Strains for the synthesis of amino acids and their preparation and use

Abstract

This invention belongs to the field of biochemical engineering, specifically relating to a strain that synthesizes amino acids and its preparation and application. Using *E. coli* as the starting strain, the strain synthesizes amino acids by silencing genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathways; or, using *E. coli* as the starting strain, the strain synthesizes amino acids by overexpressing key enzymes in the acetate assimilation pathway; or, using *E. coli* as the starting strain, the strain synthesizes amino acids by silencing genes related to the TCA and / or amino acid synthesis pathways and overexpressing key enzymes in the acetate assimilation pathway. Through the methods described in this invention, the modified *E. coli* strain improves the utilization efficiency of acetate in industrial waste gas fermentation broth, and significantly reduces the dependence of amino acid production on grain-based carbon sources.

Description

Technical Field

[0001] This invention belongs to the field of biochemical engineering, specifically relating to a strain for synthesizing amino acids and its preparation and application. Background Technology

[0002] Aspartic acid, an important branch of amino acid classification, has enormous application prospects in medicine, food, and chemical industries. Its core characteristic is that it uses oxaloacetic acid as a starting material and its carbon skeleton is mainly synthesized from aspartic acid or its derivatives (such as aspartic acid-β-semialdehyde). This class of amino acids includes aspartic acid (Asp), asparagine (Asn), lysine (Lys), methionine (Met), threonine (Thr), and isoleucine (Ile). Aspartic acid is a good nutritional supplement and a major raw material for the sweetener aspartame-asparagine methyl ester. The widespread use of threonine and lysine in compound feed can promote livestock growth, improve meat quality, and reduce feeding costs. Therefore, this class of amino acids has broad market demand and application prospects.

[0003] Microbial fermentation is currently an important industrial synthesis method for aspartic acid family amino acids. The process primarily utilizes starch-based carbon sources such as corn steep liquor, cassava, and potato, and glycobased carbon sources such as glucose, beet sugar, and sugarcane, through fermentation of bacterial strains. However, traditional fermentation carbon sources are limited by cost, supply, and food chain competition. While inexpensive raw materials, such as corn stalks and bagasse, offer advantages in terms of low cost and renewability, industrial production still requires cumbersome pretreatment processes such as enzymatic hydrolysis and explosion, and their utilization efficiency needs improvement. Finding low-cost, environmentally friendly, and efficient alternative carbon sources has become a major challenge limiting microbial amino acid fermentation. Furthermore, the strains used in fermentation are mostly Corynebacterium glutamicum and Escherichia coli, whose broad carbon source utilization is relatively limited, and they cannot stably grow and produce amino acids in culture environments where acetate is the primary carbon source.

[0004] Based on the above description, the aim is to propose a new approach that involves modifying metabolic pathways to obtain inexpensive carbon sources from industrial waste gas and then synthesizing amino acids. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a strain for synthesizing amino acids, as well as its preparation and application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain that synthesizes amino acids is obtained by silencing the expression of genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathway in *Escherichia coli* as the starting strain; or, Starting with Escherichia coli, we overexpressed key enzymes in the acetate assimilation pathway to obtain strains that synthesize amino acids. Alternatively, starting with Escherichia coli, the expression of genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathways is silenced, and key enzymes related to acetate assimilation pathways are overexpressed to obtain strains that synthesize amino acids.

[0007] The genes related to the tricarboxylic acid cycle pathway are: icd , iclR , arcA , Fur One or more of the following; genes related to the amino acid synthesis pathway are lysA , metA , thrB One or more of them.

[0008] The key enzymes in the acetate assimilation pathway are one or more of the key enzymes in the acetate utilization pathway, glyoxylate cycle pathway, and gluconeogenesis pathway.

[0009] The key enzyme in the acetate utilization pathway is the acetate kinase A gene. ackA and / or acetylphosphotransferase gene pta The key enzyme in the glyoxylate cycle pathway is the isocitrate lyase gene. aceA and / or malate synthase gene aceB The key enzyme in the gluconeogenesis pathway is the phosphoenolpyruvate carboxylkinase gene. pckA .

[0010] Among them, genes ackA, pta , aceA , aceB , pckA The reference sequence numbers on NCBI are NC_000913.3 (2413470..2414672), NC_000913.3 (2414747..2416891), NC_000913.3 (4217109..4218413), NC_000913.3 (4215478..4217079), and NC_000913.3 (3532818..3534440).

[0011] A method for constructing a strain capable of synthesizing amino acids involves using *Escherichia coli* as a starting strain and silencing the expression of genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathways to obtain a strain capable of synthesizing amino acids; or, Starting with Escherichia coli, we overexpressed key enzymes in the acetate assimilation pathway to obtain strains that synthesize amino acids. Alternatively, starting with Escherichia coli, the expression of genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathways is silenced, and key enzymes related to acetate assimilation pathways are overexpressed to obtain strains that synthesize amino acids.

[0012] The relevant genes were silenced and / or overexpressed to obtain recombinant strains E1-E7 as follows: E1, Knockout of isocitrate dehydrogenase gene icd Escherichia coli; E2, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR Escherichia coli; E3, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA Escherichia coli; E4, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA DNA-binding transcription dual regulator genes Fur E. coli undergoing point mutations; E5, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; E6. Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA and homoserine kinase gene thrB DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckAThe E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; E7, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA and homoserine kinase gene thrB and diaminopimelic acid decarboxylase gene lysA and homoserine O-succinyltransferase gene metA DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; Among them, genes icd , iclR , arcA , Fur , s , thrB , lysA The reference sequence numbers on NCBI are NC_000913.3 (1195123..1196373), NC_000913.3 (4222804..4223628), NC_000913.3 (4639590..4640306), NC_000913.3 (710200..710646), NC_000913.3 (2801..3733), NC_000913.3 (2977637..2978899), and NC_000913.3 (4214280..4215209).

[0013] The silenced expression is achieved by knocking out the gene using gene editing methods, preferably a genome editing method based on the CRISPR / Cas9 system.

