AvtA mutant and application thereof in fermentation production of L-valine

Abstract

The invention relates to the technical field of bioengineering, and discloses an avtA mutant and application thereof in fermentation production of L-valine. According to the present invention, the avtA gene is subjected to point mutation, such that the enzyme expression activity is reduced, the carbon flow metabolism balance is achieved, and the valine yield is increased. And a recombinant strain capable of remarkably improving the yield of valine is further obtained through experimental screening. A specific fermentation result shows that the method can obviously improve the yield of valine from 1.3 g / L to 1.51 g / L, the yield is improved by 16%, and a powerful strain support is provided for further industrialization.

Description

[0001] This application is a divisional application of the application with the application date of April 1, 2024, application number 2024103848302, and invention name “An avtA mutant and its application in L-valine fermentation production”. Technical Field

[0002] The present invention relates to the technical field of bioengineering, and in particular to an avtA mutant and application thereof in the fermentation production of L-valine. Background Art

[0003] L-valine, L-leucine, and L-isoleucine are collectively referred to as branched-chain amino acids (BCAAs). L-valine's chemical name is 2-amino-3-methylbutyric acid, with a chemical formula of C₅H₁₁NO₂ and a molecular mass of 117.146. At room temperature, L-valine is an odorless, bitter white crystal or powder with a melting point of 293°C and a density of 1.32 g / cm³. It is readily soluble in water and virtually insoluble in ethanol and acetone.

[0004] As a high-value-added amino acid, L-valine is widely used in the food, pharmaceutical, animal feed, and skincare industries, and holds high commercial value. To date, the industrial production methods for L-valine include chemical synthesis, protein hydrolysis extraction, and microbial fermentation. While there are multiple chemical synthesis routes for L-valine, all suffer from complex synthesis processes, high costs of chiral auxiliaries, and severe environmental pollution. Consequently, these methods are largely abandoned for industrial production. Protein hydrolysis extraction, as the name suggests, involves hydrolyzing protein-rich raw materials such as hair and cicada pupae to extract L-valine from the protein hydrolysate. Protein hydrolysis not only produces L-valine, but also other amino acids and impurities. While separation is possible using ion exchange columns, low separation efficiency, high production costs, and significant environmental pollution hinder large-scale industrial production. Microbial fermentation primarily utilizes the ability of microorganisms to synthesize L-valine. Overproduction of L-valine is achieved by altering the metabolic pathways of the microorganism through metabolic engineering, mutagenesis, or the selection of structurally analog-resistant mutants. Compared with other L-valine production methods, microbial fermentation has the advantages of mild reaction, high yield, green environmental protection, and low production cost. It is currently the main method for industrial production of L-valine.

[0005] In recent years, with the rapid development of synthetic biology and metabolic engineering, recombinant engineered strains that can efficiently produce L-valine can be obtained through genetic modification. At present, a variety of metabolic strategies have been used to improve the titer of L-valine, including screening efficient production 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 production capacity of L-valine, achieving an L-valine accumulation of 3.2 g / L. Although random mutagenesis remains an effective strategy for cultivating L-valine-producing strains, there are problems such as low production efficiency and high cost. Escherichia coli has a clear metabolic pathway and a simple genetic operating system, and has been used as a potential "microbial factory" for the production of 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 advantage of balancing metabolic influences on signal transduction and complex networks. Cofactor balance is also considered to be an important factor required for L-valine biosynthesis.

[0006] In the metabolic pathway of L-valine production in Escherichia coli, Figure 1 As shown, glucose is converted to L-valine, which is then further catalyzed by valine aminotransferase (AvtA) to produce alanine, which is then used for bacterial growth. Therefore, there is a balance between L-valine accumulation and bacterial growth. Directly knocking out the avtA gene would affect the production of alanine, which is required for bacterial growth. Since AvtA is a key enzyme controlling valine accumulation and alanine production, none of the above studies involved the study of valine aminotransferase. Therefore, regulating AvtA expression is crucial for valine-producing bacteria. Summary of the Invention

[0007] The present invention aims to provide a general method for balancing amino acid synthesis required for bacterial growth and product production during metabolic engineering. By performing point mutations in the avtA gene, the enzyme's activity is reduced, achieving a balanced carbon flux and thereby increasing valine production. Experimental screening has also yielded recombinant strains that significantly increase valine production.

[0008] The present invention provides an avtA mutant, wherein at least one of the following amino acid positions corresponding to the wild-type avtA is mutated: K76, E172, L236, G282 or E353.

