CN117327636A - Genetically engineered bacterium for producing O-succinyl-L-homoserine, construction method and application - Google Patents

Genetically engineered bacterium for producing O-succinyl-L-homoserine, construction method and application Download PDF

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CN117327636A
CN117327636A CN202311361511.1A CN202311361511A CN117327636A CN 117327636 A CN117327636 A CN 117327636A CN 202311361511 A CN202311361511 A CN 202311361511A CN 117327636 A CN117327636 A CN 117327636A
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trc
gene
homoserine
succinyl
genetically engineered
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柳志强
张晓健
李波
刘鹏
吴琦
王双慧
丁文清
陈静祥
郑裕国
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Hangzhou Youze Biotechnology Co ltd
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Abstract

The invention provides a genetic engineering bacterium for producing O-succinyl-L-homoserine, a construction method and application thereof. The O-succinyl-L-homoserine producing genetically engineered bacterium uses E.coli W3110 as chassis bacterium, and replaces the original promoters of metB and thrB genes in the genome with weak promoters M 12 Weakening the expression, then replacing the original promoter of the endogenous gene aspA, aspC, metL, thrA, lysC with a trc strong promoter to strengthen the expression, integrating the exogenous gene pyc into the genome, and finally introducing the pTrc99A plasmid integrated with the metA gene. The O-succinyl-L-homoserine production genetically engineered bacterium can realize accumulation of O-succinyl-L-homoserine in fermentation liquor in the fermentation process, the shake flask yield of O-succinyl-L-homoserine is 21.56g/L, the feed fermentation yield is 82g/L, and a foundation is laid for subsequent construction of the O-succinyl-L-homoserine production genetically engineered bacterium.

Description

Genetically engineered bacterium for producing O-succinyl-L-homoserine, construction method and application
Technical Field
The invention belongs to the technical field of microbial metabolism engineering, and particularly relates to a genetically engineered bacterium for producing O-succinyl-L-homoserine, a construction method and application thereof.
Background
L-methionine is a sulfur-containing essential amino acid in animals, participates in protein synthesis, is a main methyl donor in many physiological metabolic processes, is the first limiting amino acid for animal nutrition, and is widely applied to the fields of feed additives, cosmetics, pharmacy and the like, and the market demand is great.
At present, methionine is mainly produced by a chemical method in industry, but the development of the method is limited by the use of volatile toxic reagents in the production process. In recent years, enzyme synthesis is also gradually applied to the production of methionine, however, enzyme synthesis of methionine synthesis precursors has high cost, which is unfavorable for the industrial application of the method. In microorganisms, L-methionine biosynthesis faces various challenges, mainly including long metabolic pathways, many branched synthesis pathways, the presence of various feedback inhibition and multi-stage regulation, etc., which make it difficult to further increase the L-methionine production. The main research content of the fourth industrial revolution is the synthesis biology and enzyme engineering, and the technology platform of the synthesis biology and enzyme engineering is utilized to produce methionine by adopting a fermentation-catalysis two-step method, and the method has great attention because of low cost and high yield. It has been found that L-methionine can be formed by combining O-succinyl-L-homoserine (OSH) as a precursor with sodium methyl mercaptide by an enzymatic process. Therefore, the construction of a genetic engineering strain for producing OSH has important significance for the industrial production of methionine.
OSH is an important derivative of the downstream metabolic synthesis of L-homoserine. In the prior art, biosynthesis of L-homoserine often uses E.coli (Escherichia coli) or Corynebacterium glutamicum (Corynebacterium glutamicum) as industrial strains; compared with corynebacterium glutamicum, escherichia coli has the advantages of clear genetic background, rapid propagation and the like, and is widely selected as a chassis strain to use. In E.coli, OSH biosynthesis is catalyzed by homoserine transacetylase encoded by metA gene using L-homoserine as precursor, including activation of homoserine-gamma-hydroxy group and succinyl-CoA providing acyl group. Whereas thrB-encoded homoserine kinase can decompose L-homoserine into L-threonine, and cystathionine gamma synthase encoded by metB gene can decompose OSH into cysteine, which is unfavorable for accumulation of OSH. In addition, increasing intracellular Oxaloacetate (OAA) content by metabolic engineering means also helps to increase OSH yield.
Disclosure of Invention
In order to solve the problems of difficult preparation and low yield of L-methionine in the prior art, the invention modifies escherichia coli by metabolic engineering means to obtain a genetically engineered bacterium capable of producing an L-methionine synthesis precursor O-succinyl-L-homoserine ammonia.
