A ppc mutant and its application in the fermentative production of L-valine

Abstract

The present invention relates to the field of bioengineering technology and provides a ppc mutant and its application in the fermentative production of L-valine. By performing point mutations on the ppc gene, the expression activity of phosphoenolpyruvate carboxylase is reduced, the competition of the bypass pathway is weakened, the supply of pyruvate precursors is enhanced, and the production capacity and biomass of the valine engineering strain are further improved. It can be seen from the fermentation results that this method can significantly increase the yield of valine, from the original 1.3 g / L to 1.49 g / L, with a 14.6% increase in yield. Therefore, the present invention has application value.

Description

Technical Field

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

[0002] L-valine is one of the 20 amino acids that make up proteins, and is also one of the essential amino acids and glucogenic amino acids of mammals. L-valine has important applications in the fields of feed, food, medicine, cosmetics, antibiotics, and herbicides. Especially in the feed field, in 2018, the group standard T / CFIAS 001-2018 "Compound Feed for Piglets, Growing and Finishing Pigs" drafted by the China Feed Industry Association specifically added a minimum limit for L-valine; during the period from 2017 to 2019, L-valine showed an annual compound growth rate of nearly 100%; therefore, the application and demand of L-valine in the feed field will surely be increasing in the future, and the market potential is unlimited. L-valine can also be used as a food additive, nutritional supplement fluid, flavoring agent, etc., so it also has extensive applications in the fields of food and medicine.

[0003] With the rapid development of synthetic biology and metabolic engineering, technologies for the green, environmentally friendly, and efficient production of bulk chemicals, fine chemicals, natural products, and other compounds through microbial fermentation using renewable resources as raw materials have attracted increasing attention and gradually become an important force to replace petrochemical-based chemicals, showing a booming development trend. Currently, L-valine is mainly produced by fermentation. In terms of microbial fermentation, pyruvate produced by the glycolysis pathway of Escherichia coli is an important central metabolic intermediate for the synthesis of L-valine. It provides reducing power through the tricarboxylic acid (TCA) cycle and participates in the synthesis of NADH. Studies by Marienhagen et al. have shown that knocking out the pqo gene encoding pyruvate oxidoreductase, the pyc gene encoding alanine carboxylase, and the alaT and avtA genes encoding alanine aminotransferase can weaken the metabolic pathways of by-products such as acetic acid, oxaloacetic acid, and L-alanine, thereby increasing the metabolic flux from pyruvate to valine (Marienhagen J, et al. Metabolic function of Corynebacterium glutamicum aminotransferases AlaT and AvtA and impact on L-valine production. Appl Environ Microbiol. 2008 Dec;74(24):7457-62.). However, during the anaerobic fermentation of wild-type strains to synthesize L-valine, the competitive consumption of phosphoenolpyruvate, a pyruvate precursor, by bypass pathways such as the intracellular TCA cycle limits the accumulation of pyruvate and, to a certain extent, restricts its production capacity, resulting in a low yield of L-valine.

[0004] To address the above technical deficiencies, synthetic biology means are now used to transform L-valine-producing bacteria. By introducing point mutations into the genes of relevant branch enzymes in the metabolic pathway, the competitive pathway of phosphoenolpyruvate, a pyruvate precursor, is weakened to solve the problem of insufficient pyruvate supply, and thus the efficient production of L-valine by recombinant strains under anaerobic conditions is achieved. However, without domestication, the L-valine fermentation production capacity of the above-mentioned transformed recombinant strains still needs to be improved. Summary of the Invention <{

[0005] The object of the present invention is to provide a method for improving the supply of pyruvate, a precursor for synthesizing L-valine, during metabolic engineering transformation and further increasing the yield of L-valine. The present invention introduces point mutations into the ppc gene to reduce the expression activity of phosphoenolpyruvate carboxylase, weaken the competition of the bypass pathway, enhance the supply of pyruvate precursors, and thus achieve the goal of increasing the valine yield. Through experimental screening, recombinant strains capable of significantly increasing the valine yield are obtained.

[0006] The present invention provides a ppc mutant, in which there is a mutation at at least one of the following sites corresponding to the amino acids of the wild type of ppc: R528, S828, D277, D417, S664.

