Genetically engineered bacterium for producing L-alanine and application of genetically engineered bacterium in two-stage fermentation of L-alanine

Abstract

The invention belongs to the technical field of microorganism application, and particularly relates to a genetically engineered bacterium for producing L-alanine and application of the genetically engineered bacterium in two-stage fermentation of the L-alanine, and the genetically engineered bacterium takes vibrio natrievibrio as a chassis bacterium and can co-express an alanine dehydrogenase gene and an alanine transporter gene. An alanine dehydrogenase gene is heterologously introduced into a vibrio natriticus genome, and an inactivated gene combination with the optimal L-alanine yield is found out by randomly inactivating a key enzyme in a byproduct production pathway, so that the vibrio natriticus genetically engineered bacterium capable of efficiently producing the L-alanine through a fermentation method is constructed. According to the present invention, the L-alanine is produced through the two-stage fermentation by using the gene engineering bacteria through the temperature-sensitive control strategy so as to reduce the influence of the accumulation of the L-alanine on the cell growth, and the high production of the Xiabanmate is provided. The genetically engineered bacterium is short in growth cycle, glucose can be efficiently utilized to convert the genetically engineered bacterium into L-alanine, the L-alanine is transferred to the outside of cells, separation and purification are simple, cell disruption is not needed, and the yield and conversion rate of the L-alanine can both reach a high level.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial application, and particularly relates to a genetically engineered bacterium for producing L-alanine and application of the genetically engineered bacterium in two-stage fermentation of L-alanine. BACKGROUND

[0002] L-alanine is an important natural amino acid and is widely used in the fields of medicine, food, feed and the like. L-alanine is also used as a food additive due to its sweetness. In traditional industrial production, L-alanine is produced by decarboxylation of substrate aspartate catalyzed by immobilized cells, and the yield is greater than 90%. The substrate L-aspartate for this enzyme catalytic production is usually obtained by catalysis of aspartate ammonia lyase from fumaric acid. Fumaric acid is mainly produced from non-renewable raw material petroleum, and thus the use of L-alanine is limited to a certain extent due to high cost. Microbial fermentation method has mild reaction conditions and can use renewable raw materials such as glucose. The currently studied L-alanine production strains include recombinant Escherichia coli, recombinant Corynebacterium glutamicum, recombinant Lactococcus lactis, Clostridium sp., and Oxalobacter sp. Due to the clear genetic background and easy genetic engineering operation of Escherichia coli, Escherichia coli has become the main strain for producing L-alanine. However, the fermentation method of Escherichia coli still has problems such as long fermentation period, low yield, and many by-products.

[0003] Vibrio natriegens is the fastest growing microorganism known, with a doubling time of less than 10 minutes, which is half of that of Escherichia coli. Therefore, modification of Vibrio natriegens into a cell factory for L-alanine can greatly save fermentation time and production cost. Secondly, Vibrio natriegens does not contain alanine racemase, and thus the optical purity of alanine produced by Vibrio natriegens is high and does not spontaneously racemize. Finally, Vibrio natriegens naturally has an alanine dehydrogenase alaD gene in its genome, which catalyzes the conversion of pyruvate into L-alanine. As a new type of microbial chassis, the production capacity of Vibrio natriegens for L-alanine is unknown. SUMMARY

[0004] In view of the defect that the production capacity of Vibrio natriegens for L-alanine is unknown in the prior art, the present application provides a genetically engineered bacterium for producing L-alanine and application of the genetically engineered bacterium in two-stage fermentation of L-alanine, and the specific technical solutions are as follows.

[0005] In a first aspect, the present application provides a genetically engineered bacterium for producing L-alanine, wherein the genetically engineered bacterium takes Vibrio natriegens as a chassis bacterium, and the genetically engineered bacterium co-expresses an alanine dehydrogenase gene and an alanine transporter gene.

[0006] The alanine dehydrogenase gene is one of NCBI accession numbers EF154460.1 and PN9607945.