[0009] The mutation is molecularly docked based on the wild-type sequence of AvtA to simulate sequences of different mutation sites. The mutant protein will reduce the enzyme catalytic ability to a certain extent while retaining some protein activity, so that the strain can grow normally while increasing the accumulation of L-valine.

[0010] Specifically, at least one of the following positions corresponding to the amino acid sequence of the wild-type avtA is mutated: K76E, E172G, L236R, G282D, E353K.

[0011] The present invention provides a nucleic acid encoding the avtA mutant.

[0012] The present invention also provides a recombinant expression vector containing the encoding nucleic acid.

[0013] The present invention further provides a recombinant bacterium containing the recombinant expression vector, preferably, the recombinant bacterium is Escherichia coli.

[0014] The present invention also provides the encoding nucleic acid of the avtA mutant described above, and the use of the encoding nucleic acid, the recombinant expression vector or the recombinant bacterium in preparing L-valine.

[0015] The present invention particularly provides an L-valine producing strain, which is obtained by the following method:

[0016] S1: A universal base strain was constructed for each of the initial bacteria (specifically, E. coli Sva1024 and wild-type E. coli ATCC8739). Specifically, a 20bp specific site mutation was performed on the avtA gene to obtain the first modified strain. This is to achieve a single point mutation in the genome. According to the CRISPR-cas9 principle, the original N20 sequence corresponding to the PAM site cannot exist in the genome after successful editing, so the above base strain needs to be constructed.

[0017] S2: The encoding nucleic acid as described in claim 3 is used to perform corresponding point mutation and 20 bp specific site reversion mutation on the avtA gene in the first transformed strain to obtain a second transformed L-valine producing strain.

[0018] The present invention further provides a method for preparing L-valine, comprising the step of fermenting and culturing the recombinant bacteria to obtain L-valine, and optionally, further comprising the step of isolating the L-valine. Preferably, the fermentation and culturing is anaerobic fermentation and culturing.

[0019] 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

[0020] Figure 1 The metabolic pathway for the synthesis of L-valine in Escherichia coli;

[0021] Figure 2 This is a graph showing the change of the remaining amount of pyruvate over time;

[0022] Figure 3 The fermentation results of metabolically modified Sva1024 strain;

[0023] Figure 4 These are the fermentation results of metabolically modified wild-type ATCC8739 strain. DETAILED DESCRIPTION

[0024] 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.

[0025] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0027] Unless otherwise specified, the test materials used in the following examples are all conventional biochemical reagents.

[0028] 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.

[0029] 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.

[0030] Fermentation medium: The composition of the fermentation medium is the same as that of the seed medium, the only difference being that the glucose concentration is 50 g / L.

[0031] The initial valine aminotransferase of the present invention is derived from the laboratory-preserved strain Sva1024.

[0032] The strains and plasmids used in the present invention are shown in Table 1.

[0033] Table 1 shows the strains and plasmids used in the present invention.

[0034]

[0035]

[0036] All citations in the examples are shown in Table 2.

[0037] Table 2 shows the primers used in the present invention.

[0038]

[0039]

[0040] Example 1: Detection of enzymatic parameters of different AvtA mutants

[0041] (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, the molecular docking method was used to simulate the sequences of five different mutation sites, namely: avtAK76E, avtAE172G, avtAL236R, avtAG282D, and avtAE353K. These mutant proteins will reduce the catalytic ability of the enzyme to a certain extent, while retaining some protein activity. The simulated gene sequences were sent to Universal Biotechnology for gene synthesis and constructed into plasmid pET28a, respectively, to obtain plasmids pET28a-avtA1, pET28a-avtA2, pET28a-avtA3, pET28a-avtA4, and pET28a-avtA5. The sequence numbers of each plasmid correspond to the above-mentioned avtA mutation sites.

[0042] (2) Amplification of the avtA fragment

[0043] To obtain the avtA fragment, PCR amplification was performed using primers avtA-F / avtA-R and the Sval024 genome as a template (a positive result of 1254 bp was considered). The PCR reaction system consisted of 25 μL of KOD enzyme, 1 μL each of avtA-F / avtA-R, 1 μL of template DNA, and 22 μL of ddH2O. The PCR protocol was as follows: 95°C for 5 min; 35 cycles of 98°C for 10 s, 58°C for 5 s, and 68°C for 20 s; and 72°C for 10 min. After verification by electrophoresis, the positive amplicon was purified using a DNA product purification kit to obtain the AVT fragment.

