Alanine dehydrogenase mutant, L-alanine high-yield strain containing alanine dehydrogenase mutant and application thereof

Abstract

The invention discloses an alanine dehydrogenase mutant, an L-alanine high-yield strain containing the alanine dehydrogenase mutant and application of the alanine dehydrogenase mutant. Compared with SEQ ID NO: 1, the amino acid sequence of the alanine dehydrogenase mutant disclosed by the invention comprises one or more of the following amino acid mutations: E48T, T180I, H295A, A304E and N344I. The L-alanine high-producing strain expresses the alanine dehydrogenase mutant, can generate high-concentration L-alanine through fermentation culture, is short in required fermentation time, can reach 136.5 g / kg after being fermented for 38 hours, is up to 97% in sugar-acid conversion rate, is improved in fermentation level, is greatly shortened in fermentation time, and has the potential of industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an alanine dehydrogenase mutant, an L-alanine high-yield strain containing the mutant and applications thereof. Background Art

[0002] L-alanine has a wide range of uses. It is an important raw material for synthesizing vitamin B6 and an important food additive. It can enhance the flavor of food and beverages when combined with other amino acids. At the same time, L-alanine is also an important raw material for synthesizing amino acid surfactant MGDA.

[0003] L-alanine can be produced using aspartic acid as raw material through enzyme catalysis technology (aspartate decarboxylase). Since aspartic acid is produced using maleic anhydride as raw material, the production cost and selling price of L-alanine are heavily dependent on oil prices. Microbial fermentation has many advantages over enzyme catalysis technology in producing L-alanine: Microbial fermentation technology uses glucose as raw material. Glucose is a renewable biomass resource and is currently mainly produced from corn starch. There are many studies now devoted to extracting it from lignocellulose, and the cost can remain stable for a long time.

[0004] Chinese patent application CN103602623A discloses a strain with high yield of L-alanine, which integrates alanine dehydrogenase GsAlaD from Geobacillus stearothermophilus into the chromosome of Escherichia coli ATCC8793 and blocks the competitive pathway for L-alanine accumulation. The recombinant strain is anaerobically fermented in a 3L fermenter for 48 hours, and the L-alanine accumulation is 115g / L, and the sugar-acid conversion rate is about 95%. Chinese patent application CN107937361A discloses a mutant protein of alanine dehydrogenase from thermophilic archaea, and simultaneously knocks out the key genes in the competitive pathway for L-alanine synthesis in the Escherichia coli K12 strain, performs two-stage fermentation in a fermenter, and accumulates 153.9 g / L of L-alanine in 42 hours of fermentation, with a sugar-acid conversion rate of 81%. The fermentation time of the strains disclosed in the prior art is still relatively long, and the sugar-acid conversion rate is still relatively low. Summary of the invention

[0005] The technical problem to be solved by the present invention is the defects of the prior art L-alanine production strains, that is, the fermentation time is long and the sugar-acid conversion rate is low, and an alanine dehydrogenase mutant, an L-alanine high-yielding strain containing the same and its application are provided. The L-alanine high-yielding strain of the present invention expresses the alanine dehydrogenase mutant, has the ability to produce high-concentration L-alanine through fermentation culture, and the required fermentation time is short, the fermentation level is improved, and the fermentation time is greatly shortened, and it has the potential for industrial application.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides an alanine dehydrogenase mutant, wherein the amino acid sequence of the alanine dehydrogenase mutant comprises one or more of the following amino acid mutations compared to SEQ ID NO: 1:

[0008] E48T, T180I, H295A, A304E and N344I.

[0009] In some embodiments, the amino acid sequence of the alanine dehydrogenase mutant comprises any of the following mutations compared to SEQ ID NO: 1:

[0010] (1) E48T;

[0011] (2) T180I;

[0012] (3) H295A;

[0013] (4) A304E;

[0014] (5) N344I;

[0015] (6) E48T, T180I, H295A, A304E and N344I.

[0016] In some specific embodiments, the amino acid sequence of the alanine dehydrogenase mutant is shown in SEQ ID NO: 3.

[0017] The second aspect of the present invention provides a polynucleotide encoding the alanine dehydrogenase mutant as described in the first aspect.

[0018] In some embodiments, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 4.

[0019] The third aspect of the present invention provides an isolated cell, wherein the cell expresses the alanine dehydrogenase mutant as described in the first aspect; or, the cell comprises the polynucleotide as described in the second aspect.

