Recombinant genetically engineered bacteria for producing l-homoserine, construction method and application thereof

Abstract

The present application relates to a kind of L-homoserine production of recombinant genetically engineered bacteria construction method, and its application in the microbial fermentation preparation L-homoserine in.In the present application, the beneficial effects mainly reflect: the present application provides a kind of L-homoserine production of recombinant genetically engineered bacteria and its construction method, and its application in the microbial fermentation preparation L-homoserine;L-homoserine is produced using the recombinant genetically engineered bacteria of the present application by fermentation method, raw material glucose is low in cost, mild condition, environment friendly;Using the engineering bacteria of the present application, L-homoserine production is significantly improved, and has good application prospect.

Description

(I)TECHNICAL FIELD

[0001] The present application relates to a recombinant genetically engineered bacterium producing L-homoserine and a construction method thereof, and application of the recombinant genetically engineered bacterium in microbial fermentation for preparing L-homoserine. (II)BACKGROUND

[0002] Homoserine is also known as 2-amino-4-hydroxybutyric acid, and is divided into two isomers of L-homoserine and D-homoserine, and belongs to the L-aspartate family of non-protein amino acids. L-homoserine and its derivatives are precursors for synthesizing essential amino acids L-threonine, L-methionine and L-isoleucine. L-homoserine and its derivatives are precursors for synthesizing L-methionine, and are also platform compounds for synthesizing various C4 compounds (isobutanol, gamma-butyrolactone, 1,4-butanediol, 2,4-dihydroxybutyric acid) and L-glufosinate. In the reported synthesis routes of L-homoserine and its derivatives, the chemical method is limited in development due to factors such as high price of reagents used in the reaction process, long reaction time or complicated purification steps. The enzyme catalysis method uses pyruvic acid and formaldehyde to generate homoserine under the joint action of acetalase and L-amino acid dehydrogenase. The formaldehyde required by the process is toxic, and the price of coenzyme is expensive. The microbial fermentation method has the advantages of green and efficient, and cheap and easily available substrate, and is concerned by people.

[0003] With the continuous understanding of microbial metabolic process and the continuous development of metabolic modification technology (systematic metabolic engineering, synthetic biology, promoter engineering, protein engineering and transcriptome and metabolome), it has broad production and application prospects to use cheap glucose as a carbon source to produce various high-value products including L-homoserine. However, due to the physiological characteristics of wild-type Escherichia coli, the L-homoserine synthesized in the Escherichia coli cells can only meet the needs of its own growth and cannot be accumulated in large quantities, so it is necessary to rationally modify the synthesis pathway of L-homoserine and construct a stable and efficient cell factory. The strain with high yield of L-homoserine can be obtained by genetic engineering and metabolic engineering strategies. However, in the process of constructing the genetically engineered bacteria, the correlation between different metabolic pathways needs to be considered, and the metabolic flux of the target product should not affect the normal growth of the cells, so rational metabolic engineering modification needs to be carried out from multiple angles. At present, there are still some deficiencies in the process of producing L-homoserine and its derivatives by biological method, such as low fermentation yield or low sugar acid conversion rate. Therefore, it is still a great challenge to construct an efficient microbial cell factory for producing L-homoserine and its derivatives. (III)SUMMARY

[0004] The present application aims to provide a recombinant genetically engineered bacterium producing L-homoserine and a construction method thereof, and application of the recombinant genetically engineered bacterium in microbial fermentation for preparing L-homoserine.

[0005] The technical scheme adopted by the present application is:

[0006] The recombinant genetically engineered bacteria for producing L-homoserine are obtained by the following method: taking strain E. coli W3110 (Trc-panD Trc-ppC Trc-aspC ΔpykA ΔcycA) (denoted as strain HS1) as a chassis strain, back-supplementing the original promoter of the panD gene, relieving the feedback inhibition of the thrA gene (thrA * ), knocking out the pcK gene, the thrB gene and the metA gene, replacing the promoters of the ppC gene, the nadK gene, the rhtA gene and the metL gene into the promoter Trc respectively, over-expressing thrA * , ppC and rhtA at the fliK, ompT and ylbE pseudogene sites in the E. coli W3110 genome, over-expressing thrA * and aspB (cg) genes on the plasmid pTrc99a, constructing the plasmid pTrc99a-thrA * -aspB (cg) , and transforming the aforementioned strain to obtain the recombinant genetically engineered bacteria for producing L-homoserine.

[0007] The present application reengineers the L-homoserine synthesis network of E. coli, relieves the feedback inhibition of the aspartate kinase / homoserine dehydrogenase I encoded by thrA, overcomes the deficiency of the activity of the aspartate kinase / homoserine dehydrogenase I encoded by thrA in the original strain due to the feedback inhibition, enables aspartate to successfully synthesize L-homoserine, increases the flux of aspartate converted into L-homoserine, and constructs the strain HS3; by strengthening the L-threonine / L-homoserine exporter gene rhtA, the intracellular concentration of L-homoserine is reduced, thereby increasing the concentration of L-homoserine in the fermentation broth, and the strain HS4 is constructed; by over-expressing thrA *Strain HS5 was constructed by enhancing the aspartate to L-homoserine synthesis pathway by replacing the original metL promoter of the L-homoserine synthesis pathway with the Trc promoter, thereby increasing the carbon flux in the L-homoserine synthesis pathway. Strain HS6 was constructed by overexpressing the ppC gene encoding phosphoenolpyruvate decarboxylase to enhance the metabolic flux of phosphoenolpyruvate to oxaloacetate, thus increasing the production of L-homoserine. Strain HS7 was constructed by overexpressing the ppC gene encoding phosphoenolpyruvate decarboxylase to enhance the metabolic flux of phosphoenolpyruvate to oxaloacetate, thus increasing the precursor supply for aspartate synthesis. Strain HS8 was constructed by knocking out pcK to reduce the reversible reaction of oxaloacetate to phosphoenolpyruvate, thereby better accumulating the precursor oxaloacetate of aspartate. The L-threonine / L-homoserine efflux protein gene rhtA was expressed to further enhance the efflux of L-homoserine, thereby improving the accumulation of the target product L-homoserine, and strain HS9 was constructed. Strain HS11 was constructed by knocking out the carbon flux of the competitive pathways of threonine and O-succinylhomoserine, i.e., knocking out thrB and metA to reduce the synthesis of threonine and O-succinylhomoserine in the competitive pathways, thereby increasing the carbon flux in the L-homoserine synthesis direction. Strain HS12 was constructed by knocking out the acetate production pathway, i.e., knocking out poxB to reduce carbon loss. Subsequently, the original promoter of the nadK gene was replaced with the Trc promoter to improve the availability of intracellular NADPH, providing sufficient NADPH for the L-homoserine synthesis pathway, and strain HS13 was constructed. Finally, thrA, which relieves feedback inhibition, was overexpressed on plasmid pTrc99a. * Genes and aspB derived from Corynebacterium glutamicum (cg) Genes were used to maximize the production of L-homoserine, and plasmid pTrc99a-thrA was constructed. * -aspB (cg) The strain was then transformed into strain HS13, resulting in the strain: E. coli W3110Trc-ppCTrc-aspCΔpykAΔcycAthra*Trc-rhtAΔfliK::Trc-thrA*Trc-metLΔompT::Trc-ppCΔpcKΔylbE::Trc-rhtAΔthrBΔmetAΔpoxB Trc-nadK / pTrc99a-thrA*-aspB (cg) The strain was named HS13-A. It overcomes the problem of insufficient expression of key genes in the genome, maximizes the carbon flow towards the synthesis of L-homoserine, and increases the yield of L-homoserine.