[0044] (3) Linearization of pET28a plasmid

[0045] PCR amplification was performed using pET28a-xian-F / pET28a-xian-R as primers and the pET28a plasmid as a template (5362 bp was considered positive). The PCR reaction system included 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. The PCR reaction program was as follows: 95°C for 5 min; 35 cycles of 98°C for 10 s, 58°C for 5 s, and 68°C for 30 s; and 72°C for 10 min. The original pET28a plasmid template was digested with Dpn I, and the PCR product was purified using a DNA product purification kit to obtain a linearized pET28a fragment.

[0046] (4) Target gene cloning and identification

[0047] The AVT fragment and the linearized pET28a fragment were seamlessly ligated using the Novozymes recombination kit. 10 μL of the ligation product was transformed into DH5α competent cells, plated on kanamycin plates, and incubated inverted overnight at 37°C. Colony PCR amplification (1700 bp was considered positive) was performed using pET28a-seq-F / pET28a-seq-R primers and a single colony as a template. The PCR reaction system consisted of 10 μL of 2× Rapid Taq Master Mix, 1 μL each of pET28a-seq-F / pET28a-seq-R, and 8 μL of ddH2O. The PCR reaction program was as follows: 95°C for 5 min; 35 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 45 s; and 72°C for 10 min. After verification by electrophoresis, the recombinant plasmid pET28a-AVT was obtained.

[0048] The above-mentioned recombinant plasmids and gene synthesis plasmids pET28a-AVT1, pET28a-AVT2, pET28a-AVT3, pET28a-AVT4 and pET28a-AVT5 were respectively transformed into BL21 expression strains, spread on kanamycin-resistant plates, and cultured inverted at 37°C overnight to obtain recombinant clones.

[0049] (5) Recombinant plasmid expression

[0050] The recombinant clone obtained in (4) was inoculated into 5 mL of LB medium containing kanamycin and cultured overnight at 37°C at 200 rpm. Then, a 1% inoculum was inoculated into 100 mL of LB medium, and kanamycin was added at a final concentration of 50 mg / L. The cells were cultured at 37°C until the OD600 reached 0.6-1.0. IPTG was then added at a final concentration of 0.4 mM and induced at 18°C ​​for 16-20 h.

[0051] (6) Recombinant protein purification

[0052] The bacterial solution induced in (5) was centrifuged (centrifugation temperature: 4°C, rotation speed: 10,000 rpm, time: 5 min) to collect the cells. The collected bacterial pellet was then resuspended in Lysis Buffer at a ratio of 40:3 and supplemented with phenylmethylsulfonyl fluoride solution (PMSF) to a final concentration of 1 mM (add 100 mM PMSF isopropanol stock solution). Subsequently, the cells were disrupted on ice using an ultrasonic disruptor. The cell disruption solution was centrifuged at 4°C, 10,000 rpm for 30 min to obtain the cell disruption supernatant and precipitate.

[0053] A nickel column (Cytiva, 5 mL) was loaded onto an AKTA protein purifier and equilibrated with Lysis Buffer (see Table 3) at a flow rate of 2 mL / min. After equilibration (approximately 10 column volumes), the supernatant from the lysed cells was passed through the nickel column at a flow rate of 1 mL / min using the AKTA system. His-AvtA, His-AvtA1, His-AvtA2, His-AvtA3, His-AvtA4, and His-AvtA5 were captured by the nickel column. After the supernatant had completely entered the AKTA system, the nickel column was rinsed with Washing Buffer (see Table 3) until the UV absorbance readings on the AKTA system were roughly stable. Subsequently, the target protein was eluted from the nickel column using Elution Buffer (see Table 3) to obtain six purified proteins: His-AvtA, His-AvtA1, His-AvtA2, His-AvtA3, His-AvtA4, and His-AvtA5.

[0054] Table 3 Required formula of protein purification reagents

[0055] Table 3 Required formula of protein purification reagents

[0056] Reagent name Lysis Buffer Washing Buffer Elution Buffer Tris-HCl 25mM 25mM 25mM NaCl 500mM 500mM 150mM Imidazole 10mM 25mM 300mM pH 8.0 8.0 8.0

[0057] (7) SDS-PAGE verification

[0058] Take 40 μL of the supernatant, precipitate (resuspended in 10% Lysis Buffer) and purified product after centrifugation, add 10 μL of 5× protein electrophoresis buffer, mix well, boil in boiling water bath for 5 minutes, take 10 μL of sample for 12% SDS-PAGE, and a protein band is found near 46.7 kDa.