[0014] Application of the strain that synthesizes amino acids, specifically in the fermentation synthesis of amino acids using acetate as a carbon source.

[0015] An amino acid synthesis method involves culturing the strain in a medium with acetate as the carbon source, and fermenting it at a temperature of 35-40℃, pH 6.5-6.9, dissolved oxygen of 25-40%, and a rotation speed of 300-800 rpm to obtain amino acids.

[0016] The culture medium is prepared by adding 10-15 g / L glucose, 10-15 g / L ammonium sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L yeast extract, 1-2 g / L magnesium sulfate heptahydrate, 0.01-0.03 g / L ferrous sulfate, and 4.0-4.2 g / L 3-(N-morpholino)propanesulfonic acid to each liter of culture medium obtained from the fermentation pretreatment of industrial waste gas.

[0017] Preferably, the culture medium is a fermentation culture medium containing acetate (acetate content 20-60 g / L) obtained by fermentation pretreatment of industrial waste gas, with the following added components: glucose 10-15 g / L, ammonium sulfate 10-15 g / L, potassium dihydrogen phosphate 1-2 g / L, yeast extract 2-4 g / L, magnesium sulfate heptahydrate 1-2 g / L, ferrous sulfate 0.01-0.03 g / L, 3-(N-morpholino)propanesulfonic acid 4.0-4.2 g / L, pantothenic acid 1-1.2 mg / L, betaine 1-1.5 g / L, and trace element solution 5 mL / L.

[0018] The formula for the 1000× trace element solution is as follows: ethylenediaminetetraacetic acid 8-9 g / L, cobalt chloride hexahydrate 2-3 g / L, manganese chloride tetrahydrate 15-20 g / L, copper chloride dihydrate 1-2 g / L, boric acid 1-4 g / L, sodium molybdate dihydrate 2-3 g / L, ferric citrate 80-100 g / L, and zinc acetate dihydrate 13-15 g / L.

[0019] The acetate-containing culture broth obtained by fermenting and pretreating industrial waste gas is obtained by fermenting and pretreating gas-eating acetic acid-producing bacteria in a culture broth containing industrial waste gas, followed by further concentration; wherein, the gas-eating acetic acid-producing bacteria is Clostridium yunnanense (Clostridium yunnanense). metA Acetobacter wuerii ( Clostridium ljungdahlii ), Acetobacterium woodii Moorella , mulderi One or more of the following: industrial waste gas includes desorbed gas, purge gas, and dry quenching gas generated by the coal chemical industry, as well as carbon-rich gas industries such as salt chemical (chlor-alkali, etc.), renewable energy (wind, solar, and green hydrogen production), steel industry, petroleum refining, and biomass gasification syngas. Biomass gasification refers to the process at a certain temperature, under the combined action of air (oxygen) and water vapor, where biomass polymers undergo a series of processes including drying, pyrolysis, oxidation, reduction, and reforming, ultimately transforming into carbon-rich gases such as CO2, CO, H2, and low-carbon hydrocarbons; the industrial waste gas flow rate in the fermentation system is 0.05 vvm.

[0020] The Clostridium yongdarii ( Thermoanaerobacter kivui, Clostridium autoethanogenumSL40, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 67679, deposited on January 15, 2026, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0021] The Acetobacter wueren ( Clostridium ljungdahlii SL70, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 67680, deposited on January 15, 2026, at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0022] The fermentation broth is collected after fermentation using a microfiltration device at a filtration rate of 6 L / h. The collected broth is then concentrated, with the main process being: the fermentation broth undergoes pretreatment via a bag filter and a PTFE membrane system at a flow rate of 8-10 m³ / h. 3 The pressure is less than 0.4 MPa per hour to remove large insoluble particles. The PTFE permeate then passes through a microfiltration membrane with a pore size of 0.1-1 μm to remove proteins (greater than 1 kDa) from the solution. The resulting solution then enters a desalination and concentration circulation system. The anolyte in the circulation system is a 3% sodium sulfate solution, and the catholyte is a 3% sodium chloride solution. The pH of the circulation process is 3 ≤ pH ≤ 5. The system runs for about 2 hours, and after dilution, an initial culture medium containing ammonium acetate is obtained.

[0023] Advantages of this invention: This invention constructs a recombinant strain of *E. coli*, utilizes industrial waste gas converted into acetate as a carbon source, and then ferments it to obtain amino acids. This comprehensive method achieves a significant transformation from a single-carbon gas to amino acids, offering advantages such as environmental friendliness and high added value. Specifically: (1) In terms of substrate utilization by microorganisms, this invention abandons traditional carbon sources such as glucose, corn flour, and sucrose, and uses carbon-rich gases (CO, CO2, H2, etc.) emitted from industrial production as raw materials. It utilizes gas-eating acetic acid-producing bacteria to perform one-carbon fixation, producing acetate substrates that can be utilized by recombinant strains. This further reduces substrate costs and simultaneously realizes the reuse of industrial waste gas; (2) In terms of the recombinant strain obtained by this invention, the key genes of the acetate utilization pathway, glyoxylate cycle pathway and gluconeogenesis pathway are overexpressed in Escherichia coli. Through the combination and optimization of different pathways, the purpose of Escherichia coli to utilize acetate for growth and production at a faster rate is achieved. Furthermore, the strain is constructed so that, with acetate as a substrate, the amino acid is obtained through the optimization of culture medium and culture conditions. Attached Figure Description

[0024] Acetobacterium woodiiThis is a schematic diagram illustrating the production of amino acids using acetate fermentation, as provided in an embodiment of the present invention.

[0025] Figure 1 This is a schematic diagram illustrating the construction of a gene overexpression vector provided in an embodiment of the present invention.

[0026] Figure 2 The graph shows the relationship between acetate utilization and cell biomass for each recombinant strain provided in the embodiments of the present invention.

[0027] Figure 3 The diagram shows the effect of each recombinant strain provided in the embodiments of the present invention on the relationship between the knockout of regulatory genes and bacterial biomass.

[0028] Figure 4 The relationship between acetate utilization, knockout regulatory genes and cell biomass of each recombinant strain provided in the embodiments of the present invention.