[0008] The present invention also relates to a method for constructing the genetically engineered bacteria, the method comprising:

[0009] (1) Take strain E. coli W3110 (Trc-panD Trc-ppC Trc-aspC ApykA AcycA) as the chassis strain, and use CRISPR-Cas9 gene editing technology to back up the original promoter of the panD gene to obtain the engineering strain HS2;

[0010] (2) Use CRISPR-Cas9 gene editing technology to remove the feedback inhibition of the thrA gene of the engineering strain HS2 to obtain the engineering strain HS3;

[0011] (3) Replace the original promoter of the rhtA gene on the genome of HS3 with the strong promoter Trc to obtain the engineering strain HS4;

[0012] (4) Use CRISPR / Cas9 gene editing technology to replace the fliK gene with the feedback inhibition removed thrA gene added with the Trc promoter to obtain the engineering strain HS5; *

[0013] (5) Replace the original promoter of the metL gene on the genome of the strain HS5 with the strong promoter Trc to obtain the engineering strain HS6;

[0014] (6) Use CRISPR / Cas9 gene editing technology to replace the ompT gene with the ppC gene added with the Trc promoter to obtain the engineering strain HS7;

[0015] (7) Knock out the pcK gene on the genome of the strain HS7 to obtain the engineering strain HS8;

[0016] (8) Use CRISPR / Cas9 gene editing technology to replace the ylbE gene with the rhtA gene added with the Trc promoter to obtain the engineering strain HS9;

[0017] (9) Knock out the thrB gene on the genome of the strain HS9 to obtain the engineering strain HS10;

[0018] (10) Knock out the metA gene on the genome of the strain HS10 to obtain the engineering strain HS11;

[0019] (11) Knock out the poxB gene on the genome of the strain HS11 to obtain the engineering strain HS12;

[0020] (12) Replace the original promoter of the nadK gene on the genome of the strain HS12 with the strong promoter Trc to obtain the recombinant engineering strain HS13;

[0021] (13) Overexpress the feedback inhibition removed thrA gene on the plasmid pTrc99a * ​Genes and aspartate transaminase genes derived from the source coryneform bacteria (cg) , construct plasmid pTrc99a-thrA * -aspB (cg) and transform into strain HS13 to obtain strain HS13-A, which is the L-homoserine-producing recombinant genetically engineered bacteria.

[0022] The nucleotide sequence of the panD gene is shown as SEQ ID NO. 1, the nucleotide sequence of the thrA gene is shown as SEQ ID NO. 2, the nucleotide sequence of the rhtA gene is shown as SEQ ID NO. 3, the nucleotide sequence of the metL gene is shown as SEQ ID NO. 4, the nucleotide sequence of the ppC gene is shown as SEQ ID NO. 5, the nucleotide sequence of the pcK gene is shown as SEQ ID NO. 6, the nucleotide sequence of the thrB gene is shown as SEQ ID NO. 7, the nucleotide sequence of the metA gene is shown as SEQ ID NO. 8, the nucleotide sequence of the poxB gene is shown as SEQ ID NO. 9, the nucleotide sequence of the nadK gene is shown as SEQ ID NO. 10, the nucleotide sequence of the aspB (cg) gene is shown as SEQ ID NO. 11, the nucleotide sequence of the Trc promoter is shown as SEQ ID NO. 12, and the nucleotide sequence of the thrA * gene is shown as SEQ ID NO. 13.

[0023] The application also relates to the use of the genetically engineered bacteria in the microbial fermentation preparation of L-homoserine.

[0024] Specifically, the use is as follows: inoculate the genetically engineered bacteria strain into a fermentation medium containing kanamycin, and perform fermentation culture under the conditions of 25-37℃ and 100-300rpm until OD 600 =0.5-1.0, add IPTG with a final concentration of 0.2mM, continue to culture for 48h, and obtain L-homoserine by separating and purifying the fermentation supernatant after the fermentation is completed.

[0025] The fermentation medium is composed of the following components: glucose 10-30 g / L, (NH4)2SO4 10-20 g / L, yeast extract 1-5 g / L, KH2PO4 0.5-2.0 g / L, MgSO4 0.5-2.0 g / L, CaCO3 10-20 g / L, 0.5-2 mL / L trace element solution, and solvent is water; the trace element solution is composed of the following components: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and solvent is deionized water.

[0026] Before fermentation, the recombinant genetically engineered bacteria are inoculated into LB medium, and then cultured at 37℃ and 180 rpm overnight to prepare seed liquid, and the seed liquid is inoculated into fermentation medium at a volume concentration of 5%.

[0027] The fermentation is carried out in a fermentation tank: the recombinant genetically engineered bacteria are inoculated into an LB plate containing 50 mg / L kanamycin, and then cultured at 37℃ overnight, and then a single colony is picked up into an LB test tube containing 50 mg / L kanamycin, and then cultured at 37℃ and 150 rpm overnight to prepare seed liquid; the seed liquid is inoculated into LB medium at a volume concentration of 5%, and then cultured at 37℃ and 150 rpm overnight to obtain secondary seed liquid; the secondary seed liquid is inoculated into a 5L fermentation tank containing 2L fermentation medium at a volume concentration of 15%, and then 0.2 mM IPTG is added, and then the fermentation culture is carried out at 30℃, 500 rpm, and a ventilation amount of 0.5 V / V·min, and when the pH value is higher than 6.80 (the initial sugar consumption in the fermentation tank is completed), automatic feeding is started, and then the feeding medium is added until the pH value is lower than 6.80, and then the feeding is stopped, and then the culture is carried out for 117 h to obtain fermentation liquid containing L-homoserine; the feeding medium is composed of the following components: glucose 500 g / L, (NH4)2SO4 16 g / L, KH2PO4 12.5 g / L, NaHCO3 10 g / L, threonine 4 g / L, and methionine 0.5 g / L, and solvent is water, and 50% ammonia water is used to adjust the pH value to 6.8.

[0028] The feeding speed is 25 mL / h, and the total addition amount of the feeding medium is 1000 mL / 2L.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] Among the reported synthetic routes of L-homoserine and its derivatives, the chemical method is limited by the expensive reagents, long reaction time or complicated purification steps; the enzyme catalysis method uses pyruvic acid and formaldehyde to generate homoserine under the joint action of acetalase and L-amino acid dehydrogenase, but the raw material formaldehyde is toxic and the coenzyme is expensive; the microbial fermentation method for producing L-homoserine has low cost of raw material glucose, mild conditions and environmental friendliness. + NAD 600 ) is an essential coenzyme involved in many physiological processes, and L-aspartate is the direct precursor of L-homoserine. Additional 2mol NADPH is needed to ensure that the enzyme converting L-aspartate into L-homoserine has sufficient reducing power, and strengthening nadK can increase the availability of NADPH, which can effectively improve the production of L-homoserine. After strengthening nadK, the yield of L-homoserine is obviously improved, and the yield of L-homoserine is increased by 24%. pcK encodes phosphoenolpyruvate kinase, and knockout pcK can block the anaplerotic pathway, reduce the carbon loss of the anaplerotic pathway, effectively accumulate aspartate precursor oxaloacetate, and activate the glyoxylate cycle. After knocking out pcK, the yield of L-homoserine is increased by 20%.