[0007] The mutation is based on molecular docking of the ppc wild-type sequence to simulate the sequences of different mutation sites. The mutant protein will reduce the catalytic ability of the enzyme to a certain extent while retaining part of the protein activity, enabling the strain to accumulate L-valine while growing normally.

[0008] Specifically, there is a mutation at at least one of the following sites corresponding to the amino acids of the wild type of ppc: R528H, S828P, D277G, D417G, S664Y.

[0009] The present invention provides a coding nucleic acid for the ppc mutant described above.

[0010] The present invention also provides a recombinant expression vector containing the coding nucleic acid.

[0011] The present invention further provides a recombinant bacterium containing the recombinant expression vector, preferably Escherichia coli.

[0012] The present invention also provides the use of the coding nucleic acid for the ppc mutant as described above, the coding nucleic acid, the recombinant expression vector or the recombinant bacterium in the preparation of L-valine.

[0013] The present invention particularly provides a production strain for improving the yield of L-valine, which is obtained by the following method: using gene editing to introduce the ppc mutant into the initial strain to replace the original ppc gene.

[0014] 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.

[0015] In a specific embodiment, it can be seen from the fermentation results that the strain obtained by the transformation of the present invention can significantly improve the yield of valine. For example, the initial strain Sva1024 is increased from the original 1.3 g / L to 1.49 g / L, and the yield is increased by 14.6%. The wild-type strain E. coli ATCC8739 is increased from the original 0 to 0.1 g / L, and there is obvious accumulation of L-valine, providing strong strain support for further industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the main metabolic pathway of L-valine.

[0017] Figure 2It is the reaction progress curve of ppc and mutant enzymes.

[0018] Figure 3 It is the detection result of flask fermentation of ATCC8739-ppc and mutants.

[0019] Figure 4 It is the detection result of flask fermentation of Sval024-ppc and mutants. Specific implementation mode

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the exemplified embodiments are not used as a limitation to the present invention.

[0021] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all conventional biochemical reagents unless otherwise specified.

[0023] Escherichia coli has become one of the most important model strains in metabolic engineering transformation due to its clear genetic background, convenient gene manipulation tools, wide substrate utilization, easy cultivation, and rapid growth, and the industrialization of producing compounds such as L-alanine, D-lactic acid, and succinic acid using engineering strains has been achieved. The applicant of the present invention screened a batch of recombinant strains capable of efficiently producing L-valine through preliminary strain domestication, and performed genome sequencing on these recombinant strains respectively. Among them, wild-type Escherichia coli ATCC8739 (preserved in the laboratory) and Sval024 (obtained by transforming the original strain Escherichia coli ATCC8739) were selected as the starting bacteria. Introducing ppc gene mutations into the microbial genome will be of great significance for the construction of L-valine-producing strains and the efficient fermentation production of L-valine. In the following embodiments, wild-type Escherichia coli ATCC8739 and engineering strain Sval024 (CN 113278568 A) were used as the starting bacteria respectively, and a gene transformation method that can improve the L-valine yield of the engineering strain was provided.

[0024] Table 1 Primers and sequence information used in the present invention

[0025]

[0026]

[0027] Example 1: Construction of recombinant strains with two different chassis

[0028] Starting from the wild-type Escherichia coli ATCC8739 and the engineered strain Sval024 respectively, the Crispr-cas9 method was used to perform point mutations at different positions on the sequence of the ppc gene. Among them, the nucleotide sequence of the wild-type ppc gene is shown in SEQ ID NO.1, the amino acid sequence is shown in SEQ ID NO.2, and the NCBI gene number is: NZ_CP033020.1:4474801-4477452. The specific steps are as follows:

[0029] Step 1: Construct the pTarget-ppc-N20 plasmid

[0030] Use an online website to find the 20bp sequence before the PAM site of the ppc gene, and select the one with the highest score as the primer design reference sequence. The N20 sequences designed for the 5 different mutant genes are all: gcctggtagacaaagcactg. Using the pTarget plasmid (Yang, et al., Acta Biochim Biophys Sin, 2021; 53(5):620-627) as a template, perform PCR amplification with pTarget-F / pTarget-R to obtain pTarget-ppc-N20, transform it into DH5α competent cells, and perform PCR identification with pTarget-seq-F / R as primers to obtain a 1943bp positive plasmid, and send it to Sangon for sequencing to obtain the pTarget-ppc-N20 plasmid.

