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

Abstract

The application discloses an alanine dehydrogenase mutant, an L-alanine high-yield strain containing the same and application of the L-alanine high-yield strain. The amino acid sequence of the alanine dehydrogenase mutant of the application comprises one or more amino acid mutations of E48T, T180I, H295A, A304E and N344I compared with SEQ ID NO:1. The L-alanine high-yield strain expresses the above alanine dehydrogenase mutant, can produce high-concentration L-alanine through fermentation culture, has the ability of short fermentation time, reaches 136.5 g / kg in 38 hours of fermentation, has a sugar-acid conversion rate as high as 97%, has improved fermentation level and greatly shortened fermentation time, and has the potential of industrial application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to an alanine dehydrogenase mutant, an L-alanine high-yield strain containing the same and application thereof. BACKGROUND

[0002] L-alanine has a wide range of uses, and it is an important raw material for synthesizing vitamin B6 and an important food additive. In combination with other amino acids, L-alanine can enhance the flavor of food and beverage. Meanwhile, L-alanine is also an important raw material for synthesizing amino acid surfactant MGDA.

[0003] The production of L-alanine can be achieved by using aspartic acid as a raw material and through enzymatic catalysis technology (aspartic acid decarboxylase). Since the production of aspartic acid is based on maleic anhydride as a raw material, the production cost and selling price of L-alanine are heavily dependent on the price of oil. Compared with the enzymatic catalysis technology, the microbial fermentation method for producing L-alanine has many advantages: the microbial fermentation method is based on glucose as a raw material. Glucose belongs to renewable biomass resources, and at present, it is mainly prepared from corn starch. At present, many studies are also committed to extracting it from lignocellulose, and the cost can be kept stable for a long time.

[0004] Chinese Patent Application CN103602623A discloses a strain for producing L-alanine at a high yield. The alanine dehydrogenase GsAlaD from Geobacillus stearothermophilus is integrated into the chromosome of Escherichia coli ATCC8793, and the competitive pathway for L-alanine accumulation is blocked. The recombinant strain is subjected to anaerobic fermentation in a 3L fermenter for 48 hours, and the accumulation amount of L-alanine is 115g / L, and the sugar acid conversion rate is about 95%. Chinese Patent Application CN107937361A discloses a mutant protein of alanine dehydrogenase from a thermophilic archaea, and the key genes in the competitive pathway for synthesizing L-alanine in Escherichia coli K12 strain are knocked out. Two-stage fermentation is carried out in a fermenter, and L-alanine is accumulated for 42 hours, with a yield of 153.9 g / L, and the sugar acid conversion rate is 81%. The fermentation time of the strains disclosed in the prior art is still long, and the sugar acid conversion rate is still low. SUMMARY

[0005] The present application solves the technical problems of the prior art L-alanine production strain, i.e., long fermentation time and low sugar acid conversion rate, and provides an alanine dehydrogenase mutant, an L-alanine high-yield strain containing the same and application thereof. The L-alanine high-yield strain of the present application expresses the alanine dehydrogenase mutant, has the ability to produce high-concentration L-alanine through fermentation culture, requires a short fermentation time, and has improved fermentation level and greatly shortened fermentation time, and has the potential for industrial application.

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

[0007] A 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 contains any of the following groups of 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] A second aspect of the invention provides a polynucleotide that encodes an 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] A third aspect of the invention provides an isolated cell expressing an alanine dehydrogenase mutant as described in the first aspect; or, the cell contains a polynucleotide as described in the second aspect.

[0020] A 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 alanine dehydrogenase mutant encoding gene as described in the first aspect, and inactivates or knocks out the pyruvate oxidase gene (poxB), fumarate reductase gene (frdBC), lactate dehydrogenase gene (ldhA), pyruvate formate lyase gene (pflB), alcohol dehydrogenase gene (adhE), methylglyoxal synthase gene (mgsA), alanine racemic enzyme gene (dadX), and LacI gene (lacI).

[0021] In some embodiments, the starting strain of the genetically engineered bacterium is Escherichia coli W3110, and the genotype of the genetically engineered bacterium 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 implementations, the genetically engineered bacteria is a strain with the accession number CCTCC M 20241584.