[0031] The beneficial effects of the present application mainly include: the present application provides a recombinant genetically engineered bacterium for producing L-homoserine and a construction method thereof, and its application in microbial fermentation for preparing L-homoserine; the recombinant genetically engineered bacterium is used to produce L-homoserine by fermentation, which has low cost of raw material glucose, mild conditions and environmental friendliness; the yield of L-homoserine is significantly improved by using the engineered bacterium, which has good application prospect. (IV) DESCRIPTION OF DRAWINGS

[0032] Figure 1 The column chart of biomass OD600 and L-homoserine concentration of strains HS7-A and HS8-A, strains HS12-A and HS13-A.

[0033] Figure 2 The column chart of biomass OD600 and L-homoserine concentration of strains HS7-A and HS8-A, strains HS12-A and HS13-A. (V) PREFERRED EMBODIMENTS

[0034] The present application will be further described below in conjunction with specific embodiments, but the protection scope of the present application is not limited to this:

[0035] In the embodiments, the final concentration of kanamycin in the liquid medium and the solid medium is 50 mg / L, the final concentration of spectinomycin is 50 mg / L, and the final concentration of IPTG is 0.2 mM.

[0036] Strain E. coli W3110 (Trc-panD Trc-ppCTrc-aspC ΔpykA ΔcycA) (i.e., HS1) was constructed according to Li, B., Zhang, B., Wang, P., Cai, X., Chen, Y. Y., Yang, Y. F., Liu, Z. Q., Zheng, Y. G., 2022. Rerouting fluxes of the central carbon metabolism and relieving mechanism-based inactivation of L-aspartate-α-decarboxylase for fermentative production of β-alanine in Escherichia coli.

[0037] Table 1: Genes involved in strain modification and their corresponding pathways

[0038]

[0039] Table 2: Primers used in gene editing

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046] Example 1: Determination of L-homoserine concentration

[0047] CNBF solution preparation: 0.1894 g CNBF was dissolved in 10 mL acetonitrile.

[0048] Boric acid buffer preparation: (0.2 mol / L boric acid (H3BO3, M1) solution and 0.05 mol / L borax (Na2B4O7, M2).

[0049] Sample treatment: The sample concentration was diluted with ultrapure water to between 0.1-1 g / L.

[0050] Reaction conditions: 100 μL of sample, 500 μL of boric acid buffer and 300 μL of CNBF solution were taken respectively, and incubated at 60°C, 400 rpm for 60 min, and then filtered (polyvinylidene fluoride, organic membrane, 0.22 μm) for standby.

[0051] Detection conditions: HPLC model: Thermo Scientific Utimate 3000, HPLC detection wavelength is 250 nm. Gradient elution program was used to separate L-homoserine, in which the mobile phase A component was ultrapure water: acetonitrile: glacial acetic acid: triethylamine = 828: 170: 3: 2 (v:v:v:v); the mobile phase B component was pure acetonitrile. The elution program was 0-2.5 min 18% A, 82% B; 2.5-4.5 min A 18%→19%, B 80%→81%; 4.5-7 min A 19%→35%, B 81%→65%; 7-8 min A 35%→36% B 65%→64%; 8-12 min A 36%→50% B 65%→50%; 12-15 min A 50%-51% B 50%→49%; 15-18 min A 51%→70% B 49%→30%; 18-23 min A 70%→18% B 30%→82%.

[0052] Example 2: Construction of strains HS2-HS11

[0053] 1. Construction of strain HS2

[0054] Using CRISPR / Cas9 gene editing technology, the panD (encoding aspartate decarboxylase) 99A-Trc promoter of HS1 as the starting strain was replaced with the original promoter to reduce the competitive consumption of the precursor aspartate, and the specific operation was as follows:

[0055] (1) Construct pTarget-panD plasmid: with plasmid pTarget as template, pTarget-panD-F / pTarget-panD-R as primers to amplify, add Dpn I to the PCR product after 37℃ incubation for 1h, purify by Clean up kit, then transform into E. coli DH5α, spread on LB solid plate containing 50mg / L spectinomycin, 30℃ inverted culture for 20h, preliminary verification by colony PCR, then verify the correctness of pTarget-panD plasmid by sequencing, obtain plasmid pTarget-panD. Then take pTD-line-F / pTD-line-R as primers, pTarget-panD as template to amplify, add Dpn I to the PCR product after 37℃ incubation for 3h, recover the DNA fragment by Clean up kit, obtain linearized plasmid pTarget-panD.

[0056] (2) Construct pTD-panD plasmid containing Donor: take the genome of E. coli W3110 as template, L-panD-F / L-panD-R as primers to amplify the upstream homologous arm F1; take R-panD-F / R-panD-R as primers to amplify the downstream homologous arm R1, take L-panD-F / R-panD-R as primers to obtain the upstream and downstream homologous arms, recover the DNA fragment by Clean up kit, obtain the fused fragment. Then construct pTD-panD plasmid by one-step cloning method with the fused fragment and linearized plasmid pTarget-panD of step (1), transform into E. coli DH5α, spread on LB solid plate containing 50mg / L spectinomycin, 30℃ inverted culture for 20h, preliminary screening by colony PCR, finally verify the correctness of pTD-panD plasmid by sequencing.

[0057] (3) Transform the competent cell HS1 with plasmid pCas9, spread on LB solid plate containing 50mg / L kanamycin, 30℃ overnight culture, pick single colony to LB test tube culture medium containing 50mg / L kanamycin resistance, 30℃ overnight culture. Then inoculate into 100mL LB culture medium with 1% volume concentration, add 50mg / L final concentration of kanamycin resistance and 10mM L-arabinose, 180rpm, 30℃ culture until OD 600 =0.5, 4℃, 4000rpm centrifugation. Wash twice with 4℃ cold ultrapure water, once with 10% cold glycerol, finally resuspend with 10% glycerol, aliquot and store, obtain electrotransformation competent cells for standby.

[0058] (4) Take 2 μL of the pTD-panD plasmid constructed in step (2) and mix it with 100 μL of the electrocompetent cells prepared in step (3). Transfer the mixture into a 2 mm electroporation cuvette, incubate on ice for 45 s, and then electroporate using a MicroPluser. TM BIO-RAD electroporation was performed at a voltage of 2500V. Immediately after electroporation, 700μL of pre-cooled LB medium at 4℃ was added and mixed thoroughly. The mixture was then immediately transferred to a new sterile 1.5mL EP tube and cultured at 30℃ and 150rpm for 3 hours. The culture was then spread on LB solid medium containing 50mg / L kanamycin and 50mg / L spectinomycin and incubated upside down at 30℃ for 24 hours. Colony PCR was performed using T-panD-F / T-panD-R primers to verify the construction of the strain, and sequencing was used to verify the correctness of the strain construction. The strain HS2 was successfully constructed.