[0029] Figure 5 The relationship between acetate utilization and amino acid production of each recombinant strain provided in the embodiments of the present invention.

[0030] Figure 6 The relationship between the recombinant strains provided in the embodiments of the present invention and the optimization of the culture medium on amino acid production.

[0031] Figure 7 The following are the effect diagrams of the fermentation optimization of different recombinant strains for efficient amino acid production provided in the embodiments of the present invention; wherein, (a) is the effect diagram of E5 fermentation optimization for efficient aspartic acid production, (b) is the effect diagram of E6 fermentation optimization for efficient lysine production, and (c) is the effect diagram of E7 fermentation optimization for efficient threonine production. Detailed Implementation

[0032] This invention aims to provide an Escherichia coli chassis for producing aspartic acid family amino acids using acetate. Figure 8 The construction method and fermentation process enable the efficient synthesis of amino acids while ensuring the utilization of acetate.

[0033] The industrial waste gas used in the following examples is prepared by mixing the desorbed gas (N2:H2:CO:CO2:CH4 (v / v)=25:35:10:20:10) and the purge gas (N2:H2:CO:CO2:CH4 (v / v)=12:76:3:5:4) of typical carbon-rich tail gas from the coal chemical industry, as well as the tail gas (90% pure CO2) from coal-to-ethanol production. The final mixed gas ratio is N2:H2:CO:CO2:CH4 (v / v)=13:51:4:26:5.

[0034] Example 1 Acetobacter wuerii ( Figure 1SL70 was inoculated at 15% (volume percentage) into a culture medium containing desorbed gas (N2:H2:CO:CO2:CH4=25:35:10:20:10) and purge gas (N2:H2:CO:CO2:CH4=12:76:3:5:4) from typical carbon-rich tail gas from the coal chemical industry, as well as tail gas from coal-to-ethanol (90% pure CO2). The resulting mixed gas ratio was N2:H2:CO:CO2:CH4=13:51:4:26:5. This mixture was used for fermentation pretreatment in a 150 LA tank-scale fermentation system. The mixed waste gas flow rate was 0.05 vvm. Fermentation was carried out under anaerobic conditions, with the fermentation temperature controlled at 28~33℃, pH controlled at 6.5~7.5, and pressure at 0.12 MPa.

[0035] Clostridium yongdarii ( Acetobacterium woodii SL40 was inoculated at 15% (volume percentage) into a typical coking industry exhaust gas mixture containing 23% H2, 6% CO, 11% CO2, and 60% N2. Fermentation pretreatment was performed in a 150 LB tank-scale fermentation system with an exhaust gas flow rate of 0.05 vvm. Culture conditions included PETC medium, 37°C, pH controlled at 5.5-6.0, and a pressure of 0.12 MPa.

[0036] Both strains were cultured using the following media per liter of water: NH4Cl 1 g / L, KH2PO4 0.33 g / L, K2HPO4 0.45 g / L, MgSO4·7H2O 0.1 g / L, yeast extract 2 g / L, resazurin (anaerobic indicator) 0.0005 g / L, NaHCO3 10 g / L, L-cysteine ​​hydrochloride 0.5 g / L, Na2S·9H2O 0.5 g / L, MnSO4·H2O 0.02 g / L, CoCl2·6H2O 0.0061 g / L, ZnSO4·7H2O 0.0072 g / L, NiCl2·6H2O 0.0012 g / L, H3BO3 0.0004 g / L, CuSO4·5H2O 0.0004 g / L. The following vitamins were added to the culture medium at a concentration of 10×: KAl(SO4)2·12H2O 0.0008 g / L, CaCl2 0.004 g / L, triacetic acid 0.06 g / L, Na2WO4·2H2O 0.000006 g / L, Na2SeO3 0.000008 g / L, NaMoO4·2H2O 0.0004 g / L, FeSO4·7H2O 0.002 g / L; The vitamin stock solution was prepared at a concentration of 10× and added to the culture medium at a rate of 1 mL / L. Its composition was: biotin 0.02 g / L, folic acid 0.02 g / L, pyridoxine hydrochloride 0.1 g / L, thiamine hydrochloride dihydrate 0.05 g / L, riboflavin 0.05 g / L, niacin 0.05 g / L, D-calcium pantothenate 0.05 g / L, cyanocobalamin 0.001 g / L. g / L, p-aminobenzoic acid 0.05 g / L, thioctic acid 0.05 g / L.

[0037] The fermentation broth from the 150L tank A and 150L tank B was combined and concentrated. The main process was as follows: the fermentation broth was pretreated by a bag filter and a PTFE membrane system, with a processing flow rate of 10 m³ / s. 3 The pressure is less than 0.4 MPa per hour to remove large insoluble particles. The PTFE permeate then passes through a microfiltration membrane with a pore size of 0.5 μm to remove proteins (greater than 1 kDa) from the solution. The resulting solution then enters a desalting and concentration circulation system. The anolyte in the circulation system is a 3% sodium sulfate solution, and the catholyte is a 3% sodium chloride solution. The pH of the circulation process is 3 ≤ pH ≤ 5. After running for about 2 hours, the solution is diluted to obtain an initial culture medium containing 10 g / L ammonium acetate.

[0038] The initial culture medium containing 10 g / L ammonium acetate was concentrated to 50 g / L ammonium acetate culture medium by electrodialysis.