[0059] (8) Enzyme activity detection

[0060] Valine aminotransferase can catalyze the reaction of L-valine and pyruvate to produce L-alanine and 3-methyl-2-carbonyl-butyric acid. The activity of the recombinant protein was determined based on this reaction. First, two protein reaction substrate mother solutions were prepared, 0.5M L-valine and 10M pyruvate. 10mL of reaction solution included 3mg of recombinant protein, 2mL of L-valine mother solution, 100μL of pyruvate mother solution, 50μL of Tween-80, 0.5M NaOH to adjust the pH to 8.0, and the remaining 10mL was made up with Lysis Buffer. After mixing, shake the reaction at 37°C, and take the reaction solution for 2, 5, 10, 20, 60, 120, 180, 240 and 300 minutes respectively. The consumption of pyruvate was detected using Agilent liquid chromatography. The liquid chromatography method was consistent with the L-valine detection method. Lysis Buffer was used instead of the enzyme solution as a blank control. The changes in the remaining amount of pyruvate over time are shown in the following table. Figure 2 shown.

[0061] Judging from the residual pyruvate, the catalytic ability of the five mutant enzymes was 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 of L-valine and increase the yield, while retaining some catalytic ability of the enzyme to enable normal growth of the strain.

[0062] Example 2: Obtaining recombinant strains

[0063] The specific operations are as follows:

[0064] Construction of recombinant strains SA0 and AA0

[0065] To subsequently perform point mutations in the genome, we first constructed universal chassis strains SA0 and AA0. We then introduced the mutant N20 into the genomes of chassis strains Sva1024 and ATCC 8739, respectively. The integration was performed using CRISPR-cas9 gene editing. The specific steps are as follows:

[0066] The first step is to construct the pTarget-avtA-N20 plasmid

[0067] The mutant avtA N20 was designed using the CHOPCHOP online website. The nucleotide sequence is shown in SEQ ID NO. 2. PCR amplification was performed using the pTarget (Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang SA modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021Apr 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. The PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, and 72°C for 1.5 min, repeated for 35 cycles; and extension at 72°C for 10 min. The PCR product was treated with Dpn I at 37°C for 1 h, transformed into E. coli DH5α recipient bacteria, spread on LB solid plates containing spectinomycin hydrochloride resistance at a final concentration of 50 mg / L, and cultured at 37°C for 12 h. Single colonies were randomly picked and transferred to LB liquid medium containing spectinomycin hydrochloride resistance at a final concentration of 50 mg / L, cultured at 37°C for 12 h, and the bacteria were collected and the plasmid was extracted to obtain the pTarget-avtA-N20 vector.

[0068] The second step is to construct overlapping segments U avtA -avtA N20-1 -D avtA

[0069] Using the Sva1024 genome as a template, primers avtA-F1 / primer avtA-R1 and primers avtA-F2 / primer avtA-R2 were used for PCR amplification. The PCR reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 1 min, repeated 35 cycles; and 72°C for 10 min. The PCR products were recovered by gel chromatography to obtain fragment U. avtA (544 bp is positive) and fragment avtA N20-1 -D avtA (501bp is positive).

[0070] The above fragment U was cleaved using primers avtA-F1 and avtA-R2. avtA and avtA N20-1 -D avtAFusion PCR was performed under the following reaction conditions: 95°C for 3 min; 95°C for 15 s, 58°C for 15 s, 72°C for 1.5 min, repeated 30 cycles; and further extension at 72°C for 10 min. The PCR products were recovered by gel extraction to obtain overlapping fragments U avtA -avtA N20-1 -D avtA (1045bp is positive).

[0071] Step 3: Construct pTarget-avtA vector

[0072] The pTarget-avtA-N20 vector and the overlapping fragment UavtA-avtAN20-1-DavtA were cloned in a single step at 37°C for 30 minutes. The cloned product was transformed into E. coli DH5α recipient strains, plated onto solid LB plates containing spectinomycin hydrochloride at a final concentration of 50 mg / L, and incubated at 37°C for 12 hours. Individual colonies were randomly selected and transferred to liquid LB medium containing spectinomycin hydrochloride at a final concentration of 50 mg / L. The cells were then incubated at 37°C for 12 hours, harvested, and the plasmid extracted to obtain the pTarget-avtA vector.