[0031] Step 2: Prepare the pTarget-ppc-donor targeting plasmid

[0032] Using the genome of Sval024 strain as a template, the 500 bp sequences upstream and downstream of the target gene were amplified using the primers ppc-d1-F1 / ppc-d1-R1 and ppc-d1-F2 / ppc-d1-R2, ppc-d2-F1 / ppc-d2-R1 and ppc-d2-F2 / ppc-d2-R2, ppc-d3-F1 / ppc-d3-R1 and ppc-d3-F2 / ppc-d3-R2, ppc-d4-F1 / ppc-d4-R1 and ppc-d4-F2 / ppc-d4-R2, ppc-d5-F1 / ppc-d5-R1 and ppc-d5-F2 / ppc-d5-R2 as homologous arms respectively, and spliced by fusion PCR to obtain a donor fragment with a size of 1000 bp. Using pTarget-ppc-N20 as a template, the pTarget vector was linearized by inverse PCR using the primers pTarget-xian-F / pTarget-xian-R. The obtained donor band was ligated to the linearized ptarget vector of the corresponding gene using the Novoprotein One Step Cloning Kit C112, and the following reaction system was prepared on ice:

[0033] Reaction system Volume (μL) 5×CEII buffer 4 Linearized vector 1 Inserted fragment 2 Exnase II 2 ddH2O 11

[0034] The ligation product was transformed into competent DH5α, and the transformation method was the same as that in Example 1. The cell pellet was resuspended with the remaining medium, and gently spread evenly on an LB plate containing spectinomycin (final concentration 75 μg / mL) using a sterile spreading rod, and cultured upside down in a 37 °C incubator for 12 - 16 h. After verification, the plasmids were extracted to obtain: pTarget-ppc*1-donor, pTarget-ppc*2-donor, pTarget-ppc*3-donor, pTarget-ppc*4-donor, pTarget-ppc*5-donor.

[0035] In the third step, the above plasmids were electrotransformed into E. coli (Sval024), and the specific steps are as follows:

[0036] Take the electrocompetent cells of E. coli (Sval024) / pEcCas (Yang, et al., Acta Biochim Biophys Sin, 2021; 53(5): 620 - 627), and place them on ice bath. Add 3 μL of pTarget-ppc-donor plasmid in the ultra-clean bench, gently mix well, and place on ice bath for 1 min. Transfer it to a pre-cooled 2 mm electroporation cuvette with a pipette, and place on ice bath for 5 min. Dry the water mist on the outside of the electroporation cuvette with a tissue paper, place it in the electroporator, use the ECO 2 setting for electroporation, immediately add 1 mL of pre-cooled LB medium, tilt the electroporation cuvette, aspirate all the bacterial liquid from the mouth of the electroporation cuvette, and transfer it to a 12 mL sterile tube. Resuscitate at 37 °C and 75 rpm for 4 - 5 h. Centrifuge at 3000 rpm for 5 min, discard the supernatant, leave 100 μL, and spread all of it on the LB solid medium (containing SD+kan resistance), and culture overnight at 37 °C. Use donor-yz-F / donor-yz-R as primers and monoclonal colonies as templates for colony PCR verification. At the same time, use the original genome as a template for negative control. The size of all bands in the positive verification results is 1644 bp, and the recombinant strains ppc001, ppc002, ppc003, ppc004, and ppc005 containing double plasmids are obtained.

[0037] Step 4, plasmid elimination

[0038] Since the fermentation results will be affected when using the bacterial strains containing plasmids for fermentation, it is necessary to lose the plasmids. Inoculate the above recombinant strains into 4 mL of LB sterile tubes (kan resistance), and add 1% rhamnose. Culture at 37 °C and 180 rpm for 8 - 16 h. Streak in three zones on the LB solid medium (kan resistance), and culture overnight at 37 °C. Number the single colonies, and pick some single colonies, and streak them on the corresponding areas of the LB solid medium (SD resistance) and the LB solid medium (kan resistance) respectively, and culture overnight at 37 °C. The single colonies that cannot grow in the area corresponding to the LB solid medium (SD resistance) and can grow normally in the area corresponding to the LB solid medium (kan resistance) are the clones with successful elimination of pTarget-donor.