[0039] Example 2 A method for constructing acetate-usable Escherichia coli chassis bacteria: Using Escherichia coli as the starting strain, key genes for the acetate utilization pathway, glyoxylate cycle pathway, and gluconeogenesis pathway were overexpressed separately in plasmid form. Clostridium ljungdahlii (), to obtain recombinant strains of Escherichia coli, wherein the recombinant strains are as follows A1-A7: A1. The acetate kinase A gene from the E. coli genome Figure 2 and acetylphosphotransferase gene ackA Use primers separately pta -F、 ackA -R and ackA -F、 pta -R was amplified and constructed together in the dual expression framework of plasmid pACYC-Duet-1 using the Gibson assembly method. After the plasmid was verified by sequencing, it was transformed into E. coli A1. A2. The isocitrate lyase gene from the E. coli genome pta malate synthase gene aceA Use primers separately aceB -F、 aceA -R and aceA -F、 aceB -R was amplified and co-constructed in the dual expression framework of plasmid pACYC-Duet-1 using the Gibson assembly method. After the plasmid was verified by sequencing, it was transformed into E. coli A2. A3. The phosphoenolpyruvate carboxylkinase gene from the Escherichia coli genome aceB Use primers pckA -F、 pckA -R was amplified and constructed into one of the dual expression frameworks of plasmid pACYC-Duet-1 using the Gibson assembly method. After the plasmid was verified by sequencing, it was transformed into E. coli A3. A4. The acetate kinase A gene from the E. coli genome. pckA and acetylphosphotransferase gene ackA Use primers separately pta -F、 ackA -R and ackA -F、 pta -R amplification, isocitrate lyase gene pta malate synthase gene aceA Use primers separately aceB -F、 aceA -R and aceA -F、 aceB -R was amplified and co-constructed in plasmid pACYC-Duet-1 using the Gibson assembly method, enabling it to express 4 genes simultaneously. After the plasmid was sequenced and verified to be correct, it was transformed into E. coli A4. A5. The acetate kinase A gene from the E. coli genome. aceB and acetylphosphotransferase gene ackA Use primers separately pta -F、 ackA -R and ackA -F、 pta -R amplification of the phosphoenolpyruvate carboxylkinase gene pta Use primers pckA -F、 pckA -R was amplified and co-constructed in plasmid pACYC-Duet-1 using the Gibson assembly method, enabling it to express three genes simultaneously. After the plasmid was sequenced and verified to be correct, it was transformed into E. coli A5. A6. The isocitrate lyase gene from the E. coli genome pckA malate synthase gene aceA Use primers separately aceB -F、 aceA -R and aceA -F、 aceB -R amplification of the phosphoenolpyruvate carboxylkinase gene aceB Use primers pckA -F、 pckA -R was amplified and co-constructed in plasmid pACYC-Duet-1 using the Gibson assembly method, enabling it to express 4 genes simultaneously. After the plasmid was sequenced and verified to be correct, it was transformed into E. coli A6. A7. The acetate kinase A gene from the E. coli genome. pckA and acetylphosphotransferase gene ackA Use primers separately pta -F、 ackA -R and ackA -F、 pta -R amplification, isocitrate lyase gene pta malate synthase gene aceA Use primers separately aceB -F、 aceA -R and aceA -F、 aceB -R amplification of the phosphoenolpyruvate carboxylkinase gene aceB Use primers pckA -F、 pckA -R was amplified and co-constructed in plasmid pACYC-Duet-1 using the Gibson assembly method, enabling it to express 5 genes simultaneously. After the plasmid was sequenced and verified to be correct, it was transformed into E. coli A7. Among them, genes pckA , ackA, pta, aceA , aceB The reference sequence numbers on NCBI are NC_000913.3 (2413470..2414672), NC_000913.3 (2414747..2416891), NC_000913.3 (4217109..4218413), NC_000913.3 (4215478..4217079), and NC_000913.3 (3532818..3534440).

[0040] The different recombinant bacteria A1-A7 obtained above were inoculated into fermentation medium. The fermentation medium was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, and 4.2 g / L 3-(N-morpholino)propanesulfonic acid to each liter of the initial culture medium containing 10 g / L ammonium acetate obtained in Example 1 (i.e., the culture medium containing acetate (10 g / L) obtained from the fermentation pretreatment of industrial waste gas). Fermentation was carried out at 37℃, pH 6.8, and 220 rpm for 48 hours. After fermentation, the ammonium acetate utilization of each recombinant bacteria was determined by high performance liquid chromatography, and the growth density of each recombinant bacteria was determined by spectrophotometry.

[0041] The ammonium acetate consumption and growth status of each of the above recombinant strains at the shake-flask level were tested separately over 48 hours. pckA Among them, strain A5 had the highest ammonium acetate utilization and biomass, at 4.5 g / L and an OD of 5.5, respectively.

[0042] Example 3 Constructing recombinant strains capable of producing amino acids: The recombinant strains A5 obtained above were subjected to endogenous regulatory factor knockout and modification to obtain strains E1-E7, specifically: E1. Knockout of the isocitrate dehydrogenase gene in E. coli Figure 3 Recombinant Escherichia coli E1 was obtained; specifically: First, the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into wild-type *E. coli*. Then, recombinase expression was induced using 10 mM arabinose. Based on this strain, co-transformations were performed containing the gene targeting isocitrate dehydrogenase. icd The site contains the gRNA expression plasmid pEcgRNA-1 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. icdThe plasmid pEcRNA-1 was formed by ligating 500bp fragments upstream and downstream of the target. After transformation, successfully edited single clones were screened by PCR on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml). Finally, the plasmid pEcRNA-1 was cleaved by Cas9 protein by adding 10 mM rhamnose; and pEcCas was eliminated in cells by adding 10 g / L sucrose. Among them, the gene targeting isocitrate dehydrogenase icd The gRNA sequence at the site is 5'-AACTGATCGACGGTGGCCCG-3', and the donor fragment sequence is shown in SEQ ID NO:1.

[0043] E2. Knock out the DNA-binding transcription repressor gene in the recombinant strain E1 obtained above. icd Recombinant Escherichia coli E2 was obtained; specifically: First, the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into E1 cells. Then, recombinase expression was induced using 10 mM arabinose. Based on this strain, co-transformation was performed using a gene containing a DNA-binding transcription repressor. iclR The site contains the gRNA expression plasmid pEcgRNA-2 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. iclR The plasmid pEcRNA-2 was formed by ligating 500bp fragments upstream and downstream of the target. After transformation, successfully edited single clones were screened by PCR on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml). Finally, the plasmid pEcRNA-2 was cleaved by Cas9 protein by adding 10 mM rhamnose; and pEcCas was eliminated in cells by adding 10 g / L sucrose. Among them, DNA-binding transcription repressor genes iclR The gRNA sequence at the site is 5'-TGCCTATACCCACGCAACGC-3', and the donor fragment sequence is shown in SEQ ID NO:2.