In order to achieve the above object, the present invention provides an O-succinyl-L-homoserine producing genetically engineered bacterium in which expression of metB encoding cystathionine gamma synthase and thrB encoding homoserine kinase in the genome of Chassis E.coli W3110 is attenuated, expression of aspA encoding aspartate oxidase, aspC encoding aspartate aminotransferase, metL encoding homoserine dehydrogenase, thrA encoding homoserine dehydrogenase I and lysC encoding aspartate kinase is enhanced, and pyruvic carboxylase encoding gene pyc is introduced, and an over-expression plasmid containing metA encoding homoserine transsuccinylase is constructed. Wherein the strain e.coli W3110 is from the university of jerusalem CGSC collection Coli Genetic Stock Center, accession number cgsc#4474, which has been disclosed in patent US2009/0298135A1, US2010/0248311 A1.
The OSH biosynthetic pathway in e.coli and the engineering scheme according to the invention are shown in figure 1. Firstly, E.coli W3110 is used as a chassis strain, the original promoters of the gene metB and the gene thrB are replaced by weak promoters, the expression of the gene metB and the gene thrB is weakened, the competition of competitive amino acid metabolic pathways for carbon flux is reduced, the further metabolism of L-homoserine and OSH is reduced, and meanwhile, the generation of L-threonine auxotroph strain is avoided; secondly, the original promoter is replaced by a strong promoter to strengthen the expression of the genes aspA and aspC, so that the accumulation amount of L-aspartic acid is increased; then, the expression of genes metL, thrA and lysC is enhanced, L-homoserine which is a precursor of the catalytic synthesis of OSH by L-aspartic acid is promoted, and the carbon flux of the synthesis of OSH is increased; introducing a foreign gene pyc to increase the accumulation of Oxaloacetate (OAA), a precursor of intracellular L-aspartic acid synthesis; and finally, strengthening the key gene metA for synthesizing the OSH, promoting the synthesis of the OSH by the L-homoserine under the catalysis of homoserine transacetylase, and further improving the yield of the OSH.
Preferably, the promoters of the gene metB and the gene thrB are replaced with M, respectively 12 The promoter is weakly expressed.
Preferably, the promoters of the gene aspA, aspC, metL, thrA and lysC are replaced with Trc promoters, respectively, for overexpression.
Preferably, the gene pyc is derived from C.glutamicum Corynebacterium glutamicum.
Preferably, the overexpression plasmid containing the gene metA is pTrc99A-metA. The over-expression plasmid refers to a polynucleotide in which control sequences expressed in a host cell are operably linked, and preferably the vector of the over-expression plasmid is pTrc99A.
The invention also provides a method for constructing the genetically engineered bacterium for producing O-succinyl-L-homoserine, which comprises the following steps: (1) The CRISPR-Cas9 gene editing technology is utilized to replace the promoters of the gene metB and the gene thrB in the genome of the chassis fungus E.coli W3110 with the M12 promoter to obtain engineering bacteria E.coli W3110P M12 -metB P M12 -thrB；
(2) Using CRISPR-Cas9 gene editing technology to obtain engineering bacteria E.coli W3110P from step (1) M12 -metB P M12 The promoters of thrB gene aspA, aspC, metL, thrA and lysC are replaced by trc promoter to obtain engineering bacteria E.coli W3110P M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc -lysC；
(3) Integrating the gene pyc into engineering bacteria E.coli W3110P obtained in the step (2) M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -met P trc -thrA P trc In the genome of lysC, engineering bacteria E.coli W3110P were obtained M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc -ompT::pyc cg ；
(4) Constructing plasmid pTrc99A-metA and introducing into engineering bacterium E.coli W3110P obtained in step (3) M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc -ompT::pyc cg In the step (a), the genetically engineered bacterium E.coli W3110P for producing O-succinyl-L-homoserine is obtained M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -met P trc -thrA P trc -ompT::pyc cg /pTrc99A-metA。
Preferably, the nucleotide sequence of the gene metB is shown as SEQ ID NO.1, the nucleotide sequence of the gene thrB is shown as SEQ ID NO.2, the nucleotide sequence of the gene aspA is shown as SEQ ID NO.3, the nucleotide sequence of the gene aspC is shown as SEQ ID NO.4, the nucleotide sequence of the gene metL is shown as SEQ ID NO.5, the nucleotide sequence of the gene thrA is shown as SEQ ID NO.6, the nucleotide sequence of the gene lysC is shown as SEQ ID NO.7, and the nucleotide sequence of the gene metA is shown as SEQ ID NO. 8.