[0007] In the present application, alanine dehydrogenase gene and alanine transporter gene are introduced into Vibrio natriegens. Alanine dehydrogenase can convert pyruvate into L-alanine. The glycolysis process occurring in the cells of Vibrio natriegens will convert glucose into pyruvate, so that the recombinant Vibrio natriegens can produce L-alanine. Because the accumulation of L-alanine in the cells will cause cell toxicity and affect the normal growth and metabolism of the cells, the alanine transporter is used to transport L-alanine out of the cells in time, so as to ensure the normal production of L-alanine.

[0008] Further, the alanine dehydrogenase gene and the alanine transporter gene are expressed in the chassis in one of the following ways:

[0009] (1) expressed in the chassis in the form of a recombinant plasmid;

[0010] (2) expressed in the form of an expression cassette inserted into the genome of the chassis.

[0011] Further, the alanine dehydrogenase gene is expressed in the form of an expression cassette inserted into the genome of the chassis, and the copy number is 1-5.

[0012] Further, the copy number is 5.

[0013] Further, in the genetically engineered bacteria, one or more of the genes on the genome of the chassis that affect the synthesis of L-lactic acid, D-lactic acid, succinic acid, acetic acid, ethanol, and formic acid are functionally deleted.

[0014] Further, in the genetically engineered bacteria, one or more of the genes on the genome of the chassis that are required for the synthesis of L-lactic acid, D-lactic acid, succinic acid, acetic acid, ethanol, and formic acid are functionally deleted; the gene encoding nuclease on the genome of the chassis is functionally deleted.

[0015] The genes required for the synthesis of L-lactic acid include PN96_04755, PN96_19735, and PN96_16800; the genes required for the synthesis of D-lactic acid include PN96_16785; the genes that affect the synthesis of succinic acid or are required for the synthesis of succinic acid include PN96_09285; the genes required for the synthesis of acetic acid include PN96_03365, PN96_21510, and PN96_20020; the genes required for the synthesis of ethanol include PN96_03185 and PN96_04715; the genes required for the synthesis of formic acid include PN96_08455.

[0016] The gene encoding nuclease is the dns gene.

[0017] In the present application, the alanine dehydrogenase synthesizes L-alanine by using pyruvate, which is an important substrate and intermediate in the process of glycolysis and tricarboxylic acid cycle of Vibrio natriqutus by using glucose. However, there are many intermediates in the process of glycolysis and tricarboxylic acid cycle, and many enzymes can use these intermediates, which will compete with the reaction catalyzed by alanine dehydrogenase, so that a part of glucose is not finally converted into L-alanine, which affects the yield of the process of converting glucose into L-alanine in the present application. Therefore, the genes in the genome of Vibrio natriqutus which will affect the above process are functionally deleted to ensure the efficiency and yield of the production process in the present application.

[0018] In the present application, the gene required for synthesizing L-lactic acid refers to the coding gene of the protein for synthesizing L-lactic acid by using the intermediates in the process of glycolysis or the intermediates in the process of tricarboxylic acid cycle converted from glucose in Vibrio natriqutus. The above-mentioned protein for synthesizing L-lactic acid includes methylglyoxal synthase (PN96_04755), methylglyoxal synthase (PN96_19735) and malate dehydrogenase (PN96_16800). The methylglyoxal synthase can convert dihydroxyacetone phosphate (DHAP) into methylglyoxal, which is further synthesized into L-lactic acid in the cell; and the malate dehydrogenase can convert L-malic acid into oxaloacetic acid, which is further synthesized into L-lactic acid in the cell.

[0019] In the present application, the gene required for synthesizing D-lactic acid refers to the coding gene of the protein for synthesizing D-lactic acid by using the intermediates in the process of glycolysis converted from glucose in Vibrio natriqutus. The protein for synthesizing L-lactic acid includes lactate dehydrogenase (PN96_16785), which can convert pyruvate into D-lactic acid.

[0020] In the present application, the gene affecting the synthesis of succinic acid or the gene required for synthesizing succinic acid refers to the coding gene of succinate dehydrogenase, which can catalyze the mutual conversion of succinic acid and fumaric acid. The succinate dehydrogenase includes PN96_09285.