[0059] (5) Elimination of pTarget and pCas9 plasmids: Positive single colonies from step (4) are picked and inoculated into LB liquid medium containing 2 mM IPTG and 50 mg / L kanamycin. The culture is incubated overnight at 30°C. The bacterial culture is then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 30°C for 20 hours until single colonies appear. Single colonies are then picked onto LB solid medium containing 50 mg / L spectinomycin and incubated upside down at 30°C for 20 hours. If no single colonies appear, the pTarget plasmid has been eliminated from this strain. The strain with the pTarget plasmid eliminated is then inoculated into antibiotic-free LB liquid medium tubes and incubated at 42°C for 10 hours. The bacterial culture is then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 37°C for 10 hours. If no single colonies appear, the pCas9 plasmid has been successfully eliminated, resulting in the plasmid-free strain HS2.

[0060] 2. Construction of strain HS3

[0061] Using HS2 as the starting strain, CRISPR / Cas9 gene editing technology was employed to remove feedback inhibition from the thrA gene (encoding aspartate kinase / homoserine dehydrogenase I), overcoming the deficiency of insufficient activity of the original aspartate decarboxylase. The specific operation is as follows:

[0062] (1) Construct pTarget-thrA * Plasmid: Using plasmid pTarget as a template, pTarget-thrA * -F / pTarget-thrA *R is primer amplification, PCR product is added Dpn I after incubation at 37°C for 1 h, purified by Clean up kit, and then transformed into E. coli DH5α, coated on LB solid plate containing 50 mg / L spectinomycin, 30°C inverted culture for 20 h, colony PCR is used for preliminary verification, and then sequencing is used for verifying pTarget-thrA * The correctness of the plasmid is verified, and the plasmid pTarget-thrA is obtained * . Then, pTD-line-F / pTD-line-R is used as a primer, and the plasmid pTarget-thrA * is used as a template for amplification. After the PCR product is added Dpn I and incubated at 37°C for 3 h, the DNA fragment is recovered by Clean up kit, and the linearized plasmid pTarget-thrA * is obtained.

[0063] (2) Constructing plasmid pTD-thrA containing Donor * : The genome of E. coli W3110 is used as a template, and L-thrA * -F / L-thrA * -R is used as a primer to amplify the upstream homologous arm F1; R-thrA * -F / R-thrA * -R is used as a primer to amplify the downstream homologous arm R1. The upstream and downstream homologous arms F1 and R1 are used as templates, and L-thrA * -F / R-thrA * -R is used as a primer to fuse the upstream and downstream homologous arms. The DNA fragment is recovered by Clean up kit, and the fused fragment is obtained. Then, the fused fragment and the linearized plasmid pTarget-thrA * of step (1) are used to construct plasmid pTD-thrA by one-step cloning method * , transformed into E. coli DH5α, coated on LB solid plate containing 50 mg / L spectinomycin, 30°C inverted culture for 20 h, colony PCR is used for preliminary screening, and finally the correctness of plasmid pTD-thrA * is verified by sequencing.

[0064] (3) The plasmid pCas9 is transformed into competent cells HS2, coated on LB solid plate containing 50 mg / L kanamycin, 30°C overnight culture, and single colonies are picked into LB test tube medium containing 50 mg / L kanamycin resistance, 30°C overnight culture. Then, 1% of the volume concentration is inoculated into 100 mL of LB medium, and 50 mg / L of kanamycin resistance and 10 mM of L-arabinose are added, and the culture is carried out at 180 rpm, 30°C until OD600 =0.5, centrifuged at 4℃ and 4000rpm. Washed twice with cold ultrapure water at 4℃, then washed once with cold 10% glycerol, and finally resuspended in 10% glycerol, aliquoted and stored to obtain electrocompetent cells for later use.

[0065] (4) Take 2 μL of the pTD-thrA constructed in step (2). * The plasmid was mixed with 100 μL of electrocompetent cells prepared in step (3), transferred into a 2 mm electroporation cuvette, incubated on ice for 45 s, and then electroporated using a MicroPluser. TM BIO-RAD electroporation was performed at a voltage of 2500V. Immediately after electroporation, 700μL of pre-cooled LB medium at 4℃ was added, mixed thoroughly, and immediately transferred to a new sterile 1.5mL EP tube. The tube was incubated at 30℃ with shaking at 150rpm for 3 hours. The mixture was then spread onto LB solid medium containing 50mg / L kanamycin and 50mg / L spectinomycin, and incubated upside down at 30℃ for 24 hours. The T-thrA... * -F / T-thrA * -R is for primer colony PCR verification, and sequencing is used to verify the correctness of the strain construction. The strain HS3 was successfully constructed.

[0066] (5) Elimination of pTarget and pCas9 plasmids: A single positive colony from step (4) is picked and inoculated into a test tube containing 2 mM IPTG and 50 mg / L kanamycin. The culture is incubated overnight at 30°C. The bacterial culture is then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 30°C for 20 hours until a single colony appears. A single colony is then picked and incubated onto LB solid medium containing 50 mg / L spectinomycin and incubated upside down at 30°C for 20 hours. If no single colony appears, the pTarget plasmid has been eliminated from this strain. The strain with the pTarget plasmid eliminated is then inoculated into an antibiotic-free LB liquid medium test tube and incubated at 42°C for 10 hours. The bacterial culture is then streaked onto LB solid medium containing 50 mg / L kanamycin and incubated upside down at 37°C for 10 hours. If no single colony appears, the pCas9 plasmid has been successfully eliminated, resulting in the plasmid-free strain HS3.

[0067] 2. Construction of strain HS4

[0068] (1) Construction of pTarget-Trc-rhtA plasmid: pTarget as template, pTarget-Trc-rhtA-F / pTarget-Trc-rhtA-R as primers, PCR amplification, add Dpn I to PCR product, 37°C for 1h to eliminate methylated plasmid template. Transform into E. coli DH5α competent cells, spread on LB solid medium containing 50mg / L spectinomycin, 30°C for 20h until single colony appears, colony PCR for preliminary verification, finally sequencing to verify the correctness of the constructed plasmid. Linearized plasmid pTarget-Trc-rhtA was prepared by the method of step 1.

[0069] (2) Construction of plasmid pTD-Trc-rhtA: E. coli W3110 genome as template, L-ΔfliK::Trc-thrA * -F / L-ΔfliK::Trc-thrA * -R and R-Trc-rhtA-F / R-Trc-rhtA-R as primers, PCR amplification to obtain the upper and lower homology arms, and the upper and lower homology arms were introduced into the linearized plasmid pTarget-Trc-rhtA by the method of step 1 to obtain the plasmid pTD-Trc-rhtA.

[0070] (3) Using the method of step 1, the pCas9 plasmid was introduced into the strain HS3 of step 1 and the electrotransformation competent cells were prepared.

[0071] (4) Using the method of step 1, the plasmid pTD-Trc-rhtA was introduced into the electrotransformation competent cells of step (3) to construct the strain HS4.