Preferably, the nucleotide sequence of said gene pyc is shown in SEQ ID NO. 9.
The invention also provides application of the genetically engineered bacterium for producing O-succinyl-L-homoserine in producing O-succinyl-L-homoserine by microbial fermentation.
Preferably, the application is: inoculating the genetically engineered bacteria producing O-succinyl-L-homoserine into a fermentation culture medium, fermenting and culturing for 70-90 h at 28-37 ℃ and 100-500 rpm, and separating and purifying the supernatant of the fermentation broth after fermentation is finished to obtain O-succinyl-L-homoserine.
Preferably, the fermentation medium is composed of: glucose 10-30 g/L, ammonium sulfate 10-20 g/L, yeast extract powder 1-5 g/L, KH 2 PO 4 0.5～3g/L、MgSO 4 0.1-2.0 g/L, 0.5-5 mL/L of trace metal salt solution, 1.0-3.0 g/L of betaine, pH value of 6.5-7.0 and deionized water as solvent; the trace metal salt solution consists of: feSO 4 .4H 2 O 0.01g/L，MnSO 4 0.005g/L，ZnSO 4 0.0025 g/L, and the solvent is deionized water.
The term "enhancement" in the present invention means that the activity of an enzyme encoded by a corresponding polynucleotide is increased by over-expression of a gene or substitution of an expression regulatory sequence (promoter substitution, etc.) of the gene on the genome. The term "attenuation" in the present invention means that the activity of an enzyme encoded by a corresponding polynucleotide is reduced by repressing the expression of a gene or by replacing an expression regulatory sequence (promoter replacement, etc.) of the gene on the genome.
The vector used in the present invention may not be particularly limited as long as the vector is replicable in a host, and any vector known in the art may be used.
The invention has the beneficial effects that: compared with the prior art, the synthesis approach of knocking out the byproduct amino acid can improve the yield of the target amino acid, but the constructed strain is mostly auxotroph, such as L-lysine, L-threonine or L-methionine auxotroph, and necessary amino acid needs to be added additionally during fermentation to ensure the normal growth of thalli, so that the fermentation cost is increased, and certain difficulty is caused to the fermentation regulation and control process of the amino acid. Therefore, the present invention replaces the original promoter with the weak promoter to reduce the expression amounts of the gene metB and the gene thrB, and does not cause the generation of amino acid auxotrophs while reducing the competitive amino acid carbon flux. The O-succinyl-L-homoserine producing genetically engineered bacteria obtained through the system metabolic engineering realize the effective accumulation of OSH, the shake flask yield of OSH reaches 21.56g/L, the feed fermentation yield of a 5L fermentation tank reaches 82g/L, the sugar acid conversion rate reaches 45%, and a foundation is laid for the subsequent construction of genetically engineered bacteria with high OSH yield.
Drawings
FIG. 1 is a schematic diagram of O-succinyl-L-homoserine biosynthesis pathway in E.coli and genetic engineering related to the present invention.
Detailed Description
The following specific examples are presented to illustrate the present invention, and those skilled in the art will readily appreciate the additional advantages and capabilities of the present invention as disclosed herein. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. The methods used in the examples of the present invention are conventional methods, and the reagents used are commercially available.
In the following examples, the final concentration of kanamycin sulfate (kana) was used at 50 ng/. Mu.L. The final concentration of spectinomycin hydrochloride (SD) was 50 ng/. Mu.L. The E.coli competent cells used in the examples were commercial E.coli DH 5. Alpha. Purchased from Novain, but not limited thereto, and the one-step directed cloning kit used in the recombination reaction was purchased from Novain.
The method for constructing the genetically engineered bacterium for producing O-succinyl-L-homoserine is disclosed in the invention, and the method for constructing the genetically engineered bacterium for producing O-succinyl-L-homoserine is disclosed in the reference of Zhang, B.et al, metabolic engineering of Escherichia coli for D-pantothic acid production, food Chem,2019.294:p.267-275.