[0021] In the present application, the genes affecting the synthesis of acetic acid or required for the synthesis of acetic acid include the coding genes of acetate kinase (acetate kinase) PN96_03365, PN96_21510 and 3-oxoacid CoA-transferase (3-oxoacid CoA-transferase) PN96_20020; the acetate kinase can catalyze the mutual conversion of acetic acid and acetyl phosphate; the 3-oxoacid CoA-transferase can catalyze the mutual conversion of acetoacetate and succinic acid, thereby affecting the synthesis of acetic acid.

[0022] In the present application, the genes affecting the synthesis of ethanol or required for the synthesis of ethanol include the coding genes of acetaldehyde dehydrogenase (acetaldehyde dehydrogenase) PN96_03185, PN96_04715; the acetaldehyde dehydrogenase can convert acetaldehyde into acetic acid, and also catalyze the pathway from acetyl-CoA to acetaldehyde to ethanol, thereby affecting the synthesis of ethanol in Vibrio natriophilus.

[0023] In the present application, the genes affecting the synthesis of formic acid or required for the synthesis of formic acid include the coding gene of Pyruvate formate-lyase (pyruvate formate-lyase) PN96_08455; the pyruvate formate-lyase can catalyze the production of formic acid from pyruvic acid.

[0024] Further, the alanine transporter gene is inserted into the genome of the chassis strain in the form of an expression cassette for expression.

[0025] Still further, the alanine transporter gene is inserted between the dns genes in the genome of the chassis strain.

[0026] Further, 7-9 genes required for the synthesis of L-lactic acid, D-lactic acid, succinic acid, acetic acid, ethanol and formic acid in the genome of the chassis strain are deleted in function.

[0027] Still further, 7-9 genes among PN96_04755, PN96_19735, PN96_16800, PN96_16785, PN96_09285, PN96_03365, PN96_21510, PN96_20020, PN96_03185, PN96_04715 and PN96_08455 in the genome of the chassis strain are deleted in function.

[0028] Further, in the genetically engineered bacteria, the promoter for starting the expression of the alanine dehydrogenase gene is an inducible promoter; the inducible promoter is a lactose inducible promoter P lac , P tac and a temperature-sensitive λ P R -PL One of the promoters.

[0029] Further, the alanine transporter gene is alaE gene.

[0030] Further, the alaE gene has the accession number NP_417156 in NCBI library.

[0031] Further, the inducible promoter is temperature-sensitive lambda P R -P L Promoter.

[0032] Further, the temperature-sensitive lambda P R -P L Promoter starts the expression of the downstream alanine dehydrogenase gene (alaD gene) at 37-42℃.

[0033] In the second aspect, the application provides the use of the genetically engineered bacteria in the production of L-alanine.

[0034] In the third aspect, the application provides a two-stage method for producing L-alanine, comprising:

[0035] S1: fermenting and culturing the genetically engineered bacteria, and the fermentation and culturing process is carried out at 26-35℃;

[0036] S2: using glucose as the substrate and the genetically engineered bacteria cell cultured in S1 as the catalyst to form a reaction system, and catalyzing the production of L-alanine, and the production process is carried out at 37-42℃ under limited oxygen condition.

[0037] In the genetically engineered bacteria, the promoter that starts the expression of the alanine dehydrogenase gene is temperature-sensitive lambda P R -P L Promoter.

[0038] Further, the temperature-sensitive lambda P R -P L Promoter starts the expression of the downstream alaD gene at 37-42℃.

[0039] Further, in S2, the production process is carried out at 37℃ under limited oxygen condition.

[0040] Further, the limited oxygen condition is that the dissolved oxygen is lower than 20%.

[0041] Further, in S1, the fermentation and culturing time is 4-8h.

[0042] Further, the fermentation and culturing time is 5h.

[0043] Further, in S1, the production process is carried out under aerobic condition.

[0044] Further, in S2, the glucose concentration is 20-30 g / L, and the reaction time is 6-10 h.

[0045] Further, in S2, the glucose concentration is 25 g / L, and the reaction time is 8 h.