[0039] Pick a clone with successful elimination of pTarget-donor and inoculate it into a 10 mL LB test tube (without resistance), add sucrose with a final concentration of 10 g / L, culture at 37 °C and 180 rpm for 8 - 16 h. Streak on the LB solid medium (without resistance), and culture overnight at 37 °C. Number the single colonies, and pick some single colonies, and streak them on the corresponding areas of the LB solid medium (kan resistance) and the LB solid medium (without resistance) respectively, and culture overnight at 37 °C. The single colonies that can grow normally in the area corresponding to the LB solid medium (without resistance) and cannot grow in the area corresponding to the LB solid medium (kan resistance) are the clones with successful elimination of pEcCas.

[0040] Thus, plasmid-free recombinant strains ppc001, ppc002, ppc003, ppc004, and ppc005 can be obtained.

[0041] The process of constructing the strains using wild-type Escherichia coli ATCC8739 as the chassis is the same as that of engineering strain Sval024, and the resulting recombinant strains are named ppcA01, ppcA02, ppcA03, ppcA04, and ppcA05.

[0042] Example 2: Expression and Enzyme Activity Assay of ppc Protein and Mutant ppc* in Valine-Producing Bacteria

[0043] (1) Protein Expression of ppc and ppc*

[0044] To investigate whether the modified gene has an impact on the yield of L-valine, expression plasmids of phosphoenolpyruvate carboxylase ppc and mutant ppc* genes need to be constructed separately, and their enzymatic properties need to be compared. The effect of ppc gene mutation on L-valine yield is reflected by the magnitude of enzyme activity.

[0045] The specific steps of this method are as follows:

[0046] First step, inoculate Escherichia coli carrying the Pet-28a plasmid (purchased from Sangon Biotech Co., Ltd., B540183 - 0001) into 5 ml of LB + kan resistant liquid medium and culture overnight at 37°C. Extract the plasmid the next day. Using the plasmid as a template and Pet-28a-F / Pet-28a-R as primers, linearized Pet-28a is obtained by reverse PCR, and the band size is 5369 bp.

[0047] The amplification system is: 25 μL of Novoprotein 2×phanta master Mix, 20 ng of DNA template, 2 μL of each primer (10 μM), and supplemented with ddH2O to 50 μL.

[0048] The amplification conditions are: pre-denaturation at 95°C for 3 min (1 cycle); denaturation at 95°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 6 min (30 cycles); extension at 72°C for 10 min (1 cycle).

[0049] Step 2: Using the genomes of engineered strain Sval024 and mutant strains (the recombinant strains ppc001, ppc002, ppc003, ppc004, ppc005 obtained in Example 1) as templates, and ppc-F / ppc-R as primers, amplify the target gene ppc and the other 5 mutant genes ppc*-1, ppc*-2, ppc*-3, ppc*-4, ppc*-5. The lengths of both ppc and ppc* are 2652 bp.

[0050] The amplification system and conditions are the same as those in the first step. Note that the annealing temperature and time are adjusted according to the size of the target product.

[0051] Step 3: Recombine the linearized Pet-28a with the above-mentioned fragments ppc and ppc*-1 to 5 respectively.

[0052] Recombination system and conditions: The recombinant enzymes used are all from the ClonExpress II One Step Cloning Kit series. Recombination conditions: 37 °C, 30 min. The recombination system is as follows:

[0053] Reaction system Volume (μL) 5×CEII buffer 4 Linearized vector 1 Inserted fragment 2 Exnase II 2 ddH2O 11

[0054] Transformation of the recombinant product: Take 10 μL of the reaction system and add it to 100 μL of DH5α competent cells. Gently mix and incubate on ice for 30 min, heat shock at 42 °C for 60 s, immediately incubate on ice for 2 min, add 900 μL of LB medium, and resuscitate at 37 °C for 1 h. Centrifuge at 5000 rpm for 2 min, discard part of the supernatant, leave about 150 μL, resuspend the cell pellet, and spread it on a plate containing 50 μg / mL kanamycin. Invert the plate and incubate overnight at 37 °C.

[0055] Identification of the recombinant product: After single colonies grow on the plate, identify them by colony PCR. Pick a positive colony and inoculate it into LB medium containing 50 μg / mL kanamycin for overnight culture and then preserve the bacteria. Extract the plasmids Pet-ppc, Pet-ppc*1, Pet-ppc*2, Pet-ppc*3, Pet-ppc*4, and Pet-ppc*5 for standby.