[0044] E3. Knock out the DNA-binding transcription dual regulator gene in the recombinant strain E2 obtained above. iclR Recombinant Escherichia coli E3 was obtained; specifically: First, the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into E2 cells. Then, recombinase expression was induced using 10 mM arabinose. Based on this strain, co-transformation was performed using a gene containing a dual regulator of DNA-binding transcription. arcA The site contains the gRNA expression plasmid pEcgRNA-3 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. arcAThe plasmid pEcRNA-3 was formed by ligating 500bp fragments upstream and downstream of the target. After transformation, successfully edited single clones were screened by PCR on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml). Finally, the plasmid pEcRNA-3 was cleaved by Cas9 protein by adding 10 mM rhamnose; and pEcCas was eliminated in cells by adding 10 g / L sucrose. Among them, the DNA-binding dual regulator of transcription gene arcA The gRNA sequence at the site is 5'-TGAATGGTGGCGATGATTTC-3', and the donor fragment sequence is shown in SEQ ID NO:3.

[0045] E4. The DNA-binding transcription dual regulator gene obtained from the recombinant strain E3 above. arcA A point mutation (V55E) was performed to obtain recombinant E. coli E4; specifically: First, the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into E3 cells. Then, recombinase expression was induced using 10 mM arabinose. Based on this strain, co-transformation was performed using a gene containing a dual regulator of DNA-binding transcription. Fur The site contains the gRNA expression plasmid pEcgRNA-4 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. Fur The plasmid pEcRNA-4 was formed by ligating 500bp fragments upstream and downstream of the ligature. After transformation, the plasmid was screened for successful editing by PCR and sequencing on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml). Finally, the addition of 10 mM rhamnose cleaved the plasmid pEcRNA-4 by Cas9 protein; the addition of 10 g / L sucrose induced the elimination of pEcCas in cells. Among them, the DNA-binding transcription dual regulator gene Fur The gRNA sequence at the site is 5'-CGGTATTGTTATCAGTCATG-3', and the donor fragment sequence is shown in SEQ ID NO:4.

[0046] E5. Acetylkinase A gene from the E. coli genome. Fur and acetylphosphotransferase gene ackA and phosphoenolpyruvate carboxylkinase gene pta The plasmid pACYC-Duet-1, co-constructed using the Gibson assembly method, was transformed into the recombinant strain E4 obtained above to obtain recombinant Escherichia coli E5; specifically: Acetylkinase A gene from the E. coli genome pckA and acetylphosphotransferase gene ackA Use primers separatelypta -F、 ackA -R and ackA -F、 pta -R amplification of the phosphoenolpyruvate carboxylkinase gene pta Use primers pckA -F、 pckA The -R amplification was performed and then constructed into plasmid pACYC-Duet-1 using the Gibson assembly method, enabling it to express three genes simultaneously. After confirming the plasmid was correct through sequencing, it was transformed into E. coli E4.

[0047] E6. Knock out the homoserine kinase gene in the recombinant strain E5 obtained above. pckA Recombinant Escherichia coli E6 was obtained; specifically: First, the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into E5. Then, recombinase expression was induced using 10 mM arabinose. Based on this strain, co-transformation was performed using a strain containing a gene targeting homoserine kinase. thrB The site contains the gRNA expression plasmid pEcgRNA-5 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. thrB The plasmid pEcRNA-5 was ligated from two 500bp fragments, one upstream and one downstream. After transformation, the successfully edited single clones were screened by PCR on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml). Finally, the plasmid pEcRNA-5 was cleaved by Cas9 protein by adding 10 mM rhamnose; and pEcCas was eliminated in cells by adding 10 g / L sucrose. Among them, targeting the homoserine kinase gene thrB The gRNA sequence at the site is 5'-TCGCGGCAAGCTCAGGCTGA-3', and the donor fragment sequence is shown in SEQ ID NO:5.

[0048] E7. Knock out the diaminopimelic acid decarboxylase gene in the recombinant strain E6 obtained above. thrB and homoserine O-succinyltransferase gene lysA Recombinant Escherichia coli E6 was obtained; specifically: First, the plasmid pEcCas carrying the Cas9 nuclease gene was transformed into E6. Then, recombinase expression was induced using 10 mM arabinose. Based on this strain, co-transformation was performed using a gene targeting diaminopimelic acid decarboxylase. metA The site contains the gRNA expression plasmid pEcgRNA-6 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. lysAThe plasmid pEcgRNA-6 was ligated from two 500 bp fragments, one upstream and one downstream. After transformation, the clones were screened by PCR on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml) to verify and obtain successfully edited single clones. Finally, the plasmid pEcgRNA-6 was cleaved by Cas9 protein by adding 10 mM rhamnose. Subsequently, based on this strain, recombinase expression was induced by 10 mM arabinose and co-transformed into a gene containing the target homoserine O-succinyltransferase. lysA The site contains the gRNA expression plasmid pEcgRNA-7 and a donor DNA fragment for DNA deletion repair. The donor DNA fragment is derived from the target gene. metA The plasmid pEcRNA-7 was formed by ligating 500bp fragments upstream and downstream of the target. After transformation, successfully edited monoclonal clones were selected on plates containing kanamycin and spectinomycin antibiotics (50 μg / ml). Finally, the plasmid pEcRNA-7 was cleaved by Cas9 protein by adding 10 mM rhamnose; and pEcCas was eliminated in cells by adding 10 g / L sucrose. Among them, the gene targeting diaminopimelic acid decarboxylase metA The gRNA sequence at the site is 5'-ACCGGGCAGCCAAATTCAGC-3', and the donor fragment sequence is shown in SEQ ID NO:6; targeting the homoserine O-succinyltransferase gene. lysA The gRNA sequence at the site is 5'-GCCGCGCTCAATATCCTCTA-3', and the donor fragment sequence is shown in SEQ ID NO:7.