[0073] Step 4: Obtain recombinant strains SA0 and AA0

[0074] The pTarget-avtA vector was electroporated into Sva1024 and ATCC 8739 strains containing the pEccas vector (Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang SA modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021Apr 15; 53(5): 620-627. doi: 10.1093 / abbs / gmab036. PMID: 33764372.) using the following steps:

[0075] Electroporation: Sva1024 and ATCC 8739 strains transformed with the pEccas vector were cultured in LB medium containing 50 mg / L kanamycin and 10 mM L-arabinose at 37°C until the OD600 reached 0.6. The culture broth was then centrifuged to obtain cells. Cells were washed twice with 10% glycerol before use. Electroporation was performed at 2.5 kV.

[0076] Plasmid elimination: The electroporated bacterial solution was plated onto LB plates containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was selected as a template for PCR amplification using primers avtA-seq-F740 and avtA-seq-R (750 bp was considered positive). The verified strain was inoculated into LB medium containing 50 mg / L kanamycin and 10 mM rhamnose and cultured overnight at 37°C to remove the pTarget-avtA vector. The pTarget-avtA vector-depleted strain was then inoculated into LB medium containing 10 g / L sucrose to remove the pEccas vector, resulting in the recombinant strains SA0 and AA0.

[0077] (2) Construction of recombinant strains SA1, SA2, SA3, SA4, SA5, AA1, AA2, AA3, AA4, and AA5

[0078] The first step is to construct the pTarget-A-N20-1 vector

[0079] Using the pTarget vector as a template, primers A-N20-F and A-N20-R were used for PCR amplification to obtain the pTarget-A-N20-1 mutant vector. The PCR product was treated with Dpn I at 37°C for 1 hour, then transformed into E. coli DH5α recipient strains, plated onto solid LB plates containing spectinomycin hydrochloride at a final concentration of 50 mg / L, and incubated at 37°C for 12 hours. Individual colonies were randomly selected and transferred to LB liquid medium containing spectinomycin hydrochloride at a final concentration of 50 mg / L. The cells were then incubated at 37°C for 12 hours, and the plasmid was extracted to obtain the pTarget-A-N20-1 vector.

[0080] Step 2: Construct pTarget-A vector

[0081] Using plasmids pET28a-avtA1, pET28a-avtA2, pET28a-avtA3, pET28a-avtA4, and pET28a-avtA5 as templates, primers AF and AR were used for PCR amplification. The products were recovered by gel extraction to obtain fragments A1, A2, A3, A4, and A5 (2254 bp was considered positive). PCR reaction conditions were as follows: 95°C for 3 minutes; 35 cycles of 95°C for 15 seconds, 58°C for 15 seconds, and 72°C for 2 minutes; and extension at 72°C for 10 minutes.

[0082] The pTarget-A-N20-1 vector and fragments A1, A2, A3, A4, and A5 were cloned in one step at 37°C for 30 min. The cloned products were transformed into E. coli DH5α recipient strains, plated onto solid LB plates containing spectinomycin hydrochloride at a final concentration of 50 mg / L, and incubated at 37°C for 12 h. Single colonies were randomly selected and transferred to liquid LB medium containing spectinomycin hydrochloride at a final concentration of 50 mg / L. The cells were incubated at 37°C for 12 h, and the plasmids were harvested and extracted to obtain the pTarget-A1, pTarget-A2, pTarget-A3, pTarget-A4, and pTarget-A5 vectors.

[0083] The third step is to obtain recombinant strains SA1, SA2, SA3, SA4, SA5, AA1, AA2, AA3, AA4, AA5

[0084] The pTarget-A1, pTarget-A2, pTarget-A3, pTarget-A4, and pTarget-A5 vectors were electroporated into the SA0 and AA0 strains containing the pEccas vector, respectively. The electroporation steps were the same as described in (1).

[0085] The electroporated bacterial solution was spread onto an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin hydrochloride and cultured overnight at 37°C. A single colony was picked as a template and PCR amplified using primers A-seq-F1100 and A-seq-R (1100 bp was considered positive), followed by plasmid elimination using the same steps as described in (1). Recombinant strains SA1, SA2, SA3, SA4, SA5 and AA1, AA2, AA3, AA4, AA5 were obtained.