[0020] The fourth aspect of the present invention provides a genetically engineered bacterium, wherein the starting strain of the genetically engineered bacterium is Escherichia coli, and the genetically engineered bacterium overexpresses the gene encoding the alanine dehydrogenase mutant as described in the first aspect, and inactivates or knocks out the pyruvate oxidase gene (poxB), the fumarate reductase gene (frdBC), the lactate dehydrogenase gene (ldhA), the pyruvate formate lyase gene (pflB), the alcohol dehydrogenase gene (adhE), the methylglyoxal synthase gene (mgsA), the alanine racemase gene (dadX) and the LacI gene (lacI).

[0021] In some embodiments, the starting strain of the genetically engineered bacteria is Escherichia coli W3110, and the genotype of the genetically engineered bacteria is E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2 △frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI.

[0022] In some specific embodiments, the genetically engineered bacteria is a strain with a deposit number of CCTCC M 20241584.

[0023] The fifth aspect of the present invention provides a method for preparing a genetically engineered bacterium, the method comprising: using Escherichia coli W3110 as a starting strain, inserting nucleotides encoding the alanine dehydrogenase mutant as described in the first aspect at the pyruvate oxidase poxB gene site and the fumarate reductase frdBC gene site, respectively, and then knocking out the lactate dehydrogenase encoding gene ldhA, the pyruvate formate lyase gene pflB, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase mgsA, the alanine racemase gene dadX and the transcriptional regulatory factor LacI encoding gene lacI in sequence to obtain the genetically engineered bacterium.

[0024] In some embodiments, the method further comprises subjecting the genetically engineered bacteria to ultraviolet mutagenesis and microaerobic screening.

[0025] In some preferred embodiments, the ultraviolet wavelength of the ultraviolet mutagenesis is 254 nm, the power is 15 W, the mutagenesis distance is 15-20 cm, the ultraviolet irradiation time can be 10s, 20s, 30s, 40s, 50s or 60s, and the lethality is controlled to be above 90%.

[0026] In some preferred embodiments, when performing microaerobic screening, the volume of the shake flask culture solution is 80% to 90%, the flask is sealed with a seal such as a silicone stopper, and the rotation speed is 120-150 rpm.

[0027] In some embodiments, the nucleotide encoding the alanine dehydrogenase mutant as described in the first aspect has a sequence as shown in SEQ ID NO: 4.

[0028] The sixth aspect of the present invention provides a use of the alanine dehydrogenase mutant as described in the first aspect, the polynucleotide as described in the second aspect, the cell as described in the third aspect, or the genetically engineered bacteria as described in the fourth aspect in preparing alanine.

[0029] In some embodiments, the alanine is L-alanine.

[0030] In some embodiments, the preparation is prepared by a whole-cell synthetic system.

[0031] The seventh aspect of the present invention provides a method for preparing alanine, which comprises fermenting and culturing the genetically engineered bacteria as described in the fourth aspect to obtain alanine from the culture.

[0032] In some embodiments, the fermentation medium of the fermentation culture includes: 120-150 g / L of glucose, 3-5 g / L of yeast extract powder, 5-7 g / L of peptone, 6-8 g / L of ammonium sulfate, 2-3 g / L of potassium dihydrogen phosphate, 3-5 g / L of disodium hydrogen phosphate, 2-3 g / L of magnesium sulfate heptahydrate and 0.5-1 g / L of defoaming agent.

[0033] In some embodiments, the fermentation culture conditions are non-aerated and anaerobic.

[0034] In some embodiments, the fermentation culture has a culture temperature of 30-42° C. and a pH of 6.8-7.2.

[0035] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0036] The reagents and raw materials used in the present invention are commercially available.

[0037] The positive and progressive effects of the present invention are:

[0038] The recombinant genetically engineered bacteria of the present invention express alanine dehydrogenase mutants, and are capable of producing high-concentration L-alanine through fermentation culture with a short fermentation time, for example, reaching 136.5 g / kg after 38 hours of fermentation, and the sugar-acid conversion rate is as high as 97%, the fermentation level is improved and the fermentation time is greatly shortened, and the bacteria have the potential for industrial application.

[0039] Biomaterial Deposit Information

[0040] The Escherichia coli A129 strain of the present invention was deposited in the China Type Culture Collection on July 17, 2024, with a deposit address of Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, postal code 430072, and its deposit number is CCTCCNO: M 20241584, and its classification name is Escherichia coli A129. DETAILED DESCRIPTION

[0041] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0042] The coding genes involved in the present invention, the expression cassettes and plasmids containing these genes, and the transformants containing the plasmids can all be obtained by genetic engineering construction methods well known to those skilled in the art.

[0043] This article involves the addition amount, content and concentration of various substances, and the percentages mentioned are all mass percentages unless otherwise specified.