[0049] Among them, genes metA , icd , iclR , arcA , Fur , thrB , lysA The reference sequence numbers on NCBI are NC_000913.3 (1195123..1196373), NC_000913.3 (4222804..4223628), NC_000913.3 (4639590..4640306), NC_000913.3 (710200..710646), NC_000913.3 (2801..3733), NC_000913.3 (2977637..2978899), and NC_000913.3 (4214280..4215209).

[0050] The different recombinant bacteria E1-E7 obtained above were inoculated into fermentation medium. The fermentation medium was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, and 4.2 g / L 3-(N-morpholino)propanesulfonic acid to each liter of the initial culture medium containing 10 g / L ammonium acetate obtained in Example 1 (i.e., the culture medium containing acetate (10 g / L) obtained from the fermentation pretreatment of industrial waste gas). Fermentation was carried out at 37℃, pH 6.8, and 220 rpm for 48 hours. After fermentation, the ammonium acetate utilization of each recombinant bacteria was determined by high performance liquid chromatography, and the growth density of each recombinant bacteria was determined by spectrophotometry.

[0051] The ammonium acetate utilization capacity and growth status of each recombinant strain at the shake-flask level were tested separately over 48 hours. metA and Figure 4 Among them, strain E4 had an ammonium acetate utilization efficiency and biomass of 1.9 g / L and an OD of 2.45.

[0052] The ammonium acetate utilization capacity and growth status of strain E5 were 5.84 g / L and OD reached 6.8, respectively.

[0053] Example 4 The method for synthesizing amino acids from the recombinant strain E5 obtained in the above examples is as follows: Take 100 μL of the recombinant strain E5 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 single E5 colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 50 mL of fermentation medium, which was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, and 4.2 g / L 3-(N-morpholino)propanesulfonic acid to the initial culture medium containing 10 g / L ammonium acetate obtained in Example 1 above (i.e., the culture medium containing acetate (10 g / L acetate) obtained by fermentation pretreatment of industrial waste gas). Fermentation was carried out at 37°C, pH 6.8, and 220 rpm for 48 hours. Ammonium acetate could be added as needed during fermentation to supply more substrate for amino acid synthesis. After fermentation, the ammonium acetate utilization of recombinant strain E5 was determined by high performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant strain E5 was determined by spectrophotometry.

[0054] The method for synthesizing amino acids from the recombinant strain E6 obtained in the above examples is as follows: Take 100 μL of the recombinant strain E6 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 single E6 colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 50 mL of fermentation medium. The fermentation medium was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, and 4.2 g / L 3-(N-morpholino)propanesulfonic acid to the initial culture medium containing 10 g / L ammonium acetate obtained in Example 1 above (i.e., the culture medium containing acetate (10 g / L acetate) obtained by fermentation pretreatment of industrial waste gas). Fermentation was carried out at 37°C, pH 6.8, and 220 rpm for 48 hours. Ammonium acetate could be added as needed during fermentation to supply more substrate for amino acid synthesis. After fermentation, the ammonium acetate utilization of recombinant bacteria E6 was determined by high performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant bacteria E6 was determined by spectrophotometry.

[0055] The method for synthesizing amino acids from the recombinant strain E7 obtained in the above examples is as follows: Take 100 μL of the recombinant strain E7 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 E7 single colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 50 mL of fermentation medium. The fermentation medium was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, and 4.2 g / L 3-(N-morpholino)propanesulfonic acid to the initial culture medium containing 10 g / L ammonium acetate obtained in Example 1 above (i.e., the culture medium containing acetate (10 g / L acetate) obtained by fermentation pretreatment of industrial waste gas). Fermentation was carried out at 37°C, pH 6.8, and 220 rpm for 48 hours. Ammonium acetate could be added as needed during fermentation to supply more substrate for amino acid synthesis. After fermentation, the ammonium acetate utilization of recombinant bacteria E7 was determined by high performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant bacteria E7 was determined by spectrophotometry.

[0056] The ammonium acetate was obtained by filtering and concentrating the fermentation broth in Example 1, and then diluting it with deionized water to 10 g / L (ammonium acetate can also be obtained commercially available and diluted with deionized water to 10 g / L). After adding other components of the fermentation culture medium, it was sterilized at 115°C and autoclaved for 20 minutes. Glucose needs to be sterilized separately before being added.

[0057] Depend on Figure 5 It can be seen that the ammonium acetate utilization capacity, growth status and amino acid production capacity of different recombinant strains at the shake-flask level within 48 hours were as follows: E5 produced 10.91 g / L of aspartic acid, E6 produced 12.2 g / L of lysine, and E7 produced 12.5 g / L of threonine.

[0058] Example 5 The method for synthesizing amino acids using optimized culture conditions of the recombinant strain E5 obtained in the above examples: Take 100 μL of the recombinant strain E5 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 single E5 colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 50 mL of preferred fermentation medium, which was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, 4.2 g / L 3-(N-morpholino)propanesulfonic acid, 1 mg / L pantothenic acid, 1.5 g / L betaine, and 5 mL / L trace element solution to each liter of the initial culture medium containing 50 g / L ammonium acetate obtained in Example 1 above (i.e., the culture medium containing acetate (acetate content 50 g / L) obtained by fermentation pretreatment of industrial waste gas).

[0059] The formula for the 1000× trace element solution is as follows: 8.4 g / L ethylenediaminetetraacetic acid, 2.5 g / L cobalt chloride hexahydrate, 15 g / L manganese chloride tetrahydrate, 1.5 g / L copper chloride dihydrate, 3 g / L boric acid, 2.5 g / L sodium molybdate dihydrate, 100 g / L ferric citrate, and 13 g / L zinc acetate dihydrate.

[0060] Fermentation was carried out at 37℃, pH 6.8, and 220 rpm for 48 hours. During fermentation, ammonium acetate could be added as needed to supply more substrate for amino acid synthesis. After fermentation, the amount of ammonium acetate utilized by recombinant bacteria E5 was determined by high performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant bacteria E5 was determined by spectrophotometer.