[0023] The fifth aspect of the present invention provides a method for preparing genetically engineered bacteria, the method comprising: using Escherichia coli W3110 as the starting strain, inserting nucleotides encoding the alanine dehydrogenase mutant as described in the first aspect into the pyruvate oxidase poxB gene site and the fumarate reductase frdBC gene site respectively, and then sequentially 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 transcription factor LacI encoding gene lacI, thereby obtaining the genetically engineered bacteria.

[0024] In some implementations, the method further includes ultraviolet mutagenesis and micro-oxygen screening of the genetically engineered bacteria.

[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, and the ultraviolet irradiation time can be 10s, 20s, 30s, 40s, 50s or 60s, controlling the lethality rate to over 90%.

[0026] In some preferred embodiments, when performing micro-oxygen screening, the shake flask culture volume is 80%~90%, sealed with 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 the use of an alanine dehydrogenase mutant as described in the first aspect, a polynucleotide as described in the second aspect, a cell as described in the third aspect, or a genetically engineered bacterium as described in the fourth aspect in the preparation of alanine.

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

[0030] In some embodiments, the preparation is carried out using a whole-cell synthesis system.

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

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

[0033] In some implementation schemes, the fermentation culture is carried out under anaerobic conditions without aeration.

[0034] In some implementations, the fermentation culture is carried out at a temperature of 30-42°C and a pH of 6.8-7.2.

[0035] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0036] The reagents and raw materials used in this invention are all commercially available.

[0037] The positive and progressive effects of this invention are as follows:

[0038] The recombinant genetically engineered bacteria of the present invention expresses an alanine dehydrogenase mutant, which can produce a high concentration of L-alanine through fermentation culture with a short fermentation time. For example, it can reach 136.5 g / kg in 38 hours of fermentation, with a sugar-acid conversion rate as high as 97%. The fermentation level is improved and the fermentation time is greatly shortened, which has the potential for industrial application.

[0039] Biological material preservation information

[0040] The Escherichia coli A129 strain of the present invention was deposited on July 17, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Luojia Mountain, Wuchang District, Wuhan, Hubei Province, China 430072, China. Its accession number is CCTCCNO: M 20241584, and its classification name is Escherichia coli A129. Detailed Implementation

[0041] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] The coding genes, expression cassettes and plasmids containing these genes, and transformants containing the plasmids involved in this invention can all be obtained through 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. Unless otherwise specified, all percentage contents mentioned are mass percentage contents.

[0044] The molecular biology experiments involved in the examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, culture medium preparation, etc., and were carried out in accordance with "Molecular Cloning: A Laboratory Manual" (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 with an additional 20 g / L agar powder).

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

[0047] The shake-flask fermentation medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 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 seed culture medium consists of: 30 g / L glucose, 5 g / L yeast extract, 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, and 0.5 g / L defoamer.

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

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

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

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

[0053] HPLC determination conditions for L-alanine:

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

[0055] Mobile phases: Mobile phase A: 0.006 M dipotassium hydrogen phosphate (adjusted to pH 3 with phosphoric acid, then filtered through a 0.45 μm aqueous filter membrane, degassed by sonication for 20 min before use); Mobile phase B: methanol (chromatographic grade, degassed by sonication for 20 min before use).

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

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

[0058] Method for detecting alanine dehydrogenase activity: The activity of alanine dehydrogenase is determined based on the absorbance at 595 nm. Specifically, alanine dehydrogenase solution diluted appropriately is added to a reaction mixture (containing 50 mM L-alanine, 50 mM Tris-HCl pH 9.0, 0.625 mM NAD+, 0.064 mM methyl 5-methylphenazine sulfate (PMS), and 0.24 mM nitrotetrazole blue chloride (NBT)). After incubation at 37°C for 10 min, the absorbance at 595 nm is measured using a spectrophotometer.