[0072] (5) Plasmid elimination: using the method of step 1 to eliminate the plasmid in the strain of step (4) to obtain the plasmid-free strain HS4.

[0073] 3. Construction of strains HS5 and HS6

[0074] (1) Construction of pTarget-ΔfliK::Trc-thrA * and pTarget-metL plasmids: pTarget as template, pTarget-Trc-ΔfliK::Trc-thrA * -F / pTarget-ΔfliK::Trc-thrA *-R and pTarget-metL-F / pTarget-metL-R are primers for PCR amplification to obtain the corresponding plasmids, which are digested by Dpn I at 37°C for 2 hours, and then transformed into E. coli DH5a, inoculated into LB solid medium containing 50 mg / L spectinomycin, and cultured at 30°C to screen, and then verified by colony PCR, and finally verified by sequencing to confirm the correctness of plasmid construction, thereby successfully constructing plasmids pTarget-ΔfliK::Trc-thrA * and pTarget-metL. The linearized plasmids pTarget-ΔfliK::Trc-thrA * and pTarget-metL are prepared by the method of step 1.

[0075] (2) Construction of pTD-ΔfliK::Trc-thrA * and pTD-metL plasmids: The E. coli W3110 genome is used as a template, and L-ΔfliK::Trc-thrA * -F / L-ΔfliK::Trc-thrA * -R, ΔfliK::Trc-thrA * -F / ΔfliK::Trc-thrA * -L, R-ΔfliK::Trc-thrA * -F / R-ΔfliK::Trc-thrA * -R and L-metL-F / L-metL-R, R-metL-F / R-metL-R are primers for amplifying the corresponding upstream and downstream homologous arms, and the construction steps are shown in step 1, thereby obtaining the corresponding plasmids pTD-ΔfliK::Trc-thrA * and pTD-metL.

[0076] (3) The plasmid pCas9 is introduced into the strain HS4 of step 2 and the electrocompetent cells are prepared by the method of step 1.

[0077] (4) The pTD-ΔfliK::Trc-thrA * is transformed into the electrocompetent cells of step (3) to obtain the HS5 positive colonies.

[0078] (5) Plasmid elimination: The method of step 1 is implemented to obtain the plasmid-free strain HS5.

[0079] (6) The pTD-metL is electrotransformed into the strain HS5 of step (5) according to the methods of steps (3)-(5) to construct the plasmid-free strain HS6.

[0080] 4. Construction of strains HS7, HS8, and HS9

[0081] (1) Construction of pTarget-ΔompT::Trc-ppC, pTarget-pcK, and pTarget-ΔylbE::Trc-rhtA plasmids: PCR amplification of the corresponding plasmids was performed using pTarget as the template and pTarget-ΔompT::Trc-ppC-F / pTarget-ΔompT::Trc-ppC-R, pTarget-pcK-F / pTarget-pcK-ppC-R, and pTarget-ΔylbE::Trc-rhtA-F / pTarget-ΔylbE::Trc-rhtA-R as primers, respectively. After addition of Dpn I, the mixture was incubated at 37°C for 2 h to digest the methylated plasmid template, and then transformed into E. coli DH5α. The transformants were inoculated into LB solid medium containing 50 mg / L spectinomycin and cultured at 30°C to select for transformants. Colony PCR was used to preliminarily verify the transformants, and sequencing was used to verify the correctness of the plasmid construction. The plasmids pTarget-ΔompT::Trc-ppC, pTarget-pcK, and pTarget-ΔylbE::Trc-rhtA were successfully constructed. Linearized plasmids pTarget-ΔompT::Trc-ppC, pTarget-pcK, and pTarget-ΔylbE::Trc-rhtA were prepared using the method of step 1.

[0082] (2) Construction of pTD-ΔompT::Trc-ppC, pTD--pcK, and pTD-ΔylbE::Trc-rhtA plasmids: The corresponding upstream and downstream homology arms and replacement gene fragments were amplified using E. coli W3110 genomic DNA as the template and L-ΔompT::Trc-ppC-F / L-ΔompT::Trc-ppC-R, R-Trc-ΔompT::Trc-ppC-F / R-ΔylbE::Trc-rhtA-R, ppC-F / R, L-pcK-F / L-pcK-R, R-pcK-F / R-pcK-R, L-ΔylbE::Trc-rhtA-F / L-ΔylbE::Trc-rhtA-R, R-ΔylbE::Trc-rhtA-F / R-ΔylbE::Trc-rhtA-R, and rhtA-F / R as primers, respectively. The construction was performed according to step 1, and the corresponding plasmids pTD-ΔompT::Trc-ppC, pTD-pTD-pcK, and pTD-ΔylbE::Trc-rhtA were obtained.

[0083] (3) Using the method of step 1, the plasmid pCas9 is introduced into the strain HS6 of step 2 and the electrotransformation competent cells are prepared.

[0084] (4) Using the method of step 1, the pTD-ΔompT::Trc-ppC is transformed into the electrotransformation competent cells of step (3) to obtain the positive colonies of HS7.

[0085] (5) Plasmid elimination: the method of step 1 is implemented to obtain the plasmid-free strain HS7.

[0086] (6) According to the method of step (3)-step (5), the pTD-pcK is electrotransformed into the strain HS7 of step (5) to obtain the plasmid-free strain HS8.

[0087] (7) According to the method of step (3)-step (5), the pTD-ΔylbE::Trc-rhtA is electrotransformed into the strain HS8 of step (6) to obtain the plasmid-free strain HS9.

[0088] 5. Construction of strains HS10 and HS11

[0089] (1) Construction of pTarget-thrB and pTarget-metA plasmids: pTarget is used as a template, and pTarget-thrB-F / pTarget-thrB-R and pTarget-metA-F / pTarget-metA-R are used as primers to amplify the corresponding plasmids by PCR, respectively. After adding Dpn I, the plasmid template is digested at 37°C for 2h, and then transformed into E. coli DH5α and inoculated into LB solid medium containing 50mg / L spectinomycin. After 30°C culture and screening, colony PCR is used for preliminary verification, and sequencing is used to verify the correctness of the plasmid construction. The plasmids pTarget-thrB and pTarget-metA are successfully constructed. The linearized plasmids pTarget-thrB and pTarget-metA are prepared by the method of step 1.

[0090] (2) Construction of pTD-thrB and pTD-metA plasmids: E. coli W3110 genome is used as a template, and L-thrB-F / L-thrB-R, R-thrB-F / R-thrB-R, L-metA-F / L-metA-R, and R-metA-F / R-metA-R are used as primers to amplify the corresponding upper and lower homologous arms, respectively. The construction is shown in step 1, and the corresponding plasmids pTD-thrB and pTD-metA are constructed.

[0091] (3) Using the method of step 1, the plasmid pCas9 is introduced into the strain HS9 and the electrotransformation competent cells are prepared.

[0092] (4) Using the method of step 1, pTD-thrB was transformed into the electrocompetent cells of step (3) to obtain the HS10 positive colonies.

[0093] (5) Plasmid elimination: the method of step 1 was implemented to obtain the plasmid-free strain HS10.