[0086] Example 3: Fermentation assay of 10 mutant strains, the original strain Sva1024, and the wild-type strain ATCC 8739

[0087] Fermentation experiments were conducted on the strain Sva1024, the wild-type strain ATCC 8739, and the strains constructed in Example 2 (SA1, SA2, SA3, SA4, SA5 and AA1, AA2, AA3, AA4, AA5) to compare the effects of different mutation sites in the avtA gene on the L-valine production capacity of different strains. The fermentation experiments were conducted according to the following protocol:

[0088] (1) Seed Culture: Fresh colonies from the LB plate were inoculated into a test tube containing 4 mL of seed culture medium and cultured overnight at 37°C, 250 rpm with shaking. Subsequently, the culture was transferred to a 250 mL Erlenmeyer flask containing 30 mL of seed culture medium at a 2% (v / v) inoculum volume and cultured at 37°C, 250 rpm with shaking for 12 hours to obtain the seed culture solution for inoculation of the fermentation medium.

[0089] (2) Fermentation: The fermentation medium was inoculated with the seed culture solution in a volume of 250 mL in a 500 mL anaerobic jar to a final concentration of OD550 = 0.1. The culture was incubated at 37°C, 150 rpm, for 4 days to obtain a fermentation broth. The neutralizer was 5 M aqueous ammonia to control the pH of the fermentation jar at 7.0. No gas was introduced during the culture.

[0090] 1 mL of fermentation broth was taken from the fermenter, centrifuged at 12000 rpm for 1 min, and the supernatant was taken. After passing through a filter membrane (pore size 0.22 μm), the L-valine content in the fermentation broth was analyzed by HPLC. Finally, the amount of L-valine obtained by each strain was compared. The results are as follows: Figure 3 shown.

[0091] An Agilent high-performance liquid chromatograph was used for detection: the chromatographic column was Yuexu Ultimate HILIC Amphion II, the mobile phase was pure acetonitrile in the organic phase; the aqueous phase was 0.05 M potassium dihydrogen phosphate, pH 3.0 (preparation method: weigh 0.05 M potassium dihydrogen phosphate of the corresponding concentration mass and dissolve it in ultrapure water, adjust the pH to 3.0 with phosphoric acid, filter, and add the corresponding volume of acetonitrile in a ratio of acetonitrile: 0.05 M potassium dihydrogen phosphate = 75:25. After mixing, ultrasonication was used to remove bubbles). The parameters were set as a detection wavelength of 206 nm, an injection volume of 10 μL, a flow rate of 1 mL / min, and a detection time of 20 min.

[0092] from Figure 3 and Figure 4 As can be seen, mutations at five different sites in different chassis strains all resulted in increased L-valine production. After four days of fermentation, the initial strain, Sva1024, achieved valine production of 1.3 g / L. The highest yield increase after point mutations was observed in strain SA3, reaching 1.51 g / L, a 16.1% increase. Genetically modified wild-type strains also exhibited a similar improvement, increasing L-valine accumulation from 0 to 0.1 g / L. These fermentation results demonstrate that this method has a positive effect on L-valine accumulation and provides a metabolic engineering strategy for the subsequent construction of chassis for valine and its derivatives.

Claims

1. An avtA mutant, characterized in that The amino acid sequence thereof corresponds to the wild-type amino acid sequence of avtA with the G282D mutation present, wherein the wild-type amino acid sequence of avtA is encoded by the nucleotide sequence shown in SEQ ID No:

1.

2. The avtA mutant according to claim 1, wherein It also includes at least one of the following mutations: K76E, E172G, L236R, E353K.

3. The nucleic acid encoding the avtA mutant according to claim 1 or 2.

4. A recombinant expression vector containing the encoding nucleic acid according to claim 3.

5. A recombinant bacterium containing the recombinant expression vector according to claim 4, wherein the wild-type avtA gene in the recombinant bacterium is replaced by the avtA mutant according to claim 1 or 2, and the recombinant bacterium is Escherichia coli.

6. Use of the avtA mutant according to claim 1 or 2, the encoding nucleic acid according to claim 3, the recombinant expression vector according to claim 4 or the recombinant bacterium according to claim 5 in the preparation of L-valine.

7. An L-valine producing strain, characterized in that Obtained by the following method: S1: construct a universal base strain of the initial Escherichia coli, specifically by performing a 20 bp specific site mutation on the avtA gene to obtain the first modified strain; S2: Based on the first modified strain, the coding nucleic acid according to claim 3 is used to achieve point mutation and 20bp specific site reversion mutation of the wild-type avtA gene to obtain a second modified L-valine-producing strain.

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

9. A method for preparing L-valine, characterized in that: The method comprises the step of fermenting and culturing the L-valine-producing strain according to claim 7 to obtain L-valine.

10. The method according to claim 9, wherein The fermentation culture is anaerobic fermentation culture.

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