Example 1: m12 promoter replaces original promoters of genes metB and thrB
(1) pTarget-gRNA plasmid mutation
The pTarget mutant plasmid is amplified by PCR with pTarget as a template and F1 and R1 as mutation primers, and the 20bp homologous sequence on pTarget is mutated into the 20bp sequence before the PAM site of the target gene metB promoter. The amplification system (20. Mu.L) was as follows:
after the completion of PCR amplification, the PCR product was detected by agarose gel electrophoresis, 0.5. Mu.L of DpnI was added to the PCR product, digested at 37℃for 1 hour, 10. Mu.L of the digested product was added to 100. Mu.L of DH 5. Alpha. Competent cells for transformation experiments, and the culture broth was applied to LB solid medium (SD resistance) and incubated overnight at 37 ℃. Single colony is selected and inoculated in 10mL of LB liquid culture medium (SD resistance), cultured for more than 18h, bacterial liquid is taken to send sequence and mutant plasmid pTarget-metB-g is extracted for standby.
(2) PCR amplification of metB donor DNA and linearized pTarget-metB-g plasmid fragment
Primers F2, R2, F3 and R3 were designed, and the E.coli genome was used as a template, and about 500bp each was used as a homology arm on the upstream and downstream of the metB promoter of the target gene, and the M12 promoter sequence, together as a donor DNA. The Target-metB-g linearized plasmid fragment was amplified by PCR using the Target-metB-g as template and the primers F4, R4. The amplification system (50. Mu.L) was as follows:
after the PCR amplification was completed, the PCR product was detected by agarose gel electrophoresis, and then 1. Mu.L of DpnI was added to the linearized Target-metB-g plasmid fragment, and digested at 37℃for 1 hour. Then using DNA cleaning kit to clean up the digestion product and the PCR amplified donor DNA, and detecting the DNA concentration of the cleaned product, and preserving at-20 ℃ for standby.
(3) One-step cloning of linearized Target-metB-g plasmid fragment and donor DNA
Taking the product cleaned in the step (2), and carrying out recombination reaction by using a one-step directional cloning seamless cloning kit according to the concentration of the linearized Target-metB-g plasmid fragment and the concentration of the donor DNA. 10. Mu.L of the one-step clone was added to 100. Mu.L of DH 5. Alpha. Competent cells for transformation experiments, and the culture broth was then spread on LB solid medium (SD resistance) and incubated overnight at 37 ℃. After single colonies were grown, the single colonies were picked and inoculated with 10mL of LB liquid medium (SD resistance), and the plasmid pTarget-metB-pdg was sequenced and extracted for use.
(4) Shock transformation of pTarget-metB-pdg
Electrotransformation competence of E.coli W3110 strain (laboratory deposit) containing pCas plasmid was prepared. Taking 1 mu L of E.coli W3110 strain containing pCas plasmid, placing in ice bath, adding 2 mu L of pTarget-metB-pdg in a super clean bench, gently mixing with a pipette, and ice-bathing for 1min; the mixed solution is transferred to a precooled electric shock cup with the length of 2mm by a pipette (the electric shock cup needs to be dried in an ultra-clean bench in advance) and is subjected to ice bath for 45s. The water mist outside the electric shock cup is wiped by paper towel and placed in an electric converter, and electric shock is carried out by using the Eco 2 gear. 1mL of precooled LB culture medium is added into a groove of the electric shock cup in an ultra-clean bench, the electric shock cup is inclined, all bacterial liquid is sucked from the opening of the electric shock cup, and the bacterial liquid is transferred into a 2mL sterile EP tube. Resuscitating at 180rpm at 30 ℃ for more than 2.5 hours; 200. Mu.L of the culture medium was plated on LB solid medium (SD+kana resistance) and cultured overnight at 30 ℃.
(5) Positive clone screening and validation
The forward verification primer F5 is designed at the position 100bp outside the upstream homology arm of the target gene metB promoter, the directional primer R5 is designed on the M12 promoter, the cloned seed is selected as a template, colony PCR is carried out, and meanwhile, the original genome is taken as a template as a negative control. Colony PCR was banded and correctly sized as positive.
(6) pTarget-pdg and pCas elimination and sequencing verification
Positive clones were inoculated into 10mL LB tubes (kana resistance) and incubated with 10. Mu.L of IPTG stock at 30℃for 12h at 180 rpm. The overnight culture broth was streaked onto LB solid medium (kana resistance) and incubated overnight at 30 ℃. The overnight streak plate was taken, single colonies were numbered, and a portion of the numbered single colonies was picked, streaked onto the corresponding area on LB solid medium (SD resistance), and incubated overnight at 37 ℃. Single colonies incapable of growing in the area corresponding to LB solid medium (SD resistance) were clones successfully deleted by pTarget-metB-pdg.