[0094] (6) pTD-metA was electrotransformed into the strain HS10 of step (5) according to the method of step (3) to step (5) to construct the plasmid-free strain HS11.

[0095] Example 3: Knockout of poxB gene in acetic acid synthesis pathway to construct strain HS12

[0096] (1) Construction of plasmid pTarget-poxB: pTarget-poxB-F / pTarget-poxB-R were used as primers and pTarget plasmid as template. The PCR product was added with Dpn I to digest the methylated vectors, and then purified and transformed into E. coli-DH5a competent cells. The cells were inoculated on LB plates containing 50 mg / L spectinomycin, and screened at 30°C. The colonies were verified by PCR and sequencing to confirm the correctness. The plasmid pTarget-poxB was used as template and pTD-line-F / pTD-line-R as primers to amplify, and the PCR product was added with Dpn I and incubated at 37°C for 1 h. The purified product was recovered by Clean up kit and used for preparation of linearized plasmid pTarget-poxB.

[0097] (2) Construction of plasmid pTD-poxB: E. coli W3110 genome was used as template and L-poxB-F / L-poxB-R and R-poxB-F / R-poxB-R as primers to amplify the upper and lower homologous arms F1 and R1. The PCR products were recovered and purified by Clean up kit and used for preparation. The upper and lower homologous arms were transformed into linearized plasmid pTarget-poxB by one-step cloning method to construct plasmid pTD-poxB, which was transformed into E. coli DH5a competent cells. The cells were inoculated on LB plates containing 50 mg / L spectinomycin and cultured at 30°C. The colonies were screened by colony PCR and finally verified by sequencing to confirm the correctness of plasmid pTD-poxB.

[0098] (3) pCas9 plasmid was introduced into the strain HS11 constructed in Example 2, and coated on LB solid medium containing 50 mg / L kanamycin and incubated at 30°C overnight. Single colonies were picked into LB test tube medium containing 50 mg / L kanamycin and incubated at 30°C overnight. Then, 1% (v / v) of the inoculum was inoculated into 100 mL of LB medium, and 50 mg / L kanamycin and 10 mM L-arabinose were added to a final concentration, and incubated at 180 rpm and 30°C until OD 600 = 0.5, centrifuged at 4°C and 4000 rpm. The precipitate was washed twice with 4°C ultrapure water and once with 10% cold glycerol to obtain the strain HS11 electrotransformation competent cells, which were finally resuspended with 10% glycerol and stored at -80°C.

[0099] (4) 2 μL of the pTD-poxB plasmid constructed in step (3) was mixed with 100 μL of the HS11 electrotransformation competent cells prepared in step (3), and transferred into a 2-mm electroporation cup, and ice-bathed for 45 s. The cells were electroporated with an electroporator (MicroPluser TM , BIO-RAD) at a voltage of 2500 V. After the electroporation, 700 μL of 4°C pre-cooled LB medium was immediately added, and the mixture was immediately transferred to a new sterile 1.5 mL EP tube and incubated at 30°C and 150 rpm for 3 h. The cells were coated on LB solid medium containing 50 mg / L kanamycin and 50 mg / L spectinomycin and incubated at 30°C for 24 h until single colonies appeared. The colonies were verified by colony PCR with T-poxB-F / T-poxB-R primers and sequencing to confirm the correctness of the strain construction. The strain HS12 was successfully constructed.

[0100] (5) Elimination of plasmids pTarget and pCas9: positive single colonies were inoculated into LB test tubes containing 2 mM IPTG and 50 mg / L kanamycin and incubated at 30°C overnight. The bacterial solution was dipped and streaked on LB solid medium containing 50 mg / L kanamycin and incubated at 30°C for 20 h until single colonies appeared. The single colonies were picked and streaked on LB solid medium containing 50 mg / L spectinomycin and incubated at 30°C for 20 h. If no single colonies appeared, it indicated that the pTarget plasmid had been eliminated in the strain. The strain in which the pTarget plasmid was eliminated was inoculated into LB test tubes without antibiotics and incubated at 42°C for 10 h. The bacterial solution was dipped and streaked on LB solid medium containing 50 mg / L kanamycin and incubated at 37°C for 10 h. If no single colonies appeared, it indicated that the pCas9 plasmid was successfully eliminated. Finally, the plasmid-free strain HS12 was obtained.

[0101] Example 4: Strengthening the NADPH supply pathway nadK to construct strain HS13

[0102] (1) Construction of plasmid pTarget-nadK: pTarget plasmid as template, pTarget-nadK-F / pTarget-nadK-R as primers, PCR product was added Dpn I to digest the methylation of the carrier, purified and transformed into E. coli DH5α competent cells. Inoculated into LB plate containing 50 mg / L spectinomycin resistance, 30°C culture screening, colony PCR verification, and sequencing to verify its correctness. The method of Example 2 was used to prepare linearized plasmid pTarget-nadK.

[0103] (2) Construction of plasmid pTD-nadK: E. coli W3110 genome as template, L-nadK-F / L-nadK-R and R-nadK-F / R-nadK-R as primers to amplify the upper and lower homologous arms. The method of Example 2 was used to transfer the upper and lower homologous arms into the linearized plasmid pTarget-nadK of step (1) to construct plasmid pTD-nadK.

[0104] (3) The method of Example 1 was used to introduce pCas9 plasmid into strain HS12 in Example 3 and prepare competent cells.

[0105] (4) The method of Example 1 was used to transfer plasmid pTD-nadK into the competent cells of step (3) to construct strain HS12.

[0106] (5) Plasmid elimination: the method of Example 2 was used to obtain plasmid-free strain E. coli W3110 (Trc-ppC Trc-aspC ΔpykA ΔcycA thrA ΔfliK::Trc-thrA * Trc-rhtA ΔfliK::Trc-thrA * Trc-metL ΔompT::Trc-ppC ΔpcK ΔylbE::Trc-rhtA ΔthrB ΔmetA ΔpoxB Trc-nadK), denoted as HS13.

[0107] Example 5: Construction of overexpression plasmid pTrc99a-thrA * -aspB (cg) and construction of strains HS7-A, HS8-A, HS12-A, HS13-A and shake flask fermentation

[0108] (1) Plasmid pTrc99a-thrA * -aspB (cg)Construction: The plasmid pTrc99a was used as a template, and the pTrc99a-line-F / pTrc99a-line-R primers were used for PCR. The methylated plasmid was digested with Dpn I in the PCR product, and the linearized plasmid pTrc99a was prepared by recovery and purification. The HS3 strain and the genome of Corynebacterium glutamicum ATCC 13032 were used as templates, and the thrA * -F / thrA * -R and aspB (cg) -F / aspB (cg) -R primers, and the thrA * (SEQ ID NO. 13) and aspB (cg) (SEQ ID NO. 11) fragments were cloned by PCR, and were recovered and purified. According to the instructions of the One Step Cloning Kit, the linearized plasmid pTrc99a, the thrA * and aspB (cg) fragments were ligated, and the ligation product was transformed into E. coli DH5α competent cells to construct the vector pTrc99a-thrA * -aspB (cg) . Colony PCR was used to preliminarily verify its correctness, and sequencing was used to finally determine that the plasmid construction was successful.