[0044] The molecular biology experiments involved in the examples include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are performed with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0045] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium plus 20 g / L agar powder).

[0046] UV mutagenesis plate culture medium: 30 g / L glucose, 5 g / L yeast extract powder, 5 g / L peptone, 20 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, 3 g / L magnesium sulfate heptahydrate, containing 2% agar powder.

[0047] The components of the shake flask fermentation medium are: 30 g / L glucose, 5 g / L yeast extract powder, 5 g / L peptone, 8 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, and 3 g / L magnesium sulfate heptahydrate.

[0048] The composition of the seed tank culture medium is: glucose 30g / L, yeast extract powder 5g / L, peptone 5g / L, ammonium sulfate 20g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate 5g / L, magnesium sulfate heptahydrate 3g / L, and defoamer 0.5g / L.

[0049] The fermentation tank culture medium composition is: glucose 10g / L, yeast extract powder 5g / L, peptone 5g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate 5g / L, magnesium sulfate heptahydrate 3g / L, defoamer 0.5g / L.

[0050] The composition of feed medium 1 is: glucose 600 g / kg.

[0051] The composition of feed medium 2 is: ammonium sulfate 200 g / kg.

[0052] Conversion rate (%) calculation: the ratio of the total mass of L-alanine obtained by fermentation to the total mass of glucose consumed, that is, conversion rate (%) = concentration of L-alanine in the tank sample (g / kg) × tank weight (Kg) / total mass of glucose consumed (g) × 100%.

[0053] HPLC determination conditions of L-alanine:

[0054] Column: ODS-3, 5μm, 4.6×250mm (UP)

[0055] Mobile phase: Mobile phase A: 0.006 M potassium dihydrogen phosphate (adjust the pH to 3 with phosphoric acid, then filter with a 0.45 μm water filter and ultrasonically degas for 20 minutes before use); Mobile phase B: methanol (chromatographic grade, ultrasonically degas for 20 minutes before use)

[0056] HPLC detection conditions: detection time: 30 min; detection wavelength: 210 nm; column oven temperature: 35°C; pump mode: low pressure gradient, total flow rate of 0.5 ml / min, mobile phase A: mobile phase B 9:1; injection volume: 10 μl.

[0057] Glucose concentration detection: Glucose concentration was measured using Shenzhen Hillman M-100 biosensor.

[0058] Alanine dehydrogenase activity detection method: The enzyme activity of alanine dehydrogenase is determined based on the absorbance at 595 nm. Specifically, take a suitable dilution of alanine dehydrogenase enzyme solution, add it to the reaction mixture (including 50 mM L-alanine, 50 mM Tris-HCl pH9.0, 0.625 mM NAD+, 0.064 mM 5-methylphenazine methylsulfate (PMS), 0.24 mM nitro blue tetrazolium chloride (NBT)), keep it at 37°C for 10 min, and measure the absorbance at 595 nm in a spectrophotometer.

[0059] Example 1: Construction of wild-type alanine dehydrogenase recombinant Escherichia coli expression strain

[0060] For the alanine dehydrogenase PmAlaD from Priestia megaterium, the GenBank accession number is WP_328233963.1, and the amino acid sequence is SEQ ID NO: 1. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform full gene synthesis and optimized according to the codon preference of Escherichia coli. The optimized gene sequence is SEQ ID NO: 2. After the gene synthesis, it was cloned between the NcoI and EcoRI restriction sites of the vector pTrc99A (purchased from Biowind) to obtain the recombinant plasmid pTrc99A-PmAlaD.

[0061] The recombinant plasmid pTrc99A-PmAlaD was transformed into the expression host Escherichia coli W3110 (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) by chemical transformation to obtain the recombinant Escherichia coli PmAlaD-WT capable of expressing wild-type alanine dehydrogenase.

[0062] Example 2: Construction of mutation point library by semi-rational design

[0063] Discovery Studio software was used to predict the substrate binding site in the PmAlaD protein pocket. Using pTrc99A-PmAlaD as the starting plasmid, single-point saturation mutation and iterative saturation mutation were performed on the amino acid sites of E48, T180, H295, A304, and N344, respectively. The screening method of mutant proteins was as follows: the recombinant plasmid containing the point mutation PmAlaD protein was transformed into Escherichia coli W3110 competent cells to obtain mutant transformants. The recombinant Escherichia coli PmAlaD-WT expressing wild-type alanine dehydrogenase was used as a control and inoculated into 5 mL LB medium (containing ampicillin at a final concentration of 50 μg / ml), and cultured at 200 rpm in a 37°C shaker for 16 hours to obtain seed liquid. Each sub-liquid was inoculated into fresh LB medium (containing ampicillin at a final concentration of 50 μg / ml) at a ratio of 1%, and cultured at 200 rpm in a 37°C shaker until OD 600 0.6-0.8, add IPTG with a final concentration of 0.1 mM, cool to 25℃ and induce culture for 24 hours, centrifuge at 4℃, 10000 rpm for 5 min, and collect the bacteria.

