An avtA mutant and its application in the fermentative production of L-valine

Abstract

The present invention relates to the field of bioengineering technology, and discloses a avtA mutant and its application in the fermentative production of L-valine. By performing point mutations on the avtA gene, the present invention reduces the enzyme expression activity, achieves carbon flux metabolic balance, and further realizes the purpose of increasing the valine yield. Through further experimental screening, a recombinant strain capable of significantly increasing the valine yield is obtained. From a specific fermentation result, it can be seen that this method can significantly increase the valine yield, from the original 1.3 g / L to 1.51 g / L, with a 16% increase in yield, providing strong strain support for further industrialization.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to an avtA mutant and its application in the fermentative production of L-valine. Background Art

[0002] L-valine, L-leucine and L-isoleucine are collectively referred to as branched-chain amino acids (BCAAs). The chemical name of L-valine is 2-amino-3-methylbutyric acid, and its chemical formula is C5H 11 NO2, with a molecular mass of 117.146. At room temperature, L-valine is a white crystal or crystalline powder, odorless, bitter, with a melting point of 293 °C and a density of 1.32 g / cm 3 , soluble in water, and almost insoluble in ethanol and acetone.

[0003] As a high-value-added amino acid, L-valine has a wide range of applications in the food, pharmaceutical, animal feed, and skin care product industries and has high commercial value. So far, the industrial production methods of L-valine include chemical synthesis, protein hydrolysis extraction, and microbial fermentation. There are various chemical synthesis routes for L-valine, but they all have problems such as complex synthesis processes, high costs of chiral auxiliaries, and serious environmental pollution. Therefore, they are basically not used in industrial production. As the name implies, the protein hydrolysis extraction method hydrolyzes raw materials rich in protein such as hair and cicada pupae, and extracts L-valine from the protein hydrolysate. When proteins are hydrolyzed, not only L-valine is produced, but also other amino acids and impurities are generated. Although they can be separated by an ion exchange column, there are problems such as low separation efficiency, high production costs, and large environmental pollution, which are not conducive to large-scale industrial production. The microbial fermentation method mainly utilizes the characteristics of microorganisms to synthesize L-valine, and changes the metabolic pathway of the bacterial cells by methods such as metabolic engineering, mutagenesis treatment, or selection of structural analog-resistant mutants to achieve the purpose of overproducing L-valine. Compared with other production methods of L-valine, the microbial fermentation method has the advantages of mild reaction, high yield, environmental friendliness, and low production costs, and is the main method for industrial production of L-valine at present.

[0004] In recent years, with the rapid development of synthetic biology and metabolic engineering, genetically modified recombinant engineering strains capable of highly producing L-valine can be obtained. At present, a variety of metabolic strategies have been used to increase the L-valine titer, including screening highly productive strains through mutagenesis, modifying metabolic pathways, and enhancing extracellular transport. Recently, through adaptive evolution combined with rational design, the Corynebacterium glutamicum strain ΔppcΔaceEΔalaTΔpqo was developed to improve the ability to produce L-valine, achieving an L-valine accumulation of 3.2 g / L. Although random mutation remains an effective strategy for cultivating L-valine-producing strains, there are problems such as low production efficiency and high costs. Escherichia coli has a clear metabolic pathway and a simple genetic operating system and has been used as a potential "microbial factory" for producing L-valine. Park et al. reported that by relieving the feedback inhibition of the target product on key enzymes and enhancing the metabolic flux of L-valine, the L-valine level reached 60.7 g / L (Park JH, Jang YS, Lee JW, Lee SY. Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering. Biotechnol Bioeng. 2011 May;108(5):1140-7.). In addition to traditional metabolic engineering, cofactor regeneration is an effective strategy for altering microbial metabolic pathways, with the advantages of balancing metabolism, influencing signal transduction, and complex networks. Cofactor balance is also considered an important factor required for L-valine biosynthesis.