[0046] Further, in S1, the fermentation culture medium comprises: ammonium sulfate 5-15 g / L, sodium chloride 15-25 g / L, MOPS 20-25 g / L, potassium dihydrogen phosphate 0.5-2 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.2-0.5 g / L, calcium chloride 0.01-0.03 g / L, ferrous sulfate heptahydrate 15-18 mg / L, manganese sulfate monohydrate 5-15 mg / L, potassium sulfate pentahydrate 0.2-0.6 mg / L, zinc sulfate heptahydrate 0.5-1.5 mg / L, nickel chloride hexahydrate 0.01-0.05 mg / L, and glucose 5-10 g / L.

[0047] Further, in S1, the inoculation amount of the genetically engineered bacteria in the fermentation medium is 1-2%.

[0048] Compared with the prior art, the present application has the following beneficial effects:

[0049] In the present application, the alanine dehydrogenase gene is introduced into the genome of the Vibrio natriegens, and the key enzymes in the byproduct production pathway are inactivated randomly to find the optimal combination of inactivated genes for L-alanine production, so as to construct a Vibrio natriegens genetically engineered bacteria capable of efficiently producing L-alanine by fermentation. In the present application, the L-alanine is produced by two-stage fermentation using the genetically engineered bacteria through a temperature-sensitive control strategy, so as to reduce the influence of L-alanine accumulation on cell growth, and the production efficiency is high. The genetically engineered bacteria has a short growth cycle, can efficiently convert glucose into L-alanine, and transport the L-alanine to the extracellular, so that the separation and purification are simple, and cell disruption is not required. The yield and conversion rate of L-alanine can reach a high level. DETAILED DESCRIPTION

[0050] In order for those skilled in the art to better understand the present application, the technical solutions of the present application will be described clearly and completely in conjunction with specific examples. It should be pointed out that the following detailed description is exemplary and only a part of the embodiments of the present application, but not all the embodiments.

[0051] Based on the examples in the present application, all other examples obtained by those skilled in the art without making creative efforts shall belong to the scope of protection of the present application.

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the application pertains. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0053] In the following examples, HPLC is used to detect the content of L-alanine and glucose. The L-alanine HPLC detection conditions are as follows: mobile phase A: 10 mM Na2HPO4+10 mM Na2B4O7, pH adjusted to 8.2 with hydrochloric acid, filtered with a 0.22 μm filter membrane and used as a reserve; mobile phase B: ACN:MeOH:H2O=45:45:10, constant volume 1 L and used as a reserve; chromatographic column: Eclipse Plus C18 4.6x100mm, 3.5um; flow rate: 1.0 mL / min; column temperature: 40℃; DAD detector UV 338nm, 10nm (bandwidth). The glucose HPLC detection conditions are as follows: the mobile phase is ultrapure water, the Agilent Hi-Plex Ca chromatographic column (7.7mmx300mm), and the column temperature is 85℃; the flow rate is 0.6mL / min.

[0054] In the following examples, the alaD protein catalytic activity is determined in the following way:

[0055] The reaction system is 1 mL, which includes: 1 M pyruvate stock solution 100 μL (final concentration 100 mM), 0.4 M NADH stock solution 50 μL (final concentration 20 mM), PBS buffer 750 μL, and cell disruption solution 100 μL. The reaction solution and the cell disruption solution are preheated in a metal bath at 30℃ for 10 minutes, then they are quickly mixed, and the change in absorbance at 340 nm is monitored by a spectrophotometer.

[0056] Enzyme activity unit definition: the amount of enzyme required to generate 1 μM L-alanine per minute under standard reaction conditions.

[0057] The formula for calculating the enzyme activity of alaD is: enzyme activity (U / mL) = (△A / min) x (1 / ε) x (1 / d) x (Vt / Vs) x X, wherein △A / min represents the change in absorbance per minute; ε represents the molar extinction coefficient, which is 6.402 mL / (μmol*cm) here; d represents the cuvette optical path, which is 1 cm here; Vt represents the total reaction volume, which is 1000 μL here; Vs represents the volume of the cell disruption solution, which is 100 μL here; and X represents the dilution factor.