[0064] Example 3: Screening of alanine dehydrogenase mutants

[0065] The enzyme activity of each alanine dehydrogenase mutant obtained in Example 2 was determined. 0.5 g of the same mass of cells was taken, 2 mL of 50 mM Tris-HCl pH 9.0 buffer was added for full suspension, and the cells were crushed using an ultrasonic crusher (Shunma Instrument SM-150D) with a crushing power of 5% and a crushing time of 5 min (working for 5 s and stopping for 10 s). After crushing, the cells were centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was used for enzyme activity determination. The enzyme activity of the wild-type PmAlaD was defined as 100%, and Table 1 lists the single-point mutants and five-point mutants with the highest enzyme activity among the screened mutants.

[0066] Table 1 Screening of alanine dehydrogenase mutants

[0067]

[0068] As can be seen from Table 1, we obtained alanine dehydrogenase mutant protein PmAlaD-m6-92, the amino acid sequence of which is shown in SEQ ID NO: 3, which also contains five point mutations, namely E48T, T180I, H295A, A304E and N344I, and its coding sequence is shown in SEQ ID NO: 4. Compared with the wild-type enzyme SEQ ID NO: 1, the enzyme activity of the mutant protein PmAlaD-m6-92 is increased by 8.2 times.

[0069] Example 4: Construction of genetically engineered recombinant strain A0

[0070] In this example, the Escherichia coli CRISPR gene editing tool (Yu Jiang et al. 2015 Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Applied Environmental Microbiology. 81: 2506-2514) was used to first integrate the mutant alanine dehydrogenase mutant PmAlaD-m6-92 encoding gene from Priesteria gigantea into the pyruvate oxidase poxB gene site and the fumarate reductase frdBC gene site of the Escherichia coli W3110 chromosome, and then knock out the competing pathways of the host Escherichia coli's own pyruvate precursors, including the lactate dehydrogenase encoding gene ldhA, the pyruvate formate lyase gene pflB, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase mgsA, the alanine racemase gene dadX and the transcriptional regulatory factor LacI encoding gene lacI, to obtain the genetically engineered recombinant strain A0.

[0071] 4.1 Integration of the mutant alanine dehydrogenase PmAlaD-m6-92 gene into the pyruvate oxidase poxB locus

[0072] Escherichia coli W3110 (purchased from Biowind) was used as the starting strain to prepare electroporation competent cells, and the pCas9 plasmid (Addgene: #62225) was transformed into W3110 competent cells to obtain the W3110 strain carrying the pCas9 plasmid and further cultured to prepare electroporation competent cells using L-arabinose induction.

[0073] Using the genomic DNA of Escherichia coli W3110 as a template, primers poxB-UF / poxB-UR were used for amplification; using pTrc99A-PmAlaD-m6-92 as a template, primers Ptrc-F / T1T2-R were used for amplification; using the genomic DNA of Escherichia coli W3110 as a template, the upstream homologous arm of the poxB gene, the mutant alanine dehydrogenase Ptrc-PmAlaD-m6-92-T1T2 expression frame and the downstream homologous arm of the poxB gene were amplified using the poxB-DF / poxB-DR combination, and then the primers poxB-UF / poxB-DR combination were used for fusion PCR amplification to obtain the homologous fragment poxB-U-PmAlaD-mD that integrated the PmAlaD-m6-92 coding gene into the poxB site.

[0074] The pTargetF-poxB plasmid targeting the poxB gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-poxB-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-poxB plasmid was obtained. The homologous fragments poxB-U-PmAlaD-mD and pTargetF-poxB plasmid were co-transformed into W3110 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were simultaneously verified by PCR using primers poxB-UUF / PmAlaD-yz-R and PmAlaD-yz-F / poxB-DDR. If a 1746 bp and a 2021 bp band were amplified respectively, they were positive transformants. The positive transformants were induced to eliminate the pTargetF-poxB plasmid, and the strain WA-001 carrying the pCas9 plasmid was obtained. The mutant PmAlaD-m6-92 encoding gene was integrated into the poxB gene site on its genome.