[0005] In the metabolic pathway of Escherichia coli for producing L-valine, as Figure 1 shown, glucose is converted into L-valine, but L-valine is further catalyzed by valine transaminase (AvtA) to produce alanine for the growth of the bacteria itself. Therefore, there will be a balance problem between L-valine accumulation and bacterial growth, and directly knocking out the avtA gene will affect the production of alanine required for bacterial growth. Since the AvtA enzyme is the key node controlling valine accumulation and alanine production, the above studies have not involved the research on valine transaminase. Therefore, regulating the expression of AvtA is crucial for valine-producing bacteria. Summary of the Invention

[0006] The object of the present invention is to provide a general method for balancing amino acid synthesis required for bacterial growth and product generation during metabolic engineering transformation. The present invention performs point mutation on the avtA gene to reduce the enzyme expression activity, achieve carbon flux metabolic balance, and further achieve the purpose of increasing valine production. Through experimental screening, a recombinant strain capable of significantly increasing valine production was obtained.

[0007] The present invention provides an avtA mutant, which has a mutation at at least one of the following sites corresponding to the amino acids of the wild-type of avtA: K76, E172, L236, G282 or E353.

[0008] The mutation is based on the molecular docking of the AvtA wild-type sequence to simulate the sequences of different mutation sites. The mutant protein will reduce a certain enzymatic catalytic ability while retaining partial protein activity, enabling the strain to accumulate L-valine while being able to grow normally.

[0009] Specifically, it has a mutation at at least one of the following sites corresponding to the amino acids of the wild-type of avtA: K76E, E172G, L236R, G282D, E353K.

[0010] The present invention provides the coding nucleic acid of the avtA mutant described above.

[0011] The present invention also provides a recombinant expression vector containing the coding nucleic acid.

[0012] The present invention further provides a recombinant bacterium containing the recombinant expression vector, preferably Escherichia coli.

[0013] The present invention also provides the application of the coding nucleic acid of the avtA mutant as described above, the coding nucleic acid, the recombinant expression vector or the recombinant bacterium in the preparation of L-valine.

[0014] The present invention particularly provides a strain for producing L-valine, which is obtained by the following method:

[0015] S1: A general-purpose chassis bacterium is constructed for the initial bacteria (specifically, Escherichia coli Sva1024 and wild-type Escherichia coli ATCC8739). Specifically, a 20-bp specific site mutation is performed on the avtA gene to obtain a first modified strain. This is to achieve a single-site mutation in the genome. According to the CRISPR-cas9 principle, the original N20 sequence corresponding to the PAM site should not exist in the successfully edited genome, and the above-mentioned chassis bacterium needs to be constructed.

[0016] S2: The coding nucleic acid as described in claim 3 is used to perform corresponding point mutations and 20-bp specific site reverse mutations on the avtA gene in the first modified strain to obtain a second modified strain for producing L-valine.

[0017] The present invention further provides a method for preparing L-valine, which includes the step of fermenting and culturing the recombinant bacterium described above to obtain L-valine, and optionally further includes the step of separating the L-valine. Preferably, the fermentation and culture is anaerobic fermentation and culture.

[0018] In one embodiment, fermentation results show that the strain modified by the present invention can significantly increase the production of valine. For example, the initial strain Sva1024 increased its production from 1.3 g / L to 1.51 g / L, a 16% increase in yield. The wild-type strain E. coli ATCC8739 increased its production from 0 g / L to 0.1 g / L, with significant accumulation of L-valine, providing strong strain support for further industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The metabolic pathway for the synthesis of L-valine in Escherichia coli;

[0020] Figure 2 This is a graph showing the change of the remaining amount of pyruvate over time;

[0021] Figure 3 The fermentation results of metabolically modified Sva1024 strain;

[0022] Figure 4 These are the fermentation results of metabolically modified wild-type ATCC8739 strain. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0024] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0026] Unless otherwise specified, the test materials used in the following examples are all conventional biochemical reagents.

[0027] LB medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, deionized water as the solvent. For solid medium, add agar to a final concentration of 20 g / L.

[0028] Seed culture medium: glucose 20 g / L, corn steep liquor powder 10 g / L, KH2PO4 8.8 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 2 g / L.