The successfully eliminated pTarget-metB-pdg clone was inoculated into 10mL LB tubes (no resistance), and incubated at 37℃for 12h at 180 rpm. The overnight culture broth was streaked onto LB solid medium (no resistance) and incubated overnight at 37 ℃. The overnight culture broth was taken, single colonies were numbered, and a portion of the single colonies numbered were picked, streaked onto the corresponding area on LB solid medium (kana resistance), and cultured overnight at 30 ℃. Single colonies incapable of growing in the region corresponding to LB solid medium (kana resistance) were clones that were successfully deleted by pCas.
(7) Positive clone sequencing validation
Picking grams of pTarget-pdg and pCas that were successfully eliminatedThe clone is used as a colony PCR template to verify primers F5 and R5 to perform colony PCR, the colony PCR products are sent to sequence, positive clones are verified, and engineering bacteria E.coliW3110P is obtained M12 -metB。
TABLE 1 example 1 primer Table
According to the method, engineering bacteria E.coli W3110P are sequentially treated M12 The original promoter of the gene thrB in the metB genome is replaced by an M12 promoter, and then the original promoters of the genes aspA, aspC, metL, thrA and lysC are replaced by trc promoters in sequence to obtain engineering bacteria E.coli W3110P M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -met P trc -thrA P trc -lysC。
Wherein the nucleotide sequence of the M12 promoter is shown as SEQ ID NO.10, and the nucleotide sequence of the trc promoter is shown as SEQ ID NO. 11.
Example 2: insertion of the corynebacterium glutamicum-derived pyc Gene
(1) pTarget-gRNA plasmid mutation
Site-directed mutagenesis primers F6 and R6 were designed to mutate the 20bp sgRNA homologous sequence on the pTarget plasmid to the pre-20 bp sequence of the PAM site on the flanking sequence of the insertion position of the target gene pyc. The pTarget mutant plasmid was PCR amplified using pTarget as template, the amplification system (20. Mu.L) was as follows:
after the completion of PCR amplification, the PCR product was detected by agarose gel electrophoresis, 0.5. Mu.L of DpnI was added to the PCR product, digested at 37℃for 1 hour, 10. Mu.L of the digested product was added to 100. Mu.L of DH 5. Alpha. Competent cells for transformation experiments, and the culture broth was applied to LB solid medium (SD resistance) and incubated overnight at 37 ℃. Single colonies are picked and inoculated into 10mL of LB liquid medium (SD resistance) for more than 18h of culture, bacterial liquid is taken for sequencing, and mutant plasmid pTarget-pyc-g is extracted for standby.
(2) PCR amplification of donor DNA and linearized pTarget-pyc-g plasmid fragment
Primers F7, R7 and F8, R8 were designed, and the E.coli genome was used as template, and about 500bp sequences were used as homology arms upstream and downstream of the insertion position of the target gene pyc by PCR amplification. The sequence of the pyc gene was amplified by PCR using primers F9, R9 and the C.glutamicum genome as template.
And PCR amplifying the Target-pyc-g linearized plasmid fragment using Target-pyc-g as template. The amplification system (50. Mu.L) was as follows:
after the PCR amplification was completed, the PCR product was detected by agarose gel electrophoresis, and 1. Mu.L of DpnI was added to the linearized Target-pyc-g plasmid fragment, and digested at 37℃for 1 hour. Then using DNA cleaning kit to clean up the digestion product and the PCR amplified donor DNA, and detecting the DNA concentration of the cleaned product, and preserving at-20 ℃ for standby.
(3) One-step cloning of linearized Target-pyc-g plasmid fragments, homology arms and pyc Gene
Taking the product after cleaning in (2), and carrying out recombination reaction by using a one-step directional cloning seamless cloning kit according to the concentration of the linearized Target-pyc-g plasmid fragment, the homology arm and the pyc gene. 10. Mu.L of the one-step clone was added to 100. Mu.L of DH 5. Alpha. Competent cells for transformation experiments, and the culture broth was then spread on LB solid medium (SD resistance) and incubated overnight at 37 ℃. After single colonies were grown, the single colonies were picked and inoculated with 10mL of LB liquid medium (SD resistance), sequenced and plasmid pTarget-pyc-pdg was extracted for use.