[0075] 4.2 Integration of the mutant alanine dehydrogenase PmAlaD-m6-92 encoding gene into the fumarate reductase frdBC locus

[0076] The strain WA-001 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as a template and primers frdBC-UF / frdBC-UR were used for amplification; pTrc99A-PmAlaD-m6-92 was used as a template and primers Ptrc-F / T1T2-R were used for amplification; the genomic DNA of Escherichia coli W3110 was used as a template and frdBC-DF / frdBC-DR was used to amplify the upstream homologous arm of the frdBC gene, the mutant alanine dehydrogenase PmAlaD-m6-92 and the downstream homologous arm of the frdBC gene, and then the primers frdBC-UF / frdBC-DR were used for fusion PCR amplification to obtain the homologous fragment frdBC-U-PmAlaD-mD that integrated the PmAlaD-m6-92 coding gene into the frdBC site.

[0077] The pTargetF-frdB plasmid targeting the frdB gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-frdB-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-frdB plasmid was obtained. The homologous fragments frdBC-U-PmAlaD-mD and pTargetF-frdB plasmid were co-transformed into WA-001 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were simultaneously verified by PCR using frdBC-UUF / PmAlaD-yz-R and PmAlaD-yz-F / frdBC-DDR. If a 1608 bp and a 1855 bp band were amplified respectively, they were positive transformants. The positive transformants were induced to eliminate the pTargetF-frdB plasmid, and the strain WA-002 carrying the pCas9 plasmid was obtained, in which the frdBC gene site on its genome was integrated with a second copy of the mutant PmAlaD-m6-92 gene.

[0078] 4.3 Knockout of the lactate dehydrogenase encoding gene ldhA

[0079] The strain WA-002 carrying the pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as a template, and the primers ldhA-UF / ldhA-UR; ldhA-DF / ldhA-DR were used to amplify the upstream homology arm and downstream homology arm of the ldhA gene, and then the primers ldhA-UF / ldhA-DR were used to perform fusion PCR amplification of the ldhA gene knockout homology fragment ldhA-UD.

[0080] The pTargetF-ldhA plasmid targeting the ldhA gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-ldhA-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-ldhA plasmid was obtained. The homologous fragments ldhA-UD and pTargetF-ldhA plasmid were co-transformed into WA-002 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the ldhA-UUF / ldhA-DDR combination. If a band of 2145 bp was amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-ldhA plasmid, and the strain WA-003 carrying the pCas9 plasmid was obtained, and the ldhA gene on its genome was knocked out.

[0081] 4.4 Knockout of the pyruvate formate lyase pflB gene

[0082] The strain WA-003 carrying the pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the pflB gene were amplified using the primers pflB-UF / pflB-UR; pflB-DF / pflB-DR combination, and then the primers pflB-UF / pflB-DR combination was used for fusion PCR amplification to obtain the pflB gene knockout homology fragment pflB-UD.

[0083] The pTargetF-pflB plasmid targeting the pflB gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-pflB-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-pflB plasmid was obtained. The homologous fragments pflB-UD and pTargetF-pflB plasmids were co-transformed into WA-003 competent cells carrying the pCas9 plasmid, spread on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening, and the grown transformants were verified by PCR using pflB-UUF / pflB-DDR. If a band of 1793 bp was amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-pflB plasmid, and the strain WA-004 carrying the pCas9 plasmid was obtained, and the pflB gene on its genome was knocked out.

[0084] 4.5 Knockout of the alcohol dehydrogenase adhE gene

[0085] The strain WA-004 carrying the pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as a template, and the upstream homology arm and downstream homology arm of the adhE gene were amplified using the primers adhE-UF / adhE-UR; adhE-DF / adhE-DR combination, and then the primers adhE-UF / adhE-DR combination was used for fusion PCR amplification to obtain the ahdE gene knockout homology fragment adhE-UD.

[0086] The pTargetF-adhE plasmid targeting the adhE gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-adhE-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-adhE plasmid was obtained. The homologous fragments adhE-UD and pTargetF-adhE plasmid were co-transformed into WA-004 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the adhE-UUF / adhE-DDR combination. If a band of 2000 bp could be amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-adhE plasmid, and the strain WA-005 carrying the pCas9 plasmid was obtained, and the adhE gene on its genome was knocked out.

[0087] 4.6 Knockout of the methylglyoxal synthase gene mgsA

[0088] The strain WA-005 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the mgsA gene were amplified using the primers mgsA-UF / mgsA-UR; mgsA-DF / mgsA-DR combination, and then the mgsA gene knockout homology fragment mgsA-UD was obtained by fusion PCR amplification using the primers mgsA-UF / mgsA-DR combination.