[0109] (2) Preparation of strain HS7, HS8, HS12, HS13 competent cells. The preparation method of the competent cells is as shown in Example 2.

[0110] (3) The plasmid pTrc99a-thrA * -aspB (cg) was transformed into the strain HS7, HS8, HS12, HS13 competent cells to construct the strain HS7 / pTrc99a-thrA * -aspB (cg) , HS8 / pTrc99a-thrA * -aspB (cg) , HS12 / pTrc99a-thrA * -aspB (cg) , and HS13 / pTrc99a-thrA * -aspB (cg) , which were designated as the strains HS7-A, HS8-A, HS12-A, and HS13-A.

[0111] (4) The constructed strains HS8-A and HS13-A were verified in fermentation medium, and strains HS7-A and HS12-A were used as control strains. Four single colonies were respectively picked into LB test tube medium, and cultured at 37°C, 180 rpm overnight to prepare seed liquid. 1 mL seed liquid was inoculated into a 500 mL shake flask containing 20 mL fermentation medium, and cultured at 30°C, 180 rpm until OD 600 = 0.5, and 0.2 mM IPTG was added to a final concentration, and the culture was cultured at 30°C, 180 rpm for 48 h. After fermentation, 1 mL fermentation liquid was centrifuged at 12000 rpm for 3 min, and all supernatant was discarded. 1 mL distilled water was added to resuspend the bacteria and calcium carbonate, and the mixture was centrifuged at 12000 rpm for 3 min to discard the supernatant. 1 mL distilled water was added again to resuspend the bacteria and calcium carbonate, and the mixture was centrifuged at 12000 rpm for 3 min to discard the supernatant. Finally, 800 μL distilled water was added to resuspend the bacteria and calcium carbonate, and 200 μL acetic acid aqueous solution with a volume concentration of 20% was added. The mixture was placed at room temperature for 5 min to dissolve the calcium carbonate. 50 μL bacteria liquid in which calcium carbonate was dissolved was added to 1950 μL distilled water to dilute 40 times, and the biomass OD 600 was determined by spectrophotometer. The method of Example 1 was used, and 1 mL fermentation liquid was centrifuged at 12000 rpm for 3 min. 100 μL supernatant was added to 900 μL deionized water to dilute 10 times. 100 μL diluted sample was taken, 500 μL boric acid buffer and 300 μL CNBF solution were added, and the mixture was incubated at 60°C, 400 rpm for 60 min. The membrane was passed, and the content of L-homoserine was detected. The biomass OD 600 and the content of L-homoserine are shown in Table 1. Figure 1 The biomass OD 600 of strain HS7-A was 23, and the content of L-homoserine was 6.5 g / L. The biomass OD 600 of strain HS8-A was 22.7, and the content of L-homoserine was 7.8 g / L. The biomass OD 600 of strain HS12-A was 14.11, and the content of L-homoserine was 11.5 g / L. The biomass OD 600 of strain HS13-A was 15.62, and the content of L-homoserine was 14.2 g / L.

[0112] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water, and the pH value is natural.

[0113] Fermentation medium: glucose 20 g / L, (NH4)2SO4 17 g / L, yeast extract 4 g / L, KH2PO4 1 g / L, MgSO4 1 g / L, CaCO3 15 g / L, 1 mL / L trace element solution, without pH adjustment. The composition of the trace element solution is as follows: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, with deionized water as solvent.

[0114] By Figure 1 It can be seen that, compared with strain HS7-A, the yield of L-homoserine of strain HS8-A is increased from 6.5 g / L to 7.8 g / L, and the yield of L-homoserine is increased by 20%, so it can be seen that increasing the supply of NADPH can effectively improve the synthesis of L-homoserine. Compared with strain HS12-A, the yield of L-homoserine of strain HS13-A is increased from 11.5 g / L to 14.2 g / L, and the yield of L-homoserine is increased by 24%, so it can be seen that increasing the supply of NADPH can effectively improve the synthesis of L-homoserine.

[0115] Example 6: Fed-batch fermentation of strain HS13-A in a 5-L fermenter

[0116] The strains in Example 5 were streaked onto LB plates containing 50 mg / L kanamycin and incubated at 37°C overnight. Single colonies were picked into LB test tubes containing 50 mg / L kanamycin and incubated at 37°C, 150 rpm overnight to prepare seed liquid. The seed liquid was inoculated into 100 mL LB medium at a volume concentration of 5%, and incubated at 37°C, 150 rpm overnight to prepare secondary seed liquid. The secondary seed liquid was inoculated into a 5-L fermenter containing 2 L of fermentation medium at a volume concentration of 15%, and 0.2 mM IPTG was added. The fermentation culture was carried out at 30°C, 500 rpm, and aeration rate of 0.5 V / V·min. When the pH value was higher than 6.80 (the initial sugar in the fermenter was consumed), the automatic feeding was started, and the feeding medium was added at a rate of 25 mL / h until the pH value was lower than 6.80. The residual sugar in the fermenter was maintained at a low level, and the sugar concentration was maintained at 3 g / L. The total amount of feeding medium added was 800 mL, and the culture was carried out for 117 h. The yield of L-homoserine in the fermentation broth was detected by the high performance liquid chromatography method of Example 1, the biomass OD 600 was detected by spectrophotometry, and the sugar concentration was detected by DNS method; the yield of L-homoserine after 117 h was 69 g / L, and the final biomass OD 600The fermentation results show that the L-homoserine production strain with metabolic engineering has good production performance, and no accumulation of other amino acids is detected in the fermentation broth, which lays a foundation for industrial production of L-homoserine.

[0117] 5- The medium formula of the L-fermentation tank culture: glucose 20 g / L, (NH4)2SO4 17 g / L, yeast extract 4 g / L, KH2PO4 1 g / L, MgSO4 1 g / L, CaCO3 15 g / L, 1 mL / L trace element solution; the composition of the trace element solution: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and the solvent is deionized water.

[0118] The feed medium: glucose 500 g / L, (NH4)2SO4 16 g / L, yeast extract 4 g / L, KH2PO4 12.5 g / L, NaHCO3 10 g / L, threonine 4 g / L, methionine 0.5 g / L, and the pH is adjusted to 6.8 with 50% ammonia water.

