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.23 g / L L-threonine, 0.1 g / L L-methionine, 1 g / L succinic acid, 2 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·4H2O, 0.005 g / L MnSO4, 0.0025 g / L ZnSO4, 0.004 g / L L-arginine, 0.003 g / L L-glutamic acid, and 10 g / L CaCO3 (sterilized separately). The solvent is deionized water, and the pH value is natural.

[0075] The feed medium is composed of: 500 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 1 g / L L-threonine, 0.05 g / L L-methionine, 0.5 g / L MgSO4·7H2O, 0.005 g / L MnSO4, 0.01 g / L FeSO4·4H2O, 0.0025 g / L ZnSO4, 0.004 g / L L-arginine, and 0.003 g / L L-glutamic acid, and the solvent is natural water.

[0076] O-succinyl-L-homoserine content detection: 1 mL of fermentation broth was centrifuged and diluted 500-fold with ultrapure water. After vortex mixing, it was filtered through a water membrane and tested by post-column derivatization using a Hitachi LA8080 amino acid analyzer. The OSH production results of different strains in a 5 L fermentor are shown in Table 3.

[0077] Table 3 Comparison of OSH production of different strains

[0078]

[0079]

[0080] The test results in Table 3 indicate that the introduction of exogenous glyceraldehyde-3-phosphate dehydrogenase gapC and its mutants provides more NADPH to the metabolic pathway, thereby increasing OSH production. Expression of the glyceraldehyde-3-phosphate dehydrogenase gapC mutant provided by the present invention in recombinant E. coli increased OSH production in a 5L fermentor from 87 g / L to 135 g / L, a 55% increase.

[0081] The present invention is not limited by the above specific description. The present invention can be modified in various ways within the scope of the claims, and these modifications are all within the scope of the present invention.

Claims

1. A glyceraldehyde-3-phosphate dehydrogenase mutant, characterized in that: The mutant is obtained by mutating the 233rd arginine in the amino acid sequence shown in SEQ ID NO. 2 to alanine.

2. A gene encoding the glyceraldehyde-3-phosphate dehydrogenase mutant according to claim 1.

3. A recombinant vector containing the coding gene according to claim 2.

4. A recombinant bacterium constructed by the recombinant vector according to claim 3.

5. The recombinant bacterium according to claim 4, characterized in that The recombinant bacteria were constructed as follows: the coding gene of the glyceraldehyde-3-phosphate dehydrogenase mutant was double-digested with EcoRI and BamHI and then connected to the multiple cloning site MCS1 of the plasmid pRSFDuet-1. fbr The gene was inserted into another MCS2 site of the plasmid after double digestion with KpnI and XhoI, and then transformed into E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr Competent cells were coated on LB plates containing 50 μg / ml kanamycin, cultured at 37°C overnight, and positive transformants were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin. After cultured at 37°C overnight, recombinant bacteria were obtained; the E. coli-ΔBBIJ-TrcmetL-TrcthrA fbr After knocking out metI, metJ, metB, and thrB genes in the Escherichia coli genome, thrA fbr The promoter of metL gene was replaced by the strong Trc promoter.

6. Use of the glyceraldehyde-3-phosphate dehydrogenase mutant according to claim 1 in increasing the yield of O-succinyl-L-homoserine.

7. The use according to claim 6, characterized in that The application method comprises the following steps: inoculating the recombinant bacteria according to claim 5 into a fermentation medium containing 50 μg / mL kanamycin, culturing at 37° C. and 180 rpm for 3 h, adding lactose at a final concentration of 0.5 mmol / L, inducing and culturing at 30° C. for 48 h, centrifuging, collecting the supernatant, separating and purifying, and obtaining O-succinyl-L-homoserine; wherein the fermentation medium comprises 20 g / L glucose, 2.5 g / L yeast powder, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.23 g / L L-threonine, 0.1 g / L L-methionine, 1 g / L succinic acid, 2 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·4H2O, 0.005 g / L MnSO4, and 0.1 g / L ZnSO4. 0.0025g / L, L-arginine 0.004g / L, L-glutamic acid 0.003g / L, the solvent is deionized water, and the pH value is natural.

8. The use according to claim 7, characterized in that The recombinant bacteria are cultured in a fermenter by inoculating the recombinant bacteria into a 5-L fermenter containing 2 L of a fermentation medium containing kanamycin at a final concentration of 50 μg / ml, with an initial rotation speed of 400 rpm and an initial ventilation flow rate of 1.0 L / min. As the bacterial concentration increases, the rotation speed and ventilation flow rate are adjusted to maintain a dissolved oxygen value between 20% and 30% of air saturation. The pH is adjusted to a stable value of 6.8-7.0 using 50% concentrated ammonia water and 50% phosphoric acid by volume. After culturing at 37°C for 8-12 hours, lactose at a final concentration of 0.5 mmol / L is added, the temperature is lowered to 30°C to induce expression, and the culture is continued for 100-120 hours. During the fermentation process, a feed medium is added to maintain a glucose concentration of 1-5 g / L. After the fermentation is completed, the fermentation broth is centrifuged, the supernatant is retained, and separation and purification are performed to obtain O-succinyl-L-homoserine. The fermentation medium is composed of: 20 g / L glucose, 2.5 g / L yeast powder, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.23 g / L L-threonine, 0.1 g / L L-methionine, 1 g / L succinic acid, 2 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.01 g / L FeSO4·4H2O, 0.005 g / L MnSO4, 0.0025 g / L ZnSO4, 0.004 g / L L-arginine, 0.003 g / L L-glutamic acid, and 10 g / L CaCO3; the solvent is deionized water; and the pH value is natural. The feed medium is composed of: 500 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 1 g / L L-threonine, 0.05 g / L L-methionine, 0.5 g / L MgSO4·7H2O, 0.005 g / L MnSO4, 0.01 g / L FeSO4·4H2O, 0.0025 g / L ZnSO4, 0.004 g / L L-arginine, and 0.003 g / L L-glutamic acid, and the solvent is natural water.

9. The use according to claim 8, characterized in that Before the recombinant bacteria are inoculated into the fermentation tank, slant activation and seed expansion culture are first performed, and the seed solution is inoculated into the fermentation tank culture medium at an inoculum concentration of 5-10% by volume. The seed solution is prepared as follows: (1) Plate culture: Use an inoculating loop to pick the recombinant bacteria from the glycerol tube and streak inoculate on a LB solid plate with a final concentration of 50 μg / ml kanamycin resistance. Seal the plate with sealing film and place it in a 37°C constant temperature incubator for 12-16 h. The final concentration of the LB solid plate medium is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, the solvent is deionized water, pH 7.

0. (2) Slant liquid culture: Use an inoculation loop to pick a single colony from the plate in step (1) and inoculate it into LB liquid medium containing a final concentration of 50 μg / ml kanamycin, and place it in a constant temperature shaking incubator at 37°C and 150 rpm for 8-12 hours for activation; the final concentration of the LB liquid medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7.0; (3) Seed culture: The activated bacterial solution from step (2) was inoculated into LB liquid culture medium containing kanamycin at a final concentration of 50 μg / ml at a volume concentration of 2%, and cultured in a 37°C constant temperature shaker for 8-12 hours to obtain seed solution.

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