[0089] The pTargetF-mgsA plasmid targeting the mgsA gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-mgsA-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-mgsA plasmid was obtained. The homologous fragments mgsA-UD and pTargetF-mgsA plasmid were co-transformed into WA-005 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the mgsA-UUF / mgsA-DDR primer combination. If a band of 2249 bp could be amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF-mgsA plasmid, and the strain WA-006 carrying the pCas9 plasmid was obtained, and the mgsA gene on its genome was knocked out.

[0090] 4.7 Knockout of alanine racemase gene dadX:

[0091] The strain WA-006 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the dadX gene were amplified using the primers dadX-UF / dadX-UR; dadX-DF / dadX-DR combination, and then the dadX gene knockout homology fragment dadX-UD was obtained by fusion PCR amplification using the primers dadX-UF / dadX-DR combination.

[0092] The pTargetF-dadX plasmid targeting the dadX gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-dadX-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-dadX plasmid was obtained. The homologous fragments dadX-UD and pTargetF-dadX plasmid were co-transformed into WA-006 competent cells carrying the pCas9 plasmid, and plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR using the dadX-UUF / dadX-DDR primer combination. If a band of 2196 bp could be amplified, it was a positive transformant. The obtained positive transformants were induced to eliminate the pTargetF-dadX plasmid, and the strain WA-007 carrying the pCas9 plasmid was obtained, and the dadX gene on its genome was knocked out.

[0093] 4.8 lacI gene knockout

[0094] The strain WA-007 with pCas9 plasmid was used as the starting strain to prepare electroporation competent cells. The genomic DNA of Escherichia coli W3110 was used as the template, and the upstream homology arm and downstream homology arm of the lacI gene were amplified using the primers lacI-UF / lacI-UR; lacI-DF / lacI-DR combination, and then the dadX gene knockout homology fragment dadX-UD was obtained by fusion PCR amplification using the primers lacI-UF / lacI-DR combination.

[0095] The pTargetF-lacI plasmid targeting the lacI gene was constructed. The pTargetF plasmid (Addgene: #62226) was used as a template and the primer combination pT-lacI-F / pT-R was used for PCR amplification. The PCR product was digested with DpnI and transformed into Escherichia coli Top10 competent cells. The cells were spread on LB+50 μg / mL spectinomycin plates and cultured overnight. The transformants were picked and sequenced to verify that the correct pTarget-lacI plasmid was obtained. The homologous fragments lacI-UD and pTargetF-lacI plasmid were co-transformed into WA-007 competent cells carrying the pCas9 plasmid, and the cells were plated on LB+50 μg / mL kanamycin and 50 μg / mL spectinomycin plates for culture and screening. The grown transformants were verified by PCR. The grown transformants were verified by PCR using primers lacI-UUF / lacI-DDR. If a band of 2041 bp could be amplified, it was a positive transformant. The positive transformants were induced to eliminate the pTargetF- plasmid, and the strain WA-008 carrying the pCas9 plasmid was obtained, and the lacI gene on its genome was knocked out.

[0096] The WA-008 strain was further inoculated into LB liquid culture medium without any antibiotics and cultured overnight at 42°C and 200 rpm in a shaking incubator to eliminate the pCas9 plasmid and obtain a genetically engineered recombinant strain A0 that does not carry a plasmid and has no resistance marker. The genotype of the A0 strain is E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2 △frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI.

[0097] Table 2. Primers and sequences

[0098]

[0099]

[0100] Example 5: A0 strain was subjected to ultraviolet mutagenesis shake flask screening to obtain A129 strain

[0101] The genetically engineered recombinant strain A0 was cultured in LB medium to OD600=0.4-0.6, and after gradient dilution with physiological saline, it was spread on UV mutagenesis plate medium for UV mutagenesis. The UV wavelength was 254nm, the power was 15W, the mutagenesis distance was 15-20 cm, and different UV irradiation times (10s, 20s, 30s, 40s, 50s, 60s) were set to control the lethality to be above 90%. The plate was wrapped with tin foil and placed in a 37°C incubator for culture. 24 monoclones were selected from the grown monoclones for shake flask culture screening. First, 20 monoclones were inoculated into a 10 mL LB liquid test tube, cultured at 37°C, 200 rpm to OD = 2-3 to obtain seed liquid, and further inoculated into shake flask fermentation medium at a ratio of 1%. The 250 mL shake flask was filled with 220 mL of liquid, and the bottle mouth was plugged with a rubber stopper. It was cultured at 37°C, 150 rpm for 24 hours. The fermentation liquid cell OD, residual glucose concentration and L-alanine concentration were measured. The screening results are shown in Table 3. Among them, the monoclone with the highest OD and the highest L-alanine concentration was A129. The A129 strain was deposited in the China Center for Type Culture Collection on July 17, 2024, and its deposit number is CCTCC M 20241584.