[0058] Example 1 Screening of alanine dehydrogenase

[0059] Six wild-type alanine dehydrogenase genes (alaD) from different sources (as shown in Table 1) were selected, wherein alaD2 from Vibrio natriophilus was obtained by PCR cloning, and the remaining five alaD genes were codon-optimized and synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. lac The promoters and alaD genes were constructed on the pDonor plasmid, and six alaD gene expression plasmids and strains were obtained. The strains were cultured overnight at 30℃ in 5mL LB3 (yeast powder 5g / L, protein peptone 10g / L, sodium chloride 15g / L) medium, then transferred to 50mL LB3 medium at a transfer amount of 2%, and cultured at 30℃ for 2 hours, then 5g / L lactose was added to induce the expression of alaD gene, and cultured at 30℃ for 12 hours, then the bacterial cells were collected by centrifugation. 9mL of bacterial cells collected were resuspended with 3mL PBS (pH 7.5), and the cells were broken by ultrasonic treatment (ultrasonic treatment for 3s, pause for 7s, for a total of 5 minutes) to obtain the broken cell solution.

[0060] The enzyme activity of the broken cell solution was determined, and the results are shown in Table 1. The alaD1 from Geobacillus stearothermophilus had the highest activity, reaching 8.93U / mL, followed by the endogenous alaD2 from Vibrio natriophilus, with an activity of 6.39U / mL. Therefore, the alaD1 was selected as the candidate gene for the subsequent examples, and the expression plasmid of alaD1 was named pDonor-lac-alaD1, and the strain was named VnAla1 (Vn / pDonor-lac-alaD1).

[0061] Table 1: Enzyme activity of alaD broken cell solution

[0062] alaD number Gene source Accession number Enzyme activity U / mL alaD1 Geobacillus stearothermophilus EF154460.1 8.93 alaD2 Vibrio natriegens PN96_07945 6.39 alaD3 Lysinibacillus sphaericus NZ_CP019980.1 0.00 alaD4 Bacillus subtilis BSU31930 3.92 alaD5 Natronobacterium gregoryi AFZ72932.1 2.81 alaD6 Lactobacillus casei BAN75115.1 2.17

[0063] Example 2: Genomic integration of alanine transporter alaE

[0064] The accumulation of a large amount of L-alanine in the cell can cause cell toxicity and affect the normal growth and metabolism of the cell, so it is very important to transport L-alanine to the outside of the cell in time. The dns gene of Vibrio natriophilus genome encodes a Dns nuclease, which can make the exogenous DNA unstable, so the alaE gene is inserted into the middle of the dns gene to achieve the purpose of introducing the alaE gene and destroying the dns gene.

[0065] The specific primer was designed to clone the E. coli alaE expression cassette (NCBI-Protein ID: NP_417156), which was constructed on the pMDTsimple-dnsaLR-specR plasmid backbone using the method disclosed in the literature (Frontiers in Microbiology 2021 Vol. 12 Issue 147), and the pMDTsimple-dnsaLR-alaE-specR plasmid was obtained. Then, alaE was integrated into the dns site by the MuGENT method disclosed in the literature (ACS Synth Biol 2017 Vol. 6 Issue 9 Pages 1650-1655). The strain with the genomic integration of the E. coli-derived alaE L-alanine efflux protein was constructed, and the pDonor-lac-alaD1 plasmid was then transformed into the strain, and the constructed strain was named VnAla2 (Vn::alaE / pDonor-lac-alaD1).