[0061] The method for synthesizing amino acids using optimized culture conditions of the recombinant strain E6 obtained in the above examples: Take 100 μL of the recombinant strain E6 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 single E6 colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 50 mL of preferred fermentation medium, which was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, 4.2 g / L 3-(N-morpholino)propanesulfonic acid, 1 mg / L pantothenic acid, 1.5 g / L betaine, and 5 mL / L trace element solution to each liter of the initial culture medium containing 50 g / L ammonium acetate obtained in Example 1 above (i.e., the culture medium containing acetate (acetate content 50 g / L) obtained by fermentation pretreatment of industrial waste gas).

[0062] The formula for the 1000× trace element solution is as follows: 8.4 g / L ethylenediaminetetraacetic acid, 2.5 g / L cobalt chloride hexahydrate, 15 g / L manganese chloride tetrahydrate, 1.5 g / L copper chloride dihydrate, 3 g / L boric acid, 2.5 g / L sodium molybdate dihydrate, 100 g / L ferric citrate, and 13 g / L zinc acetate dihydrate.

[0063] Fermentation was carried out at 37℃, pH 6.8, and 220 rpm for 48 hours. During fermentation, ammonium acetate could be added as needed to supply more substrate for amino acid synthesis. After fermentation, the amount of ammonium acetate utilized by recombinant bacteria E6 was determined by high performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant bacteria E6 was determined by spectrophotometer.

[0064] The method for synthesizing amino acids using optimized culture conditions of the recombinant strain E7 obtained in the above examples: Take 100 μL of the recombinant strain E7 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 E7 single colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 50 mL of preferred fermentation medium, which was prepared by adding 10 g / L glucose, 15 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 2 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 0.02 g / L ferrous sulfate, 4.2 g / L 3-(N-morpholino)propanesulfonic acid, 1 mg / L pantothenic acid, 1.5 g / L betaine, and 5 mL / L trace element solution to each liter of the initial culture medium containing 50 g / L ammonium acetate obtained in Example 1 above (i.e., the culture medium containing acetate (acetate content 50 g / L) obtained by fermentation pretreatment of industrial waste gas).

[0065] The formula for the 1000× trace element solution is as follows: 8.4 g / L ethylenediaminetetraacetic acid, 2.5 g / L cobalt chloride hexahydrate, 15 g / L manganese chloride tetrahydrate, 1.5 g / L copper chloride dihydrate, 3 g / L boric acid, 2.5 g / L sodium molybdate dihydrate, 100 g / L ferric citrate, and 13 g / L zinc acetate dihydrate.

[0066] Fermentation was carried out at 37℃, pH 6.8, and 220 rpm for 48 hours. During fermentation, ammonium acetate could be added as needed to supply more substrate for amino acid synthesis. After fermentation, the amount of ammonium acetate utilized by recombinant bacteria E7 was determined by high performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant bacteria E7 was determined by spectrophotometer.

[0067] Depend on Figure 6 It can be seen that under optimized culture conditions, different recombinant strains exhibited varying levels of ammonium acetate utilization, growth status, and amino acid production capacity at the shake-flask level within 48 hours. The aspartic acid production of E5 was 14.9 g / L, the lysine production of E6 was 15.6 g / L, and the threonine production of E7 was 16.1 g / L.

[0068] Example 6 The fermentation and synthesis of amino acids by *E. coli* was scaled up. Recombinant strains E5, E6, and E7 were cultured in a 5 L reactor to obtain amino acids. Specifically... The method for synthesizing amino acids from the recombinant strain E5 obtained in the above examples: Take 100 μL of the recombinant strain E5 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 single E5 colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 150 mL of the preferred fermentation medium of Example 5 above containing antibiotics (20 μg / mL of chloramphenicol resistance was added to the preferred medium described above) and cultured at 220 rpm and 37°C for 10 hours. 150 mL of seed culture was inoculated into 3 L of optimized fermentation medium. The temperature was 37°C, dissolved oxygen was set to 30%, pH was set to 6.8, and the fermentation speed was 300-800 rpm, correlated with dissolved oxygen. During fermentation, ammonium acetate (200 g / L) obtained from the filtered, concentrated, and sterilized fermentation broth in Example 1 was used as a fed-batch carbon source to maintain acetate concentration in the medium at 5-10 g / L. After fermentation, the ammonium acetate utilization of recombinant strain E5 was determined by high-performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant strain E5 was determined by spectrophotometry.

[0069] The method for synthesizing amino acids from the recombinant strain E6 obtained in the above examples: Take 100 μL of the recombinant strain E6 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 single E6 colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 150 mL of optimized fermentation medium (containing 20 μg / mL chloramphenicol resistance) and cultured at 220 rpm and 37 °C for 10 hours. 150 mL of seed culture was inoculated into 3 L of optimized fermentation medium. The temperature was 37°C, dissolved oxygen was set to 30%, pH was set to 6.8, and the fermentation speed was 300-800 rpm, correlated with dissolved oxygen. During fermentation, ammonium acetate (200 g / L) obtained from the filtered, concentrated, and sterilized fermentation broth in Example 1 was used as a fed-batch carbon source to maintain acetate concentration in the medium at 5-10 g / L. After fermentation, the ammonium acetate utilization of recombinant strain E6 was determined by high-performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant strain E6 was determined by spectrophotometry.