[0029] Fermentation medium: The fermentation medium and the seed medium have the same composition, the only difference being that the glucose concentration is 50 g / L. The initial valine aminotransferase of the present invention is derived from the laboratory-preserved strain Sva1024.

[0030] The strains and plasmids used in the present invention are shown in Table 1.

[0031] Table 1 shows the strains and plasmids used in the present invention

[0032]

[0033] All references in the examples are shown in Table 2.

[0034] Table 2 shows the primers used in the present invention

[0035]

[0036] Example 1: Detection of enzymatic parameters of different mutants of AvtA

[0037] (1) Using the wild-type sequence of AvtA (the nucleotide sequence of the wild-type avtA gene is shown in SEQ ID NO.1, NCBI gene number NZ_CP043852.1: 3427520-3428773) as the object, five sequences with different mutation sites were simulated by the molecular docking method, which are: avtA K76E 、avtA E172G 、avtA L236R 、avtA G282D 、avtA E353K . These mutant proteins will reduce the enzymatic catalytic ability to a certain extent while retaining part of the protein activity. The simulated gene sequences were sent to General Biosystems for gene synthesis and constructed into the plasmid pET28a to obtain plasmids pET28a-avtA1, pET28a-avtA2, pET28a-avtA3, pET28a-avtA4, and pET28a-avtA5 respectively. The serial numbers of each plasmid correspond to the above-mentioned avtA mutation sites.

[0038] (2) Amplification of the avtA fragment

[0039] Obtaining the avtA fragment: Using avtA-F / avtA-R as primers and the genome of Sval024 as the template, PCR amplification was carried out (1254bp was positive). PCR reaction system: 25 μL of KOD enzyme, 1 μL of each of avtA-F / avtA-R, 1 μL of template DNA, 22 μL of ddH2O. PCR reaction program: 95 °C for 5 min; 98 °C for 10 s, 58 °C for 5 s, 68 °C for 20 s, 35 cycles; 72 °C for 10 min. After electrophoresis verification, the positive amplicon was purified using a DNA product purification kit to obtain the fragment AVT.

[0040] (3) Linearization of the pET28a plasmid

[0041] Using pET28a-xian-F / pET28a-xian-R as primers and the pET28a plasmid as a template, PCR amplification was performed (5362bp was positive). PCR reaction system: 25 μL of KOD enzyme, 1 μL each of pET28a-xian-F / pET28a-xian-R, 1 μL of pET28a plasmid, and 22 μL of ddH2O. PCR reaction program: 95°C for 5 min; 98°C for 10 s, 58°C for 5 s, 68°C for 30 s, 35 cycles; 72°C for 10 min. The original pET28a plasmid template was digested with DpnⅠ enzyme, and the PCR product was purified using a DNA product purification kit to obtain a linearized pET28a fragment.

[0042] (4) Cloning and identification of the target gene

[0043] The AVT fragment and the linearized pET28a fragment were seamlessly ligated using the Novoprotein recombination kit. 10 μL of the ligation product was transformed into DH5α competent cells and spread on a kanamycin plate, and cultured overnight at 37°C in an inverted position. Using pET28a-seq-F / pET28a-seq-R as primers and a single colony as a template, colony PCR amplification was performed (1700bp was positive). PCR reaction system: 10 μL of the verification enzyme 2×Rapid Taq Master Mix, 1 μL each of pET28a-seq-F / pET28a-seq-R, and 8 μL of ddH2O. PCR reaction program: 95°C for 5 min; 95°C for 15 s, 58°C for 15 s, 72°C for 45 s, 35 cycles; 72°C for 10 min. After electrophoresis verification, the recombinant plasmid pET28a-AVT was obtained.

[0044] The above recombinant plasmid and the gene synthesis plasmids pET28a-AVT1, pET28a-AVT2, pET28a-AVT3, pET28a-AVT4, and pET28a-AVT5 were respectively transformed into the BL21 expression strain, spread on a kanamycin-resistant plate, and cultured overnight at 37°C in an inverted position to obtain recombinant clones.