(4) Shock transformation of pTarget-pyc-pdg
Preparation of E.coli W3110P containing pCas plasmid M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc Electrotransport competence of lysC. 1 μl of the above electric transduction competent sample was takenPlacing engineering bacteria in ice bath, adding 2 mu L of pTarget-pyc-pdg into an ultra-clean bench, gently mixing with a pipettor, and ice-bathing for 1min; the mixed solution is transferred to a precooled electric shock cup with the length of 2mm by a pipette (the electric shock cup needs to be dried in an ultra-clean bench in advance) and is subjected to ice bath for 45s. The water mist outside the electric shock cup is wiped by paper towel and placed in an electric converter, and electric shock is carried out by using the Eco 2 gear. 1mL of precooled LB culture medium is added into a groove of the electric shock cup in an ultra-clean bench, the electric shock cup is inclined, all bacterial liquid is sucked from the opening of the electric shock cup, and the bacterial liquid is transferred into a 2mL sterile EP tube. Resuscitating at 180rpm at 30 ℃ for more than 2.5 hours; 200. Mu.L of the culture medium was plated on LB solid medium (SD+kana resistance) and cultured overnight at 30 ℃.
(5) Positive clone screening and validation
Forward and reverse verification primers F10 and R10 are designed at the position 100bp outside the upstream and downstream homology arms of the target site, then the clonotypes are selected as templates, colony PCR is carried out, and meanwhile, the original genome is taken as a template to serve as negative control. Colony PCR bands were larger than control and correctly sized as positive.
(6) pTarget-pdg and pCas elimination and sequencing verification
Positive clones were inoculated into 10mL LB tubes (kana resistance) and incubated with 10. Mu.L of IPTG stock at 30℃for 12h at 180 rpm. The overnight culture broth was streaked onto LB solid medium (kana resistance) and incubated overnight at 30 ℃. The overnight streak plate was taken, single colonies were numbered, and a portion of the numbered single colonies was picked, streaked onto the corresponding area on LB solid medium (SD resistance), and incubated overnight at 37 ℃. Single colonies which failed to grow in the region corresponding to LB solid medium (SD resistance) were those which were successfully deleted by pTarget-pyc-pdg.
The successfully eliminated pTarget-pyc-pdg clone was inoculated into 10mL LB tubes (no resistance), and incubated at 37℃for 12h at 180 rpm. The overnight culture broth was streaked onto LB solid medium (no resistance) and incubated overnight at 37 ℃. The overnight culture broth was taken, single colonies were numbered, and a portion of the single colonies numbered were picked, streaked onto the corresponding area on LB solid medium (kana resistance), and cultured overnight at 30 ℃. Single colonies incapable of growing in the region corresponding to LB solid medium (kana resistance) were clones that were successfully deleted by pCas.
(7) Positive clone sequencing validation
Selecting a clone successfully eliminated by pTarget-pdg and pCas as a colony PCR template, carrying out colony PCR by using a verification primer, sequencing a colony PCR product, and verifying positive clones to obtain an engineering strain E.coli W3110P M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc -ompT::pyc cg 。
TABLE 2 example 2 primer tables
Example 3: construction of pTrc99A-metA plasmid
The pTrc99A plasmid (laboratory deposit) is used as a template, and primers F11 and R11 are used for amplifying the linearized plasmid fragment by PCR; meanwhile, the genome of Escherichia coli is used as a template, and primers F12 and R12 are used for amplifying the metA fragment by PCR. The amplified linearized plasmid fragment was digested with DpnI, then the digested linearized pTrc99A vector fragment and metA fragment were cleaned using clean up kit, and then the reaction product was transformed into E.coli competent cells by performing recombination reaction using one-step directed cloning seamless cloning kit, and then coated on LB plates containing kana. Using the verification primer, colony PCR verifies the monoclonal, and positive clones are sequenced by sequencing company to determine the successfully ligated plasmid pTrc99A-metA. The constructed plasmid pTrc99A-metA was transformed into strain E.coli W3110P M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -met P trc -thrA P trc -ompT::pyc cg . Screening positive clones to obtain strain E.coli W3110P M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -met P trc -thrA P trc -ompT::pyc cg /pTrc99A-metA。
TABLE 3 example 3 primer tables
Example 4: fermentation of engineering strains
Activating strains: collecting strain preserved at-80deg.C, namely strain E.coli W3110P prepared in example 3 M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc -ompT::pyc cg pTrc99A-metA (designated OSH 5), and the starting strain E.coli W3110 were streaked onto an activation medium (solid LB medium), and cultured overnight at 37 ℃.