[0056] Step 4: Prepare the expression-competent BL21. Transform the 6 plasmids extracted in the previous step into BL21 competent cells respectively. The transformation method is the same as that in the third step. Leave 150 μL, resuspend the cell pellet, and spread it on a plate containing 50 μg / mL kanamycin. Incubate overnight at 37 °C. The clone identification is the same as above. The finally obtained recombinant strains are denoted as P-ppc, P-ppc*-1, P-ppc*-2, P-ppc*-3, P-ppc*-4, and P-ppc*-5.

[0057] Step 5: Inoculate the above recombinant strain into LB liquid medium containing kanamycin (final concentration: 50 μg / mL) at an inoculation amount of 1%, and culture it at 37°C and 220 rpm until OD600 = 0.6. Add IPTG (isopropyl β-D-thiogalactopyranoside) to a final concentration of 0.1 mM, and perform low-temperature induction culture at 16°C and 150 rpm for 20 h. Centrifuge the induced bacterial solution to collect the cells, then resuspend them in Lysis Buffer, and supplement with PMSF solution to a final concentration of 1 mM. Subsequently, use an ultrasonic cell disruptor to lyse the cells on ice. Ultrasonically disrupt the bacteria or cells (ice bath, power 20% or 200 W, ultrasound for 3 s, interval 10 s, repeat 30 times) according to the ratio of the number of bacteria or cells (104): extraction solution volume (mL) of 1000:1. Centrifuge at 10000 g and 4°C for 10 min, take the supernatant, and place it on ice for further measurement.

[0058] (2) Enzyme activity assay of ppc and ppc*

[0059] The ppc gene encodes phosphoenolpyruvate carboxylase (PEPC), which irreversibly catalyzes the reaction of phosphoenolpyruvate (PEP) and carbon dioxide (in the form of bicarbonate) to generate oxaloacetate and orthophosphate (in the form of bicarbonate). In this experiment, the ammonium molybdate method for phosphorus determination was used to detect the content of orthophosphate in the solution.

[0060] First, prepare the phosphorus determination reagent as shown in the following table:

[0061]

[0062] Then, plot the phosphorus standard curve: Prepare aqueous solutions of potassium dihydrogen phosphate with concentrations of 0 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM respectively. Perform the phosphorus determination reaction on the aqueous solutions of potassium dihydrogen phosphate with different concentrations as follows: Add 150 μL of freshly prepared reagent C to a 96-well transparent plate, then add 50 μL of aqueous solutions of potassium dihydrogen phosphate with different concentration gradients. After standing for 5 min, add 150 μL of reagent D. After standing for another 15 min, read the OD655 value of the sample using a continuous wavelength microplate reader. Plot the OD655 readings against the concentration of orthophosphate to obtain the phosphorus standard curve. Determine the OD655 reading of a certain solution after the phosphorus determination reaction, and calculate the content of orthophosphate in the solution based on the phosphorus standard curve.

[0063] Finally, draw the enzyme reaction progress curve: 10 mg of monopotassium phosphoenolpyruvate (PEP-K) is converted to a net amount of 0.049 mmol (molecular weight 206.13). The powder is dissolved in water to a final volume of 2.18 mL to obtain a stock solution of monopotassium phosphoenolpyruvate with a final concentration of 22.5 mM. The stock solution is aliquoted into 50 μL portions and stored in a -80 °C refrigerator. When in use, one aliquot of the stock solution is taken out and added to 450 μL of Elution buffer (composition: 25 mM Tris-HCl, 150 mM NaCl, 300 mM imidazole, pH = 8), and diluted 10-fold to form a 2.25 mM reaction stock solution. Similarly, prepare a 2.25 mM dipotassium hydrogen carbonate reaction stock solution (dissolved in Elution buffer).

[0064] Prepare the following reaction system to explore the optimal PEPC concentration and reaction progress curve.

[0065]

[0066] Definition of unit enzyme activity: The amount of phosphate generated per mg of protein per unit time is defined as one enzyme activity unit.