[0045] (5) Expression of the recombinant plasmid

[0046] The recombinant clones obtained in (4) were inoculated into 5 mL of LB medium containing kanamycin and cultured overnight at 37°C with 200 rpm. Then, they were inoculated into 100 mL of LB medium at an inoculation amount of 1%, and kanamycin antibiotic with a final concentration of 50 mg / L was added. The culture was continued at 37°C until the OD 600 reached 0.6 - 1.0, and IPTG with a final concentration of 0.4 mM was added, and induced at 18°C for 16 - 20 h.

[0047] (6) Purification of the recombinant protein

[0048] Centrifuge the induced bacterial solution in (5) (centrifugation temperature: 4°C, rotation speed: 10,000 rpm, time 5 min), collect the bacterial cells, and then resuspend the collected bacterial cell precipitate in Lysis Buffer at a ratio of 40:3, and add phenylmethylsulfonyl fluoride solution (PMSF) to a final concentration of 1 mM (add 100 mM PMSF isopropanol stock solution). Subsequently, use an ultrasonic cell disruptor to break the cells on ice. Centrifuge the cell lysate at 4°C and 10,000 rpm for 30 min to obtain the supernatant and precipitate of the cell lysate.

[0049] Load a nickel column (Cytiva, 5 mL) onto an AKTA protein purifier, and use Lysis Buffer (see Table 3) to equilibrate the nickel column at a flow rate of 2 mL / min. After the nickel column is equilibrated (about 10 column volumes), use the AKTA system to pass the supernatant of the cell lysate through the nickel column at a flow rate of 1 mL / min, and His-AvtA, His-AvtA1, His-AvtA2, His-AvtA3, His-AvtA4, and His-AvtA5 will be captured by the nickel column. After all the supernatant enters the AKTA system, use Washing Buffer (see Table 3) to wash the nickel column until the UV absorption recorded value of AKTA is roughly stable. Subsequently, use Elution Buffer (see Table 3) to elute the target protein in the nickel column to obtain six purified proteins - His-AvtA, His-AvtA1, His-AvtA2, His-AvtA3, His-AvtA4, and His-AvtA5.

[0050] Table 3 Formulas required for protein purification reagents

[0051]

[0052] (7) SDS-PAGE verification

[0053] Take 40 μL of the supernatant, precipitate (resuspended in 10% Lysis Buffer), and the purified product after centrifugation respectively, add 10 μL of 5× protein electrophoresis buffer, mix well, boil in a water bath for 5 min, and take 10 μL of the sample for 12% SDS-PAGE. There are protein bands near 46.7 kDa.

[0054] (8) Enzyme activity detection

[0055] Valine transaminase can catalyze the reaction of L-valine and pyruvate to produce L-alanine and 3-methyl-2-oxobutanoic acid. The activity of the recombinant protein was measured according to this reaction. First, prepare 2 kinds of protein reaction substrate stock solutions, 0.5 M L-valine and 10 M pyruvate. The 10 mL reaction solution contains 3 mg of recombinant protein, 2 mL of L-valine stock solution, 100 μL of pyruvate stock solution, 50 μL of Tween-80, and the pH is adjusted to 8.0 with 0.5 M NaOH, and the rest is made up to 10 mL with Lysis Buffer. After mixing, react on a shaker at 37 °C. Take the reaction solutions at 2, 5, 10, 20, 60, 120, 180, 240, and 300 minutes respectively, and use Agilent liquid phase to detect the consumption of pyruvate. The liquid phase method is the same as the L-valine detection method, and Lysis Buffer is used to replace the enzyme solution as a blank control. The results of the remaining amount of pyruvate changing with time are as Figure 2 shown.

[0056] Judging from the remaining situation of pyruvate, the catalytic abilities of the 5 mutant enzymes are all reduced, but not completely inhibited, which is consistent with the results required by the present invention. The reduction of enzyme activity will reduce the degradation amount of L-valine, increase the yield, and at the same time retain part of the catalytic ability of the enzyme to enable the normal growth of the strain.