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

[0060] The alanine dehydrogenase PmAlaD from *Priestia megaterium* (GenBank accession number WP_328233963.1, amino acid sequence SEQ ID NO: 1) was synthesized by Suzhou Genewise Biotechnology Co., Ltd. The gene sequence was optimized according to the codon bias of *E. coli*, resulting in SEQ ID NO: 2. After gene synthesis, the gene was cloned into 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 recombinant Escherichia coli PmAlaD-WT that can express wild-type alanine dehydrogenase.

[0062] Example 2: Constructing a mutation point library through semi-rational design

[0063] The substrate binding sites in the PmAlaD protein pocket were predicted using Discovery Studio software. Using pTrc99A-PmAlaD as the starter plasmid, single-point saturation mutagenesis and iterative saturation mutagenesis were performed at amino acid sites E48, T180, H295, A304, and N344. The mutant proteins were screened by transforming recombinant plasmids containing point-mutated PmAlaD protein into *E. coli* W3110 competent cells to obtain mutant transformants. Recombinant *E. coli* PmAlaD-WT expressing wild-type alanine dehydrogenase was used as a control. Seed cultures were obtained by inoculating each culture in 5 mL of LB medium (containing ampicillin at a final concentration of 50 μg / mL) at 37°C with shaking at 200 rpm for 16 hours. Each seed culture was then inoculated at a 1% ratio into fresh LB medium (containing ampicillin at a final concentration of 50 μg / mL) and cultured at 37°C with shaking at 200 rpm until OD (digestive activity) was reached. 600 Add 0.6-0.8 g of IPTG to a final concentration of 0.1 mM, cool to 25°C and induce culture for 24 hours. Centrifuge at 10,000 rpm for 5 min at 4°C and collect the bacterial cells.

[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 bacterial cells was taken from each mutant and thoroughly resuspended in 2 mL of 50 mM Tris-HCl pH 9.0 buffer. The cells were then disrupted using an ultrasonic disruptor (Shunma Instruments SM-150D) at 5% power for 5 min (5 s on, 10 s off). After disruption, the cells were centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was used for enzyme activity determination. The enzyme activity of wild-type PmAlaD was defined as 100%. Table 1 lists the single-point mutant and five-point mutant with the highest enzyme activity among the screened mutants.

[0066] Table 1 Screening of alanine dehydrogenase mutants

[0067]

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

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

[0070] This embodiment utilizes 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). First, the gene encoding the alanine dehydrogenase mutant PmAlaD-m6-92 from the selected mutant Priestella giantiformis was integrated into the pyruvate oxidase poxB gene site and the fromate reductase frdBC gene site on the E. coli W3110 chromosome, respectively. Then, the competitive pathways of the host E. coli's own pyruvate precursors were sequentially knocked out, including the lactate dehydrogenase gene ldhA, the pyruvate formate lyase gene pflB, the alcohol dehydrogenase gene adhE, the methylglyoxal synthase mgsA, the alanine racemase gene dadX, and the transcription factor LacI 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 site

[0072] Using Escherichia coli W3110 (purchased from BioWind) as the starting strain, electrotransformation competent cells were prepared. The pCas9 plasmid (Addgene: #62225) was transformed into W3110 competent cells to obtain W3110 strains carrying the pCas9 plasmid. The cells were then further cultured and induced to produce electrotransformation competent cells using L-arabinose.

[0073] Using Escherichia coli W3110 genomic DNA as a template, amplification was performed using primers poxB-UF / poxB-UR; using pTrc99A-PmAlaD-m6-92 as a template, amplification was performed using primers Ptrc-F / T1T2-R; using Escherichia coli W3110 genomic DNA as a template, amplification was performed using primers poxB-DF / poxB-DR to obtain the upstream homologous arm of the poxB gene, the expression cassette of the mutant alanine dehydrogenase Ptrc-PmAlaD-m6-92-T1T2, and the downstream homologous arm of the poxB gene. Fusion PCR amplification was then performed using primers poxB-UF / poxB-DR to obtain the homologous fragment poxB-U-PmAlaD-mD that integrates the PmAlaD-m6-92 encoding gene into the poxB site.