[0066] The L-alanine fermentation medium was prepared: ammonium sulfate 10 g / L, sodium chloride 15 g / L, MOPS 21 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.25 g / L, calcium chloride 0.01 g / L, ferrous sulfate heptahydrate 16.4 mg / L, manganese sulfate monohydrate 10 mg / L, potassium sulfate pentahydrate 0.3 mg / L, zinc sulfate heptahydrate 1 mg / L, nickel chloride hexahydrate 0.02 mg / L, and glucose 5 g / L. The strains VnAla1 and VnAla2 were activated in 5 mL of LB3 medium, and 2% of the inoculum was transferred to 50 mL of fermentation medium, which was incubated at 30°C for 2 hours. 5 g / L of lactose was added to induce the expression of the alaD gene, and the incubation was continued at 30°C for 8 hours. The L-alanine yield in the fermentation supernatant was determined by high performance liquid chromatography (HPLC).

[0067] The fermentation results of the strains are shown in Table 2. When the Vibrio natriegens introduced the exogenous alaE alanine efflux protein, the L-alanine yield in the fermentation supernatant reached 1.37 g / L, and the conversion rate was 26.86%.

[0068] Table 2: Genomic integration of L-alanine efflux protein alaE

[0069] Strain L-alanine production g / L Glucose consumption g / L Conversion rate g / g ATCC14048 0.15 4.98 3.01％ VnAla1 0.87 4.89 17.79％ VnAla2 1.37 5.10 26.86％

[0070] Example 3: Blocking of the byproduct synthesis pathway

[0071] In the L-alanine synthesis pathway, the synthesis of byproducts L-lactic acid, D-lactic acid, succinic acid, acetic acid, ethanol or formic acid will divert carbon sources, resulting in low conversion rate of L-alanine. In the Vibrio natriophilus genome, a total of 11 genes are involved in byproduct synthesis, in which the key enzymes for L-lactic acid synthesis include PN96_04755, PN96_19735, PN96_16800, the key enzymes for succinic acid synthesis include PN96_09285 (sdhB), the key enzymes for D-lactic acid synthesis include PN96_16785, the key enzymes for formic acid synthesis include PN96_08455, the key enzymes for ethanol synthesis include PN96_03185, PN96_04715, and the key enzymes for acetic acid synthesis include PN96_03365, PN96_21510, PN96_20020.

[0072] Taking the VnAla2 strain as the starting strain, a crRNA array targeting the 11 sites was designed, and the CRISPR-associated transposase method VchCAST (Biotechnol Bioeng 2024 121 (3): 1163-1172) was used to randomly block the 11 genes, and a small strain library of 26 inactivated different genes was obtained (as shown in Table 3).

[0073] An L-alanine fermentation medium was prepared: ammonium sulfate 10 g / L, sodium chloride 15 g / L, MOPS 21 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.25 g / L, calcium chloride 0.01 g / L, ferrous sulfate heptahydrate 16.4 mg / L, manganese sulfate monohydrate 10 mg / L, potassium sulfate pentahydrate 0.3 mg / L, zinc sulfate heptahydrate 1 mg / L, nickel chloride hexahydrate 0.02 mg / L, glucose 15 g / L. The strain was activated in 5 mL LB3 medium, 2% inoculation amount was transferred to 50 mL fermentation medium, 30°C culture for 2 hours, 5 g / L lactose was added to induce alaD expression, 30°C continued to culture for 8 hours, high performance liquid chromatography (HPLC) was used to detect the amount of L-alanine and glucose in the supernatant of fermentation broth, and the cell density OD600 was detected, and the results are shown in Table 4.

[0074] Table 3 Knockout of byproduct synthesis genes in different strains

[0075]

[0076]

[0077] Table 4 Synthesis of L-alanine by strains with different byproduct synthesis genes knocked out

[0078]

[0079] Example 4 Strengthening and regulating alaD gene expression

[0080] To explore the effect of different promoters of alaD gene on L-alanine production, the selected promoters include arabinose-inducible promoter P BAD , lactose-inducible promoter P lac , P trc , P tac , and temperature-sensitive λ P R -P L promoter. Among them, strain VnAla12 is P lac lactose-inducible promoter. Strains with other inducible promoters are constructed by PCR and Gibson assembly (as shown in Table 5).

[0081] The medium is prepared according to the medium formula in Example 3, the strain is transferred and cultured at 30°C for 2 hours, then the inducer is added according to the concentration in Table 5 or the culture temperature is increased, and after induction, the culture is continued for 8 hours. HPLC is used to detect the content of L-alanine and glucose in the supernatant of the fermentation broth, and a spectrophotometer is used to measure the density of the fermentation broth, and the conversion rate is calculated.