[0070] The method for synthesizing amino acids from the recombinant strain E7 obtained in the above examples: Take 100 μL of the recombinant strain E7 from the cryopreservation tube, dilute it appropriately, streak it on LB solid medium (containing 20 μg / ml chloramphenicol resistance), and incubate it overnight at 37°C in a constant temperature incubator. Pick 3–5 E7 single colonies and incubate them in 5 mL LB liquid medium (containing 20 μg / ml chloramphenicol resistance) at 220 rpm and 37°C for 8 hours to activate the strain; 1 mL of seed culture was inoculated into 150 mL of the preferred fermentation medium of Example 5 above containing antibiotics (20 μg / mL of chloramphenicol resistance was added to the preferred medium described above) and cultured at 220 rpm and 37°C for 10 hours. 150 mL of seed culture was inoculated into 3 L of optimized fermentation medium. The temperature was 37°C, dissolved oxygen was set to 30%, pH was set to 6.8, and the fermentation speed was 300-800 rpm, correlated with dissolved oxygen. During fermentation, ammonium acetate (200 g / L) obtained from the filtered, concentrated, and sterilized fermentation broth in Example 1 was used as a fed-batch carbon source to maintain acetate concentration in the medium at 5-10 g / L. After fermentation, the ammonium acetate utilization of recombinant strain E7 was determined by high-performance liquid chromatography (HPLC), the types and yields of amino acids produced were determined by HPLC, and the growth density of recombinant strain E7 was determined by spectrophotometry.

[0071] Depend on Figure 7 Figure 8 It can be seen that, within 48 hours, the utilization capacity of ammonium acetate, the growth status, and the amino acid production capacity were as follows: E5 produced 112.4 g / L of aspartic acid, E6 produced 123.7 g / L of lysine, and E7 produced 124.5 g / L of threonine.

[0072] Although the present invention has been described in detail and specific embodiments have been elaborated above, those skilled in the art can still make some obvious modifications or improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention should be considered as part of the protection scope of the present invention.

Claims

1. A strain that synthesizes amino acids, characterized in that: Starting with Escherichia coli, Starting with Escherichia coli, genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathways were silenced, and key enzymes related to acetate assimilation pathways were overexpressed to obtain strains that synthesize amino acids. The relevant genes are silenced and / or overexpressed to obtain recombinant strains such as E5, E6, and E7. E5, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; E6. Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA and homoserine kinase gene thrB DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; E7, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA and homoserine kinase gene thrB and diaminopimelic acid decarboxylase gene lysA and homoserine O-succinyltransferase gene metA DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; The gene Fur The reference sequence number on NCBI is from position 2801 to position 3733 of NC_000913.3, where the 55th amino acid changes from V to E.

2. A method for constructing a strain capable of synthesizing amino acids as described in claim 1, characterized in that: Starting with Escherichia coli, genes related to the tricarboxylic acid cycle (TCA) and / or amino acid synthesis pathways were silenced, and key enzymes related to acetate assimilation pathways were overexpressed to obtain strains that synthesize amino acids. The relevant genes were silenced and / or overexpressed to obtain recombinant strains E5, E6, and E7 as follows: E5, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; E6. Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA and homoserine kinase gene thrB DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; E7, Knockout of isocitrate dehydrogenase gene icd DNA-binding transcriptional repressor genes iclR DNA-binding transcription dual regulator genes arcA and homoserine kinase gene thrB and diaminopimelic acid decarboxylase gene lysA and homoserine O-succinyltransferase gene metA DNA-binding transcription dual regulator genes Fur Point mutations were performed, and the gene containing the acetate kinase A gene from the E. coli genome was transformed. ackA and acetylphosphotransferase gene pta and phosphoenolpyruvate carboxylkinase gene pckA The E. coli plasmid pACYC-Duet-1 was co-constructed using the Gibson assembly method; The gene Fur The reference sequence number on NCBI is from position 2801 to position 3733 of NC_000913.3, where the 55th amino acid changes from V to E.

3. The application of the strain for synthesizing amino acids according to claim 1, characterized in that: The strains are used in the fermentation synthesis of amino acids using acetate as a carbon source; E5 is used to produce aspartic acid, E6 is used to produce lysine, and E7 is used to produce threonine.

4. A method for synthesizing amino acids, characterized in that: The strain of claim 1 is cultured in a medium with acetate as the carbon source and fermented at a temperature of 35-40℃, pH 6.5-6.9, dissolved oxygen of 25-40%, and a rotation speed of 300-800 rpm to obtain amino acids; E5 is used to produce aspartic acid, E6 is used to produce lysine, and E7 is used to produce threonine.

5. The amino acid synthesis method according to claim 4, characterized in that: The culture medium is prepared by adding 10-15 g / L glucose, 10-15 g / L ammonium sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L yeast extract, 1-2 g / L magnesium sulfate heptahydrate, 0.01-0.03 g / L ferrous sulfate, and 4.0-4.2 g / L 3-(N-morpholino)propanesulfonic acid per liter of acetate culture broth obtained from the fermentation pretreatment of industrial waste gas.

6. The amino acid synthesis method according to claim 4, characterized in that: The culture medium is prepared by adding 10-15 g / L glucose, 10-15 g / L ammonium sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L yeast extract, 1-2 g / L magnesium sulfate heptahydrate, 0.01-0.03 g / L ferrous sulfate, 4.0-4.2 g / L 3-(N-morpholino)propanesulfonic acid, 1-1.2 mg / L pantothenic acid, 1-1.5 g / L betaine, and 5 mL / L trace element solution to each liter of acetate-containing culture broth obtained from the fermentation pretreatment of industrial waste gas. The formula for the 1000× trace element solution is as follows: ethylenediaminetetraacetic acid 8-9 g / L, cobalt chloride hexahydrate 2-3 g / L, manganese chloride tetrahydrate 15-20 g / L, copper chloride dihydrate 1-2 g / L, boric acid 1-4 g / L, sodium molybdate dihydrate 2-3 g / L, ferric citrate 80-100 g / L, and zinc acetate dihydrate 13-15 g / L.

7. The amino acid synthesis method according to any one of claims 4-6, characterized in that: The acetate-containing culture broth obtained from the fermentation pretreatment of industrial waste gas is obtained by fermenting gas-eating acetic acid-producing bacteria in the culture broth containing industrial waste gas, followed by further concentration; among them, the gas-eating acetic acid-producing bacteria is Clostridium yunnanense (Clostridium yunnanense). Clostridium ljungdahlii Acetobacter wuerii ( Acetobacterium woodii ), Moorella mulderi , Thermoanaerobacter kivui, Clostridium autoethanogenum One or more of them.

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