[0074] The pTargetF-poxB plasmid targeting the poxB gene was constructed. Using the pTargetF plasmid (Addgene: #62226) as a template, PCR amplification was performed using the primer combination pT-poxB-F / pT-R. The PCR product was digested with DpnI and transformed into E. coli Top10 competent cells. The cells were plated on LB plates with 50 μg / mL spectinomycin and cultured overnight. Transformants were picked and sequenced to verify the correct pTarget-poxB plasmid. The homologous fragments poxB-U-PmAlaD-mD and pTargetF-poxB plasmid were co-transformed into W3110 competent cells carrying the pCas9 plasmid. The cells were plated on LB plates containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin for screening. The transformed cells 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, the cells were considered positive transformants. The positive transformants were induced to eliminate the pTargetF-poxB plasmid, resulting in strain WA-001 carrying the pCas9 plasmid. The poxB gene locus in the WA-001 genome integrated the mutant PmAlaD-m6-92 encoding gene.

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

[0076] Using strain WA-001 carrying the pCas9 plasmid as the starting strain, electrotransformation competent cells were prepared. Genomic DNA of *E. coli* W3110 was used as a template for amplification using primers frdBC-UF / frdBC-UR; pTrc99A-PmAlaD-m6-92 was used as a template for amplification using primers Ptrc-F / T1T2-R; and frdBC-DF / frdBC-DR was used as a template 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. Fusion PCR was then performed using primers frdBC-UF / frdBC-DR to obtain the homologous fragment frdBC-U-PmAlaD-mD, which integrates the PmAlaD-m6-92 encoding gene into the frdBC site.

[0077] The pTargetF-frdB plasmid targeting the frdB gene was constructed. Using the pTargetF plasmid (Addgene: #62226) as a template, PCR amplification was performed using the primer combination pT-frdB-F / pT-R. The PCR product was digested with DpnI and transformed into E. coli Top10 competent cells. The cells were plated on LB plates with 50 μg / mL spectinomycin and cultured overnight. Transformants were picked and sequenced to verify the correct pTarget-frdB plasmid. Homologous fragments frdBC-U-PmAlaD-mD and pTargetF-frdB plasmids were co-transformed into WA-001 competent cells carrying the pCas9 plasmid. The cells were plated on LB plates containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin for screening. 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 considered positive transformants. Positive transformants were induced to eliminate the pTargetF-frdB plasmid, resulting in strain WA-002 carrying the pCas9 plasmid. The second copy of the mutant PmAlaD-m6-92 gene was integrated into the frdBC gene locus in the WA-002 genome.

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

[0079] Electrotransformation competent cells were prepared using strain WA-002 carrying the pCas9 plasmid as the starting strain. Using genomic DNA from *E. coli* W3110 as a template, the upstream and downstream homologous arms of the ldhA gene were amplified using primers ldhA-UF / ldhA-UR and ldhA-DF / ldhA-DR. Fusion PCR was then performed using primers ldhA-UF / ldhA-DR to amplify the ldhA gene knockout homologous fragment ldhA-UD.

[0080] The pTargetF-ldhA plasmid targeting the ldhA gene was constructed. Using the pTargetF plasmid (Addgene: #62226) as a template, PCR amplification was performed using the primer combination pT-ldhA-F / pT-R. The PCR product was digested with DpnI and transformed into E. coli Top10 competent cells. The cells were plated on LB plates with 50 μg / mL spectinomycin and cultured overnight. Transformants were picked and sequenced to verify that the correct pTarget-ldhA plasmid was obtained. Homologous fragments ldhA-UD and pTargetF-ldhA plasmid were co-transformed into WA-002 competent cells carrying the pCas9 plasmid. The cells were plated on LB plates containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin for screening. Transformants were verified by PCR using the ldhA-UUF / ldhA-DDR combination. A 2145 bp band was amplified, indicating a positive transformant. Positive transformants were induced to eliminate the pTargetF-ldhA plasmid, resulting in strain WA-003 carrying the pCas9 plasmid, in which the ldhA gene was knocked out.

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

[0082] Electrotransformation competent cells were prepared using strain WA-003 carrying the pCas9 plasmid as the starting strain. Using genomic DNA from *Escherichia coli* W3110 as a template, the upstream and downstream homologous arms of the pflB gene were amplified using primers pflB-UF / pflB-UR and pflB-DF / pflB-DR. Fusion PCR was then performed using primers pflB-UF / pflB-DR to amplify the pflB gene knockout homologous fragment pflB-UD.