[0082] As can be seen from Table 5, when P BAD , P trc , P tac promoters are used, the production of L-alanine is not as good as that of strain VnAla12, which may be due to the fact that these three promoters are not suitable for the expression of alaD gene. When the P lac promoter is replaced by the temperature-sensitive λ P R -P L promoter, the production of L-alanine and the conversion rate are improved, and the regulation of the temperature-sensitive promoter does not require additional inducer, only the culture temperature needs to be increased from 30°C to 37°C.

[0083] Table 5 Effect of different alaD promoters on L-alanine production

[0084]

[0085] Example 5 Different ways of inserting alaD gene

[0086] The retention and replication of a free plasmid in a host requires antibiotic selection, while the expression of a genome-integrated gene does not require additional antibiotics due to the stability of the genome, and the gene dose can be strictly regulated. Therefore, the present application explores the effect of integrating the temperature-sensitive λ P R -P L -alaD expression cassette into Vibrio natriq genome on L-alanine production.

[0087] λ P R -PL The integration of the alaD expression cassette used the CRISPR-associated transposase method VchCAST (Biotechnol Bioeng 2024 121(3):1163-1172) disclosed in the literature, and the integration site of the genome was 8 non-coding regions of the Vibrio natriq genome. Eight engineering bacteria were constructed, each of which integrated 1-8 copies of the alaD expression cassette. R -P L The alaD expression cassette is shown in Table 6.

[0088] According to the method in Example 3, shake flask fermentation was carried out, and it can be seen from Table 6 that as the number of integrated copies increases, the yield and conversion rate of L-alanine gradually increase, and the strain VnAla37 with 5 copies has the highest yield and conversion rate. When the number of copies reaches 6 or more, the yield and conversion rate of L-alanine decrease sharply, which may be because the excessive number of L-alanine copies affects the intracellular resource allocation, resulting in a decrease in the overall efficiency of the engineering bacteria. Therefore, the number of copies of alaD is not the higher the better.

[0089] Table 6 Integration of alaD gene

[0090]

[0091] Example 6 Two-stage fermentation for producing L-alanine

[0092] The VnAla37 strain prepared in Example 5 was used to produce L-alanine by a two-stage process.

[0093] (1) First stage:

[0094] The fermentation medium was prepared as follows: ammonium sulfate 10 g / L, sodium chloride 15 g / L, MOPS 21 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.25 g / L, calcium chloride 0.01 g / L, ferrous sulfate heptahydrate 16.4 mg / L, manganese sulfate monohydrate 10 mg / L, potassium sulfate pentahydrate 0.3 mg / L, zinc sulfate heptahydrate 1 mg / L, nickel chloride hexahydrate 0.02 mg / L, and glucose 5 g / L. The seed liquid of VnAla37 strain was inoculated into 50 mL of fermentation medium at an inoculation amount of 1%, and cultured at 30°C for 5 hours to accumulate cell amount.

[0095] (2) Second stage:

[0096] Glucose was added to the fermentation broth to a final concentration of 25 g / L, and the temperature was raised to 37°C. The culture was incubated for 8 hours, which was the stage for converting glucose to L-alanine.

[0097] The contents of glucose and L-alanine in the fermentation supernatant were detected by HPLC, and the results are shown in Table 7.

[0098] Example 1 - Two-stage fermentation for production of L-alanine

[0099] Example 6 - One-stage fermentation for production of L-alanine using VnAla37 strain prepared in Example 5

[0100] The fermentation medium was prepared as follows: ammonium sulfate 10 g / L, sodium chloride 15 g / L, MOPS 21 g / L, potassium dihydrogen phosphate 1 g / L, potassium phosphate dibasic 1 g / L, magnesium sulfate 0.25 g / L, calcium chloride 0.01 g / L, ferrous sulfate heptahydrate 16.4 mg / L, manganese sulfate monohydrate 10 mg / L, cobalt sulfate pentahydrate 0.3 mg / L, zinc sulfate heptahydrate 1 mg / L, nickel chloride hexahydrate 0.02 mg / L. The seed culture of VnAla37 strain was inoculated into 50 mL of the fermentation medium at an inoculation amount of 1%, and incubated at 30°C for 2 hours. Then, glucose was added to the fermentation broth to a final concentration of 25 g / L, and the temperature was raised to 37°C. The fermentation was carried out for 11 hours.