[0102] Table 3 OD, residual glucose concentration and L-alanine concentration of each mutant strain cultured in UV-induced shake flasks

[0103]

[0104] Example 6: Fermentation and accumulation of L-alanine by strain A129 in a 15L fermenter

[0105] (1) Seed preparation: Inoculate the frozen A129 strain glycerol tube onto the LB plate, activate and culture at 37°C for 12 hours, scrape a loop and inoculate into a 500ml Erlenmeyer flask containing 100ml liquid LB medium, culture at 37°C and 200rpm for 2.5h until OD600 is 2-3 to obtain shake flask seeds. Inoculate the shake flask seeds into a 15L seed tank containing 10L seed medium at a 1% inoculum, at 37°C, tank pressure 0.05Mpa, ventilation ratio 1VVM, pH 6.8 controlled by ammonia water, 300-700rpm, control DO ≥ 30% by speed, culture for about 4h until OD600 is 8 to obtain seed liquid.

[0106] (2) Fermentation culture: The seed liquid was inoculated at a 10% inoculation rate into a 15-L fermentation tank containing a fermentation medium with a feed volume of 10 L. The fermentation was carried out at 37°C with ammonia water to control the pH at 6.8 and a rotation speed of 150 rpm without ventilation. The residual sugar and ammonia nitrogen contents in the fermentation liquid were detected in real time by online near-infrared. The residual sugar was controlled at 5-10 g / L by feeding medium 1, and the ammonia nitrogen was controlled at 0.1%-0.15% by feeding medium 2. The total glucose concentration was 150 g / L. When all the sugar was consumed (residual sugar ≤ 0.5 g / L), the tank was released. The fermentation time was 34 hours. The liquid phase detection showed that the L-alanine content reached 136.5 g / kg, and the sugar-acid conversion rate was as high as 97%.

[0107] Comparative Example 1: A0 strain was fermented in a 15L fermenter to accumulate L-alanine

[0108] The A0 strain was subjected to seed culture and fermentation culture. The seed preparation and fermentation culture conditions were the same as those in Example 6. When all the sugar was consumed (residual sugar ≦ 0.5 g / L), the tank was released. The fermentation time was 45 hours. The liquid phase detection showed that the L-alanine content in the tank was 129.5 g / kg, and the sugar-acid conversion rate was 95.0%.

[0109] Sequence information

[0110] SEQ ID NO: 1 Wild-type alanine dehydrogenase PmGlaD from Priestia megaterium, GenBank accession number WP_328233963.1

[0111] MKIGVPKEVKNNENRVAITPAGVTALVNGGHDVYIETQAGEGSGFHDEDYTNAGASIVNTAKEAWDAEMVMKVKEPISEEYGYFREGLILFTYLHLAAEEALTKALIDKKVVGIAYETVQLSNKSLPLLTPMSEVAGRMSSQIGAQFLEKTKGGKGILLSGVPGVQRGKVTIIGGGVAGTNAAKIA VGLGADVTIIDLNADRLRQLDDLFGKEVTTLMSNHYNIAQSVKESDLVIGAVLIPGAKAPKLVTEEMVQTMSKGSVVVDIAIDQGGIFETTDRITTHDNPTYSKHEVLHYAVANMPGAVPRTSTLALTNVTVPYALQIANQGYKKACLKNPSLLQGINTLNGFVTYAAVADAHGLTYADATTLLEQA

[0112] SEQ ID NO: 2 The encoding gene of wild-type alanine dehydrogenase PmGlaD from Priestia megaterium, which is optimized according to the codon preference of Escherichia coli

[0113]

[0114] SEQ ID NO: 3 Amino acid sequence of the screened alanine dehydrogenase mutant PmAlaD-m6-92

[0115] MKIGVPKEVKNNENRVAITPAGVTALVNGGHDVYIETQAGEGSGFHDTDYTNAGASIVNTAKEAWDAEMVMKVKEPISEEYGYFREGLILFTYLHLAAEEALTKALIDKKVVGIAYETVQLSNKSLPLLTPMSEVAGRMSSQIGAQFLEKTKGGKGILLSGVPGVQRGKVTIIGGGVAGINAAKIA VGLGADVTIIDLNADRLRQLDDLFGKEVTTLMSNHYNIAQSVKESDLVIGAVLIPGAKAPKLVTEEMVQTMSKGSVVVDIAIDQGGIFETTDRITTHDNPTYSKHEVLAYAVANMPGEVPRTSTLALTNVTVPYALQIANQGYKKACLKNPSLLQGIITLNGFVTYAAVADAHGLTYADATTLLEQA

[0116] SEQ ID NO: 4 The coding gene sequence of the screened alanine dehydrogenase mutant PmAlaD-m6-92

[0117]

Claims

1. An alanine dehydrogenase mutant, characterized in that The amino acid sequence of the alanine dehydrogenase mutant comprises one or more of the following amino acid mutations compared to SEQ ID NO: 1: E48T, T180I, H295A, A304E and N344I.