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

[0084] 4.5 Knockout of the alcohol dehydrogenase adhE gene

[0085] Electrotransformation competent cells were prepared using strain WA-004 carrying the pCas9 plasmid as the starting strain. Using genomic DNA from *E. coli* W3110 as a template, the upstream and downstream homologous arms of the *adhE* gene were amplified using primers adhE-UF / adhE-UR and adhE-DF / adhE-DR. Fusion PCR was then performed using primers adhE-UF / adhE-DR to amplify the *ahdE* gene knockout homologous fragment, adhE-UD.

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

[0087] 4.6 Methylglyoxal synthase gene mgsA knockout

[0088] Electrotransformation competent cells were prepared using strain WA-005 carrying the pCas9 plasmid as the starting strain. Using genomic DNA from *E. coli* W3110 as a template, the upstream and downstream homologous arms of the mgsA gene were amplified using primers mgsA-UF / mgsA-UR and mgsA-DF / mgsA-DR. Fusion PCR was then performed using primers mgsA-UF / mgsA-DR to amplify the mgsA gene knockout homologous fragment mgsA-UD.

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

[0090] 4.7 Knockout of the alanine racemase gene dadX:

[0091] Electroporation competent cells were prepared using strain WA-006 carrying the pCas9 plasmid as the starting strain. Using genomic DNA from *E. coli* W3110 as a template, the upstream and downstream homologous arms of the dadX gene were amplified using primers dadX-UF / dadX-UR and dadX-DF / dadX-DR. Fusion PCR was then performed using primers dadX-UF / dadX-DR to obtain the dadX gene knockout homologous fragment dadX-UD.

[0092] The pTargetF-dadX plasmid targeting the dadX gene was constructed. Using the pTargetF plasmid (Addgene: #62226) as a template, PCR amplification was performed using the primer combination pT-dadX-F / pT-R. The PCR product was digested with DpnI and transformed into E. coli Top10 competent cells. The cells were plated on LB plates with 50 μg / mL spectinomycin and cultured overnight. Transformants were picked and sequenced to verify the correct pTarget-dadX plasmid. Homologous fragments dadX-UD and pTargetF-dadX plasmids were co-transformed into WA-006 competent cells carrying the pCas9 plasmid. The cells were plated on LB plates containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin for screening. Transformants were verified by PCR using the dadX-UUF / dadX-DDR primer combination. A 2196 bp band was amplified, indicating a positive transformant. The positive transformants were then induced to eliminate the pTargetF-dadX plasmid, resulting in strain WA-007 carrying the pCas9 plasmid, in which the dadX gene was knocked out.

[0093] 4.8 lacI gene knockout

[0094] Using strain WA-007 carrying the pCas9 plasmid as the starting strain, electrotransformation competent cells were prepared. Using genomic DNA from *Escherichia coli* W3110 as a template, the upstream and downstream homologous arms of the *lacI* gene were amplified using primer combinations lacI-UF / lacI-UR and lacI-DF / lacI-DR. Fusion PCR was then performed using primer combinations lacI-UF / lacI-DR to amplify the dadX gene knockout homologous fragment dadX-UD.

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

[0096] The WA-008 strain was further inoculated into LB liquid medium without any antibiotics and cultured overnight at 42°C and 200 rpm to eliminate the pCas9 plasmid, obtaining a genetically engineered recombinant strain A0 without plasmid and without antibiotic marker. The genotype of strain A0 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: Strain A129 was obtained by UV mutagenesis and shake-flask screening of strain A0.