[0057] Example 2. Obtaining recombinant strains

[0058] The specific operation is as follows:

[0059] Construct recombinant strains SA0 and AA0

[0060] In order to perform point mutations in the genome subsequently, first construct a general chassis strain SA0 and AA0, that is, introduce the mutant N20 into the genomes of the chassis bacteria Sva1024 and ATCC 8739 respectively. The integration method adopts the CRISPR-cas9 gene editing method. The specific operation method is as follows:

[0061] The first step is to construct the pTarget-avtA-N20 plasmid

[0062] Design the mutant avtA N20 using the CHOPCHOP online website. The nucleotide sequence is shown in SEQ ID NO.2. Using the pTarget (Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021 Apr 15;53(5):620-627. doi: 10.1093 / abbs / gmab036. PMID: 33764372.) vector as a template and avtA-N20-F / avtA-N20-R as primers, perform PCR amplification. The PCR reaction conditions are as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 1.5 min, repeat 35 cycles; continue to extend at 72°C for 10 min. After treating the PCR product with Dpn I at 37°C for 1 h, transform it into the E. coli DH5α recipient bacteria, coat it on an LB solid plate containing 50 mg / L spectinomycin hydrochloride resistance, culture it at 37°C for 12 h, randomly pick a single colony and transfer it to an LB liquid medium containing 50 mg / L spectinomycin hydrochloride resistance, culture it at 37°C for 12 h, collect the bacteria and extract the plasmid to obtain the pTarget-avtA-N20 vector.

[0063] Second step, construct the overlapping fragment U avtA -avtA N20-1 -D avtA

[0064] Using the Sva1024 genome as a template, respectively use primer avtA-F1 / primer avtA-R1 and primer avtA-F2 / primer avtA-R2, and perform PCR amplification. The PCR reaction conditions are as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 1 min, repeat 35 cycles; continue to extend at 72°C for 10 min. Recover the PCR product by gel extraction to obtain fragment U avtA (544 bp is positive) and fragment avtA N20-1 -D avtA (501 bp is positive).

[0065] Use primer avtA-F1 and primer avtA-R2 for the above-mentioned fragment U avtA and avtA N20-1 -D avtAPerform fusion PCR with the following PCR reaction conditions: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 1.5 min, repeat for 30 cycles; continue extension at 72°C for 10 min. Recover the PCR product by gel extraction to obtain the overlapping fragment U avtA -avtA N20-1 -D avtA (1045 bp is positive).

[0066] Step 3: Construct the pTarget-avtA vector

[0067] Mix the above pTarget-avtA-N20 vector and the overlapping fragment U avtA -avtA N20-1 -D avtA Perform one-step cloning with the reaction program: 37°C for 30 min. Transform the cloning product into E. coli DH5α recipient bacteria and spread them on an LB solid plate containing 50 mg / L spectinomycin hydrochloride resistance. Incubate at 37°C for 12 h. Randomly pick single colonies and transfer them to an LB liquid medium containing 50 mg / L spectinomycin hydrochloride resistance. Incubate at 37°C for 12 h, collect the bacteria, and extract the plasmid to obtain the pTarget-avtA vector.

[0068] Step 4: Obtain the recombinant strains SA0 and AA0

[0069] Electrotransform the above pTarget-avtA vector into Sva1024 and ATCC 8739 strains containing the pEccas vector (Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021 Apr 15; 53(5): 620-627. doi: 10.1093 / abbs / gmab036. PMID: 33764372.) with the following operating steps:

[0070] Electroporation: Culture the Sva1024 and ATCC 8739 strains transformed with the pEccas vector in an LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD 600 reaches 0.6. Centrifuge the bacterial solution to obtain the bacteria. Wash the bacteria twice with 10% glycerol for use. Perform electroporation at 2.5 kV.

[0071] Plasmid elimination: Spread the electrotransformed bacterial solution onto an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride resistance, and culture overnight at 37°C. Pick a single colony as a template and perform PCR amplification with primer avtA-seq-F740 and primer avtA-seq-R (750 bp is positive). Inoculate the verified correct strain into an LB medium containing 50 mg / L kanamycin and 10 mM rhamnose, culture overnight at 37°C to remove the pTarget-avtA vector. Then inoculate the strain without the pTarget-avtA vector into an LB medium containing 10 g / L sucrose to remove the pEccas vector, and the recombinant strains SA0 and AA0 can be obtained.