[0067] Through exploration of the reaction process, finally draw the enzyme reaction progress curve. As shown by Figure 2 it can be seen that compared with the wild-type ppc, the enzyme activities after mutation are all reduced to varying degrees. This indicates that the modification of the ppc gene in the present invention can, to a certain extent, reduce its activity, limit the reaction catalyzed by this enzyme, and cause more phosphoenolpyruvate to flow towards the formation of pyruvate, that is, increase the production of L-valine.

[0068] Example 3: Fermentation tests of wild-type Escherichia coli ATCC8739, engineered strain Sval024, and recombinant strain I. Seed culture

[0069] Inoculate fresh colonies on the LB plate into a test tube containing 4 mL of seed medium, and culture overnight at 37 °C with shaking at 250 rpm. Then, transfer the culture to a 250 mL Erlenmeyer flask containing 30 mL of seed medium at an inoculation amount of 2% (V / V), and culture with shaking at 37 °C and 250 rpm for 12 hours to obtain a seed culture solution for inoculating the fermentation medium.

[0070] II. Fermentation culture

[0071] The volume of the fermentation medium in a 500 mL anaerobic jar is 250 mL. The seed culture solution is inoculated according to the final concentration OD 550Inoculate into the fermentation medium at an inoculum size of 0.1, ferment at 37 °C and 150 rpm for 4 days to obtain the fermentation broth. The neutralizing agent is 5 M ammonia water to control the pH of the fermenter at 7.0. No gas is introduced during the cultivation process.

[0072] III. Liquid-phase detection of L-valine production

[0073] Centrifuge the bacterial liquid after 48 h at 12,000 rpm for 2 min, take the supernatant for dilution, dilute the supernatant 50-fold (take 20 μL of the supernatant and mix it evenly with 980 μL of pure water), vortex and mix well, then use a 1 mL syringe to filter through a 0.22 μm aqueous filter membrane into an EP tube. Place an inner cannula in the sample vial, vortex and mix well, take 200 μL of the sample and add it, cover the lid, and then shake it vigorously to prevent air bubbles. The standard is L-valine (purchased from Macklin).

[0074] Chromatographic column: ZORBAX Eclipse AAA 4.6 mm × 75 mm 3.5-Micron; detection wavelength: 338; injection volume: 5 μL; flow rate: 1 mL / min; column temperature: 40 °C; retention time: 7.734 min; mobile phase ratio:

[0075] Time (min) B(％) D(％) Flow rate (mL / min) Maximum pressure limit (bar) 0 100 0 1 400 1 100 0 1 400 11 43 57 1 400 14 0 100 1 400 17 100 0 1 400

[0076] After comparison of the liquid-phase results and data analysis, the results are as Figure 3 and Figure 4 shown.

[0077] Among them, it can be found that the yields of the recombinant strains ppcA01, ppcA02, ppcA03, ppcA04, and ppcA05 are 0.08 g / L, 0.07 g / L, 0.08 g / L, 0.11 g / L, and 0.09 g / L respectively. As Figure 3 shown, compared with the yield of 0 g / L of the initial strain wild-type Escherichia coli ATCC8739, there are varying degrees of improvement.

[0078] The yields of the recombinant strains ppc001, ppc002, ppc003, ppc004, and ppc005 are 1.41 g / L, 1.35 g / L, 1.43 g / L, 1.49 g / L, and 1.32 g / L respectively. As Figure 4 shown, compared with the yield of 1.30 g / L of the initial strain Sval024, there are varying degrees of improvement, indicating that the ppc gene modification of the present invention can improve the yield of L-valine to a certain extent for both wild bacteria and the engineered strain Sval024.

[0079] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A ppc mutant, characterized in that, It has a D417G mutation in the amino acid corresponding to the wild type of ppc; the amino acid sequence of the wild type of ppc is shown by SEQ ID NO.

2.

2. The coding nucleic acid of the ppc mutant according to 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 ppc in the recombinant bacterium is replaced by the ppc mutant according to claim 1.

5. The recombinant bacterium according to claim 4, characterized in that, The recombinant bacterium is Escherichia coli.

6. The application of the ppc 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.

7. An L-valine producing strain, characterized in that, It is obtained by the following method: using gene editing, the ppc mutant according to claim 1 is introduced into the initial strain to replace the original ppc gene.

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

9. A method for preparing L-valine, characterized in that, It includes 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, characterized in that, The fermentation culture is anaerobic fermentation culture.

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