[0101] The contents of glucose and L-alanine in the fermentation supernatant were determined by HPLC, and the results are shown in Table 7.

[0102] Table 7 - Fermentation results of Example 6 and Comparative Example 1

[0103]

[0104] As can be seen from Table 7, the two-stage fermentation method for production of L-alanine provided by the present application has a much higher fermentation yield and conversion rate than the one-stage fermentation.

Claims

1. A genetically engineered bacterium that produces L-alanine, characterized in that, The genetically engineered bacteria use sodium-dependent Vibrio as the substrate bacteria and co-express the alanine dehydrogenase gene and the alanine transporter gene. The alanine dehydrogenase gene is one of the accession numbers EF154460.1 or PN96_07945 in the NCBI database.

2. The genetically engineered bacterium according to claim 1, characterized in that, The alanine dehydrogenase gene and the alanine transporter gene are expressed in *Chachiobacter* in one of the following ways: (1) Expressed in the form of recombinant plasmids in the basal bacteria; (2) It is inserted into the genome of the chassis bacteria in the form of an expression cassette for expression.

3. The genetically engineered bacteria according to claim 2, characterized in that, The alanine dehydrogenase gene is inserted into the genome of the basal bacteria in the form of an expression cassette, with a copy number of 1 to 5.

4. The genetically engineered bacterium according to claim 1, characterized in that, In the genetically engineered bacteria, one or more genes in the genome of *Bacillus subtilis* required for the synthesis of L-lactic acid, D-lactic acid, succinic acid, acetic acid, ethanol, and formic acid are absent; genes encoding nucleases in the genome of *Bacillus subtilis* are absent. The genes required for the synthesis of L-lactic acid include PN96_04755, PN96_19735, and PN96_16800; the gene required for the synthesis of D-lactic acid includes PN96_16785; the gene required for the synthesis of succinic acid includes PN96_09285; the genes required for the synthesis of acetic acid include PN96_03365, PN96_21510, and PN96_20020; the genes required for the synthesis of ethanol include PN96_03185 and PN96_04715; and the gene required for the synthesis of formic acid includes PN96_08455. The gene encoding the nuclease is the dns gene.

5. The genetically engineered bacterium according to claim 3, characterized in that, Seven to nine genes in the genome of the *Trichoderma* required for the synthesis of L-lactic acid, D-lactic acid, succinic acid, acetic acid, ethanol, and formic acid are missing.

6. The genetically engineered bacterium according to claim 1, characterized in that, In genetically engineered bacteria, the promoter for initiating alanine dehydrogenase gene expression is an inducible promoter; the inducible promoter is the lactose-inducible promoter P. lac P tac and temperature-sensitive λP R -P L One type of promoter.

7. The use of the genetically engineered bacteria as described in any one of claims 1 to 6 in the production of L-alanine.

8. A two-stage method for producing L-alanine, characterized in that, include: S1: The genetically engineered bacteria as described in claim 1 are fermented and cultured at 26-35°C. S2: Using glucose as a substrate and genetically engineered bacteria cultured in S1 as a catalyst, the reaction system is composed of L-alanine to be produced. The production process is carried out at 37-42℃ under oxygen-limited conditions. In genetically engineered bacteria, the promoter for initiating alanine dehydrogenase gene expression is the thermosensitive λP. R -P L Promoter.

9. The method according to claim 8, characterized in that, In S1, the fermentation time is 4–8 hours.

10. The method according to claim 8, characterized in that, In S2, the glucose concentration is 20–30 g / L, and the reaction time is 6–10 h.

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