[0072] Construct recombinant strains SA1, SA2, SA3, SA4, SA5, AA1, AA2, AA3, AA4, AA5

[0073] First step, construct the pTarget-A-N20-1 vector

[0074] Using the pTarget vector as a template, perform PCR amplification with primer A-N20-F and primer A-N20-R to obtain the pTarget-A-N20-1 mutant vector. After treating the PCR product with Dpn I at 37°C for 1 h, transform it into E. coli DH5α recipient bacteria, spread it on an LB solid plate containing a final concentration of 50 mg / L spectinomycin hydrochloride resistance, and culture at 37°C for 12 h. Randomly pick a single colony and transfer it to an LB liquid medium containing a final concentration of 50 mg / L spectinomycin hydrochloride resistance, culture at 37°C for 12 h, collect the bacteria and extract the plasmid to obtain the pTarget-A-N20-1 vector.

[0075] Second step, construct the pTarget-A vector

[0076] Using plasmids pET28a-avtA1, pET28a-avtA2, pET28a-avtA3, pET28a-avtA4, pET28a-avtA5 as templates respectively, perform PCR amplification with primer A-F and primer A-R, and recover the product by gel extraction to obtain A1, A2, A3, A4, A5 fragments (2254 bp is positive). The PCR reaction conditions are as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 2 min, repeat 35 cycles; continue to extend at 72°C for 10 min.

[0077] Perform one-step cloning on the pTarget-A-N20-1 vector and fragments A1, A2, A3, A4, and A5 respectively. The reaction program: 30 min at 37°C. Transform the cloning products into E. coli DH5α recipient bacteria, and spread them on an LB solid plate containing 50 mg / L spectinomycin hydrochloride resistance. Incubate at 37°C for 12 h. Randomly pick single colonies and transfer them to an LB liquid medium containing 50 mg / L spectinomycin hydrochloride resistance, incubate at 37°C for 12 h, collect the bacteria, and extract the plasmids to obtain the pTarget-A1, pTarget-A2, pTarget-A3, pTarget-A4, and pTarget-A5 vectors.

[0078] Step 3: Obtain the recombinant strains SA1, SA2, SA3, SA4, SA5, AA1, AA2, AA3, AA4, and AA5

[0079] Electrotransform the pTarget-A1, pTarget-A2, pTarget-A3, pTarget-A4, and pTarget-A5 vectors into the SA0 and AA0 strains containing the pEccas vector respectively. The electrotransformation steps are the same as those described in (1).

[0080] Spread the electrotransformed bacterial solution on an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride resistance, and incubate at 37°C overnight. Pick single colonies as templates and perform PCR amplification with primer A-seq-F1100 and primer A-seq-R (1100 bp is positive). Subsequently, perform plasmid curing according to the steps described in (1) to obtain the recombinant strains SA1, SA2, SA3, SA4, SA5 and AA1, AA2, AA3, AA4, AA5.

[0081] Example 3: Fermentation detection of 10 mutant strains, the original strain Sva1024, and the wild-type strain ATCC 8739

[0082] Perform fermentation experiment tests on the chassis strain Sva1024, the wild-type strain ATCC 8739, and the strains constructed in Example 2 above (SA1, SA2, SA3, SA4, SA5 and AA1, AA2, AA3, AA4, AA5) to compare the effects of different mutation sites of the avtA gene on the ability to produce L-valine among different chassis strains. The fermentation experiment is carried out according to the following protocol:

[0083] (1) Seed culture: Fresh clones on the LB plate were inoculated into a test tube containing 4 mL of seed medium and cultured overnight at 37 °C with shaking at 250 rpm. Then, the culture was transferred to a 250 mL Erlenmeyer flask containing 30 mL of seed medium at an inoculation amount of 2% (V / V) and cultured at 37 °C with shaking at 250 rpm for 12 hours to obtain a seed culture solution for inoculating the fermentation medium.