[0101] The genetically engineered recombinant strain A0 was cultured in LB medium until OD600 = 0.4-0.6. After serial dilution with physiological saline, it was spread on UV mutagenesis plates for UV mutagenesis. The UV wavelength was 254 nm, the power was 15 W, the mutagenesis distance was 15-20 cm, and different UV irradiation times (10 s, 20 s, 30 s, 40 s, 50 s, 60 s) were set to control the lethality rate to be above 90%. The plates were wrapped in aluminum foil and incubated at 37°C. From the resulting single colonies, 24 single colonies were selected for shake-flask culture and screening. First, 20 single colonies were inoculated into 10 mL LB liquid test tubes and cultured at 37°C and 200 rpm until OD=2-3 to obtain seed culture. This seed culture was then inoculated at a ratio of 1% into shake-flask fermentation medium (220 mL in 250 mL shake flasks). The flasks were sealed with rubber stoppers and cultured at 37°C and 150 rpm for 24 hours. The OD, residual glucose concentration, and L-alanine concentration of the fermentation broth were measured. The screening results are shown in Table 3. The single colony with the highest OD and the highest L-alanine concentration was A129. Strain A129 was deposited at the China Center for Type Culture Collection on July 17, 2024, with accession number 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 culture of strain A129 in a 15L fermenter to accumulate L-alanine

[0105] (1) Seed preparation: The frozen A129 strain glycerol tubes were inoculated onto LB plates and activated at 37°C for 12 h. A loopful was scraped and inoculated into a 500 ml Erlenmeyer flask containing 100 ml of liquid LB medium. The flasks were incubated at 37°C and 200 rpm for 2.5 h until the OD600 reached 2-3 to obtain shake flask seeds. The shake flask seeds were inoculated at a rate of 1% into a 15 L seed tank containing 10 L of seed medium. The tanks were incubated at 37°C, 0.05 MPa, 1 VVM, pH 6.8 controlled with ammonia, and 300-700 rpm. The DO was controlled to be ≥30% by rotating the tank. The culturing was carried out for about 4 h until the OD600 reached 8 to obtain the seed solution.

[0106] (2) Fermentation culture: The seed liquid was inoculated into a 15L fermenter containing 10L of fermentation medium at a 10% inoculation rate. The fermentation was carried out at 37℃, with the pH controlled at 6.8 by ammonia water and the rotation speed at 150 rpm. The fermentation was carried out in an anaerobic environment without aeration. The residual sugar and ammonia nitrogen content in the fermentation liquid were detected in real time by online near-infrared spectroscopy. 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 fermentation was discharged. 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: Strain A0 accumulated L-alanine during fermentation in a 15L fermenter.

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

[0109] Sequence information

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

[0111] MKIGVPKEVKNNENRVAITPAGVTALVNGGHDVYIETQAGEGSGFHDEDYTNAGASIVNTAKEAWDAEMVMKVKEPISEEYGYFREGLILFTYLHLAAEEALTKALIDKKVVGIAYETVQLSNKSLPLLTPMSEVAGRMSSQIGAQFLEKTKGGKGILLSGVPGVQRGKVTIIGGGVAGTNAAKIA VGLGADVTIIDLNADRLRQLDDLFGKEVTTLMSNHYNIAQSVKESDLVIGAVLIPGAKAPKLVTEEMVQTMSKGSVVVDIAIDQGGIFETTDRITTHDNPTYSKHEVLHYAVANMPGAVPRTSTLALTNVTVPYALQIANQGYKKACLKNPSLLQGINTLNGFVTYAAVADAHGLTYADATTLLEQA

[0112] SEQ ID NO: 2 is derived from the encoding gene of wild-type alanine dehydrogenase PmGlaD from *Priestia megaterium*, which has been optimized based on the codon bias of *Escherichia coli*.

[0113]

[0114] The amino acid sequence of the alanine dehydrogenase mutant PmAlaD-m6-92 obtained by screening SEQ ID NO: 3.

[0115] MKIGVPKEVKNNENRVAITPAGVTALVNGGHDVYIETQAGEGSGFHDTDYTNAGASIVNTAKEAWDAEMVMKVKEPISEEYGYFREGLILFTYLHLAAEEALTKALIDKKVVGIAYETVQLSNKSLPLLTPMSEVAGRMSSQIGAQFLEKTKGGKGILLSGVPGVQRGKVTIIGGGVAGINAAKIA VGLGADVTIIDLNADRLRQLDDLFGKEVTTLMSNHYNIAQSVKESDLVIGAVLIPGAKAPKLVTEEMVQTMSKGSVVVDIAIDQGGIFETTDRITTHDNPTYSKHEVLAYAVANMPGEVPRTSTLALTNVTVPYALQIANQGYKKACLKNPSLLQGIITLNGFVTYAAVADAHGLTYADATTLLEQA

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

[0117]

Claims

1. An alanine dehydrogenase mutant, characterized in that, The amino acid sequence of the alanine dehydrogenase mutant is modified compared to SEQ ID NO: 1 by any of the following groups of mutations: (1) A304E; (2) E48T, T180I, H295A, A304E and N344I.