2. The alanine dehydrogenase mutant according to claim 1, characterized in that The amino acid sequence of the alanine dehydrogenase mutant comprises any of the following mutations compared to SEQ ID NO: 1: (1) E48T; (2) T180I; (3) H295A; (4) A304E; (5) N344I; (6) E48T, T180I, H295A, A304E and N344I; Preferably, the amino acid sequence of the alanine dehydrogenase mutant is shown in SEQ ID NO:

3.

3. A polynucleotide, characterized in that The polynucleotide encodes the alanine dehydrogenase mutant according to claim 1 or 2; Preferably, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

4.

4. An isolated cell, characterized in that The cell expresses the alanine dehydrogenase mutant according to claim 1 or 2; or, the cell comprises the polynucleotide according to claim 3.

5. A genetically engineered bacterium, characterized in that: The starting strain of the genetically engineered bacteria is Escherichia coli, and the genetically engineered bacteria overexpress A gene encoding an alanine dehydrogenase mutant as claimed in claim 1 or claim 2, and inactivating or knocking out the pyruvate oxidase gene (poxB), the fumarate reductase gene (frdBC), the lactate dehydrogenase gene (ldhA), the pyruvate formate lyase gene (pflB), the alcohol dehydrogenase gene (adhE), the methylglyoxal synthetase gene (mgsA), the alanine racemase gene (dadX) and the LacI gene (lacI); Preferably, the starting strain of the genetically engineered bacteria is Escherichia coli W3110, and the genotype of the genetically engineered bacteria is E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2 △frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI.

6. The genetically engineered bacterium according to claim 5, characterized in that The genetically engineered bacteria is a strain with a deposit number of CCTCCM 20241584.

7. A method for preparing genetically engineered bacteria, characterized in that: The method comprises: using Escherichia coli W3110 as a starting strain, inserting nucleotides encoding the alanine dehydrogenase mutant according to claim 1 or 2 at the pyruvate oxidase poxB gene site and the fumarate reductase frdBC gene site, respectively, and then knocking out the lactate dehydrogenase encoding gene ldhA, the pyruvate formate lyase gene pflB, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase mgsA, the alanine racemase gene dadX and the transcriptional regulatory factor LacI encoding gene lacI in sequence to obtain the genetically engineered bacteria.

8. The method according to claim 7, characterized in that The nucleotide encoding the alanine dehydrogenase mutant according to claim 1 or 2 has the sequence shown in SEQ ID NO: 4; And / or, the method further comprises subjecting the genetically engineered bacteria to ultraviolet mutagenesis and microaerobic screening; Preferably, the ultraviolet wavelength of the ultraviolet mutagenesis is 254 nm, the power is 15 W, the mutagenesis distance is 15-20 cm, the ultraviolet irradiation time can be 10s, 20s, 30s, 40s, 50s or 60s, and the lethality is controlled to be above 90%; and / or, during microaerobic screening, the shake flask culture liquid volume is 80%-90%, sealed, and the rotation speed is 120~150 rpm.

9. Use of the alanine dehydrogenase mutant according to claim 1 or 2, the polynucleotide according to claim 3, the cell according to claim 4, or the genetically engineered bacteria according to claim 5 or 6 in the preparation of alanine; Preferably, the alanine is L-alanine; and / or, the preparation is prepared by a whole-cell synthesis system.

10. A method for preparing alanine, characterized in that: The method comprises fermenting and culturing the genetically engineered bacteria as claimed in claim 5 or 6 to obtain alanine from the culture; Preferably, the fermentation medium of the fermentation culture comprises: 120-150 g / L of glucose, 3-5 g / L of yeast extract powder, 5-7 g / L of peptone, 6-8 g / L of ammonium sulfate, 2-3 g / L of potassium dihydrogen phosphate, 3-5 g / L of disodium hydrogen phosphate, 2-3 g / L of magnesium sulfate heptahydrate and 0.5-1 g / L of defoaming agent; and / or, the fermentation culture conditions are aeration-free and anaerobic; and / or, the fermentation culture temperature is 30-42°C and the pH is 6.8-7.2.

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