[0084] (2) Fermentation culture: The volume of the fermentation medium in a 500 mL anaerobic jar was 250 mL. The seed culture solution was inoculated into the fermentation medium at an inoculation amount with a final concentration of OD 550 = 0.1, and fermented at 37 °C and 150 rpm for 4 days to obtain a fermentation broth. The neutralizing agent was 5 M ammonia water to control the pH of the fermentation jar at 7.0, and no gas was introduced during the culture process.

[0085] Take 1 mL of the fermentation broth from the fermentation jar, centrifuge at 12000 rpm for 1 min, take the supernatant, filter through a filter membrane (pore size 0.22 μm), and then use HPLC to analyze the content of L-valine in the fermentation broth. Finally, compare the amount of L-valine obtained by each strain. The results are as Figure 3 shown.

[0086] Detection using an Agilent high performance liquid chromatograph: The chromatographic column was Yuexu Ultimate HILIC Amphion II. Mobile phase: Organic phase, pure acetonitrile; aqueous phase, 0.05 M potassium dihydrogen phosphate, pH 3.0 (Preparation method: Weigh the corresponding mass of potassium dihydrogen phosphate at 0.05 M concentration and dissolve it in ultrapure water, adjust the pH to 3.0 with phosphoric acid, filter by suction, and then add the corresponding volume of acetonitrile according to the ratio of acetonitrile: 0.05 M potassium dihydrogen phosphate = 75:25, mix well and ultrasonically remove bubbles). Set the parameters as detection wavelength 206 nm, injection volume 10 μL, flow rate 1 mL / min, and detection time 20 min.

[0087] From Figure 3 and Figure 4 it can be seen that mutations at 5 different sites in different chassis strains will increase the yield of L-valine. After 4 days of fermentation of the initial strain Sva1024, the valine yield reached 1.3 g / L. After point mutation, the highest yield increase was for the SA3 strain, reaching 1.51 g / L, an increase of 16.1%. The same improvement effect was observed for the genetic modification of wild bacteria, and the L-valine accumulation increased from 0 to 0.1 g / L. The fermentation results show that this method plays a certain positive role in L-valine accumulation and provides a metabolic engineering idea for the subsequent chassis construction of valine and its derivatives.

Claims

1. A avtA mutant, characterized in that There is a K76E mutation in its amino acid sequence corresponding to the amino acid sequence of the wild-type of avtA, wherein the amino acid sequence of the wild-type of avtA is encoded by the nucleotide sequence shown in SEQ ID No:

1.

2. The avtA coding nucleic acid of the mutant as claimed in claim 1.

3. A recombinant expression vector containing the coding nucleic acid according to claim 2.

4. A recombinant bacterium containing the recombinant expression vector according to claim 3, wherein the wild-type avtA gene in the recombinant bacterium is replaced by the coding nucleic acid according to claim 2.

5. The recombinant bacterium according to claim 4, wherein It is Escherichia coli.

6. The use of the mutant according to claim 1, the coding nucleic acid according to claim 2, the recombinant expression vector according to claim 3, or the recombinant bacterium according to claim 4 or 5 in the preparation of L-valine. avtA ​ 7. An L-valine producing strain, characterized in that, Obtained by the following method: S1: Construct a general chassis bacterium from the initial Escherichia coli, specifically perform a 20-bp specific-site mutation on the avtA gene to obtain the first modified strain; S2: Based on the first engineered strain, according to the coding nucleic acid described in claim 2, perform site-directed mutagenesis on the wild-type avtA gene and 20-bp specific-site reversion mutagenesis to obtain a second engineered L-valine producing strain.

8. The L-valine producing strain according to claim 7, characterized in that, The initial Escherichia coli is wild Escherichia coli or Escherichia coli capable of producing L-valine.

9. The L-valine-producing strain according to claim 7, wherein The initial Escherichia coli is Escherichia coli Sva1024 or wild-type Escherichia coli ATCC8739.

10. A method for preparing L-valine, characterized in that, It includes the step of fermenting and culturing the L-valine-producing strain according to any one of claims 7 to 9 to obtain L-valine.

11. The method according to claim 10, wherein It further includes the step of separating the L-valine.

12. The method according to claim 10 or 11, characterized in that, The fermentation culture is anaerobic fermentation culture.

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