Claims

1. A recombinant genetically engineered bacterium for producing L-homoserine, which is obtained by the following method: (1) Take strain E. coli W3110 Trc- panD Trc- ppC Trc- aspC Δ pykA Δ cycA as the chassis strain, use CRISPR / Cas9 gene editing technology to backfill panD gene original promoter, get engineering bacteria HS2; (2) Using CRISPR / Cas9 gene editing technology, the engineered bacteria HS2 were... thrA The gene is de-inhibited by feedback inhibition, resulting in a gene containing the de-inhibited feedback inhibition. thrA * Genetically engineered bacteria HS3; (3) On the HS3 genome rhtA The original gene promoter was replaced with the strong promoter Trc to obtain the engineered bacterium HS4; (4) Using CRISPR / Cas9 gene editing technology to remove feedback inhibition by adding a Trc promoter. thrA * Gene replacement engineered bacteria HS4 fliK Genes were used to obtain engineered bacteria HS5; (5) The original promoter of the gene on the genome of the strain HS5 is replaced by a strong promoter Trc to obtain an engineering strain HS6. metL (5) The original promoter of the gene on the genome of the strain HS5 is replaced by a strong promoter Trc to obtain an engineering strain HS6. (6) Using CRISPR / Cas9 gene editing technology to add the Trc promoter ppC Gene replacement engineered bacteria HS6 ompT Genes were used to obtain engineered bacteria HS7; (7) knockout of the gene on the genome of strain HS7 to obtain engineering strain HS8; pcK knockout of the gene on the genome of strain HS7 to obtain engineering strain HS8; (8) using CRISPR / Cas9 gene editing technology to add the Trc promoter to the rhtA gene of the engineering bacteria HS8 to obtain engineering bacteria HS9; ylbE gene of the engineering bacteria HS8 to obtain engineering bacteria HS9; (9) The genome of strain HS9 thrB Gene knockout yielded engineered bacteria HS10; (10) Knocking out the gene on the genome of strain HS10 to obtain engineering strain HS11; metA Knocking out the gene on the genome of strain HS10 to obtain engineering strain HS11; (11) knocking out the gene on the genome of the strain HS11 to obtain an engineering strain HS12; poxB knocking out the gene on the genome of the strain HS11 to obtain an engineering strain HS12; (12) The genome of strain HS12 nadK The original gene promoter was replaced with the strong promoter Trc to construct the recombinant genetically engineered bacterium HS13. (13) overexpressing the feedback-inhibited thrA * gene and the aspartate transaminase gene derived from the source C. glutamicum aspB （cg） , constructing plasmid pTrc99a- thrA * - aspB （cg） and transforming into strain HS13 to obtain strain HS13-A, which is the L-homoserine-producing recombinant genetically engineered bacteria; the panD gene nucleotide sequence is shown as SEQ ID NO. 1, the rhtA gene nucleotide sequence is shown as SEQ ID NO. 3, the metL gene nucleotide sequence is shown as SEQ ID NO. 4, the ppC gene nucleotide sequence is shown as SEQ ID NO. 5; the pcK a gene nucleotide sequence as shown in SEQ ID NO. 6, thrB a gene nucleotide sequence as shown in SEQ ID NO. 7, said metA a gene nucleotide sequence as shown in SEQ ID NO. 8, said poxB a gene nucleotide sequence as shown in SEQ ID NO. 9, said nadK a gene nucleotide sequence as shown in SEQ ID NO. 10, said aspB （cg） a gene nucleotide sequence as shown in SEQ ID NO. 11, said Trc promoter nucleotide sequence as shown in SEQ ID NO. 12, said thrA * a gene nucleotide sequence as shown in SEQ ID NO.

13.

2. A method for constructing the genetically engineered bacterium of claim 1, which comprises: (1) Take strain E. coli W3110Trc- panD Trc- ppC Trc- aspC Δ pykA Δ cycA as the chassis strain, CRISPR / Cas9 gene editing technology is applied to backfill panD gene original promoter, and obtain engineering bacteria HS2; (2) using CRISPR / Cas9 gene editing technology, the feedback inhibition of the thrA gene of the engineering bacteria HS2 is removed, and engineering bacteria HS3 containing the feedback inhibition-removed thrA * gene is obtained. (3) On the HS3 genome rhtA The original gene promoter was replaced with the strong promoter Trc to obtain the engineered bacterium HS4; (4) Using CRISPR / Cas9 gene editing technology to remove feedback inhibition by adding a Trc promoter. thrA * Gene replacement engineered bacteria HS4 fliK Genes were used to obtain engineered bacteria HS5; (5) The original promoter of the gene on the genome of the strain HS5 is replaced by a strong promoter Trc to obtain an engineering strain HS6. metL (5) The original promoter of the gene on the genome of the strain HS5 is replaced by a strong promoter Trc to obtain an engineering strain HS6. (6) Using CRISPR / Cas9 gene editing technology to add the Trc promoter ppC Gene replacement engineered bacteria HS6 ompT Genes were used to obtain engineered bacteria HS7; (7) knockout of the gene on the genome of strain HS7 to obtain engineered strain HS8; pcK gene knockout, to obtain engineered strain HS8; (8) using CRISPR / Cas9 gene editing technology to add the Trc promoter to the rhtA gene of the engineering bacteria HS8 to obtain engineering bacteria HS9; ylbE gene of the engineering bacteria HS8 to obtain engineering bacteria HS9; (9) The genome of strain HS9 thrB Gene knockout yielded engineered bacteria HS10; (10) The genome of strain HS10 metA Gene knockout yielded engineered bacteria HS11; (11) knocking out the gene on the genome of the strain HS11 to obtain an engineering strain HS12; poxB knocking out the gene on the genome of the strain HS11 to obtain an engineering strain HS12; (12) The genome of strain HS12 nadK The original gene promoter was replaced with the strong promoter Trc to construct the recombinant genetically engineered bacterium HS13. (13) overexpressing the feedback-inhibited thrA * gene and the aspartate transaminase gene derived from the source C. glutamicum aspB （cg） , constructing plasmid pTrc99a- thrA * - aspB （cg） and transforming into strain HS13 to obtain strain HS13-A, which is the L-homoserine-producing recombinant genetically engineered bacteria; the panD gene nucleotide sequence is shown as SEQ ID NO. 1, the rhtA gene nucleotide sequence is shown as SEQ ID NO. 3, the metL gene nucleotide sequence is shown as SEQ ID NO. 4, the ppC gene nucleotide sequence is shown as SEQ ID NO. 5; the pcK a gene nucleotide sequence as shown in SEQ ID NO. 6, thrB a gene nucleotide sequence as shown in SEQ ID NO. 7, said metA a gene nucleotide sequence as shown in SEQ ID NO. 8, said poxB a gene nucleotide sequence as shown in SEQ ID NO. 9, said nadK a gene nucleotide sequence as shown in SEQ ID NO. 10, said aspB （cg） a gene nucleotide sequence as shown in SEQ ID NO. 11, said Trc promoter nucleotide sequence as shown in SEQ ID NO. 12, said thrA * a gene nucleotide sequence as shown in SEQ ID NO.

13.

3. Use of the genetically engineered bacterium of claim 1 in microbial fermentation for preparing L-homoserine.

4. The use according to claim 3, wherein The application is: the genetically engineered bacteria strain is inoculated into fermentation medium containing kanamycin, and fermentation culture is carried out at 25-37 DEG C, 100-300 rpm, until OD 600 = 0.5-1.0, IPTG with a final concentration of 0.2 mM is added, and the culture is continued for 48 h, and after the fermentation is finished, L-homoserine is obtained by separating and purifying the fermentation supernatant.

5. The use according to claim 4, wherein The fermentation medium is composed of 10-30 g / L glucose, 10-20 g / L (NH4)2SO4, 1-5 g / L yeast extract, 0.5-2.0 g / L KH2PO4, 0.5-2.0 g / L MgSO4, 10-20 g / L CaCO3, 0.5-2 mL / L trace element solution, and deionized water as solvent; the components of the trace element solution are as follows: 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and deionized water as solvent.

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