2. The alanine dehydrogenase mutant as described in claim 1, characterized in that, 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 as described in claim 1 or 2.

4. The polynucleotide as described in claim 3, characterized in that, The polynucleotide has a nucleotide sequence as shown in SEQ ID NO:

4.

5. An isolated cell, characterized in that, The cell expresses the alanine dehydrogenase mutant as described in claim 1 or 2; or, the cell contains the polynucleotide as described in claim 3 or 4.

6. A genetically engineered bacterium, characterized in that, The starting strain of the genetically engineered bacteria is Escherichia coli. The genetically engineered bacteria overexpress the alanine dehydrogenase mutant encoding gene as described in claim 1 or 2, and inactivate or knock out the following genes: pyruvate oxidase gene poxB, fumarate reductase gene frdBC, lactate dehydrogenase gene ldhA, pyruvate formate lyase gene pflB, alcohol dehydrogenase gene adhE, methylglyoxal synthase gene mgsA, alanine racemic enzyme gene dadX, and LacI gene lacI.

7. The genetically engineered bacteria as described in claim 6, characterized in that, The starting strain of the genetically engineered bacterium is *Escherichia coli* W3110, and the genotype of the genetically engineered bacterium is *E. coli W3110 △poxB::Ptrc-PmAlaD-m6-92-T1T2△frdBC::Ptrc-PmAlaD-m6-92-T1T2 △ldhA △pflB △adhE △mgsA △dadX △lacI*, wherein *PmAlaD-m6-92* is the gene encoding a mutant alanine dehydrogenase, and its nucleotide sequence is shown in SEQ ID NO:

4.

8. The genetically engineered bacteria as described in claim 6 or 7, characterized in that, The genetically engineered bacteria is a strain with the preservation number CCTCC NO: M 20241584.

9. A method for preparing genetically engineered bacteria, characterized in that, The method includes: using Escherichia coli W3110 as the starting strain, integrating nucleotides encoding the alanine dehydrogenase mutant as described in claim 1 or 2 into the pyruvate oxidase poxB gene site and the fumarate reductase frdBC gene site, respectively, and then sequentially 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 racemic enzyme gene dadX, and the transcription factor LacI encoding gene lacI to obtain the genetically engineered bacteria.

10. The method as described in claim 9, characterized in that, The nucleotide encoding the alanine dehydrogenase mutant as described in claim 1 or 2 has the sequence shown in SEQ ID NO:

4.

11. The use of the alanine dehydrogenase mutant as described in claim 1 or 2, the polynucleotide as described in claim 3 or 4, the cell as described in claim 5, or the genetically engineered bacteria as described in any one of claims 6-8 in the preparation of alanine.

12. The application as described in claim 11, characterized in that, The alanine is L-alanine; and / or, the preparation is carried out by a whole-cell synthesis system.

13. A method for preparing alanine, characterized in that, The method includes fermenting and culturing the genetically engineered bacteria as described in any one of claims 6-8 to obtain alanine from the culture.

14. The method as described in claim 13, characterized in that, The fermentation medium for the fermentation culture comprises: 120-150 g / L glucose, 3-5 g / L yeast extract, 5-7 g / L peptone, 6-8 g / L ammonium sulfate, 2-3 g / L potassium dihydrogen phosphate, 3-5 g / L disodium hydrogen phosphate, 2-3 g / L magnesium sulfate heptahydrate, and 0.5-1 g / L antifoaming agent; and / or, the fermentation culture conditions are non-aerated and anaerobic; and / or, the fermentation culture temperature is 30-42℃, and the pH is 6.8-7.2.

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