Seed culture: the activated seeds of the strains OSH5 and E.coli W3110 were picked up by inoculating loops, respectively, and inoculated in a test tube containing 10mL of seed medium (LB medium), and cultured overnight at 37℃and 200 rpm;
shaking and fermenting: respectively inoculating seed solutions of strains OSH5 and E.coli W3110 according to 5% inoculum size into a 500mL conical flask filled with 20mL fermentation medium, performing shake culture at 37 ℃ at 200rpm/min, and fermenting for 48h;
fermenting in a fermentation tank: seed culture media of the strains OSH5 and E.coli W3110 were inoculated into 3 flasks of 500mL conical flask containing 100mL LB medium, respectively, and cultured with shaking at 37℃at 200rpm/min for 12 hours. The seed solution of the strain OSH5 and E.coli W3110 was inoculated into a 5L fermenter containing 2L of fermentation medium at a fermentation temperature of 30℃at 500rpm/min and a fermentation pH of 6.8 by 40% aqueous ammonia. The fermentation process adopts a pH-star mode to control the feed, and samples are taken every 4 hours to detect the biomass, residual sugar and OSH.
The shake flask fermentation medium comprises the following components: 25g/L glucose, 16g/L ammonium sulfate, 2.5g/L yeast powder and KH 2 PO 4 1 g/L，MgSO 4 0.5 g/L，CaCO 3 15 g/L, 1mL/L of salt solution, caCO 3 Separately packaging and sterilizing (0.3 g each part). CaCO is added during inoculation 3 And IPTG (final concentration 0.025 mM).
The fermentation medium consisted of: 25g/L glucose, 16g/L ammonium sulfate, 2.5g/L yeast powder and KH 2 PO 4 1 g/L，MgSO 4 0.5 g/L, betaine 2.0g/L, and trace metal salt solution 1mL/L.
The feed medium consisted of: glucose 500g/L, sulfurAmmonium acid 10g/L, yeast extract 5g/L, KH 2 PO 4 14 g/L、MgSO 4 8 g/L, 1mL/L of saline solution.
After the fermentation, OSH was detected using a hitachi L8080 amino acid analyzer, see the L8080 manual for specific methods.
TABLE 4 production of OSH by fermentation of genetically engineered bacteria
After genetic engineering operation, the genetically engineered bacterium OSH5, namely the genetically engineered bacterium E.coli W3110P for producing O-succinyl-L-homoserine M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -met P trc -thrA P trc -ompT::pyc cg After fermentation of pTrc99A-metA by shaking flask, OSH yield was 21.56g/L and 5L fermenter fed-batch fermentation OSH yield was 82g/L. Therefore, the OSH genetic engineering bacteria constructed by the invention can realize effective accumulation of OSH in fermentation liquor in the fermentation process, and lay a foundation for constructing genetic engineering strains for constructing high-yield OSH.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope of the present invention without departing from the design spirit of the present invention.

Claims (10)

1. An O-succinyl-L-homoserine producing genetically engineered bacterium, wherein the O-succinyl-L-homoserine producing genetically engineered bacterium is obtained by attenuating expression of gene metB encoding cystathionine gamma synthase and gene thrB encoding homoserine kinase in genome of Chassis E.coli W3110, and by constructing an overexpression plasmid containing gene metA encoding homoserine transsuccinylase by enhancing expression of gene aspA encoding aspartate oxidase, gene aspC encoding homoserine dehydrogenase, gene metL encoding homoserine dehydrogenase I, gene thrA encoding homoserine dehydrogenase I and gene lysC encoding aspartokinase and introducing gene pyc encoding pyruvate carboxylase.
2. The O-succinyl-L-homoserine producing genetically engineered bacterium according to claim 1, wherein the promoters of metB and thrB are replaced with M, respectively 12 The promoter is weakly expressed.
3. The genetically engineered O-succinyl-L-homoserine producing bacterium of claim 1, wherein the promoters of aspA, aspC, metL, thrA, lysC are replaced with trc promoters, respectively, for overexpression.
4. The genetically engineered O-succinyl-L-homoserine producing bacterium of claim 1, wherein the gene pyc is derived from corynebacterium glutamicum Corynebacterium glutamicum.
5. The genetically engineered O-succinyl-L-homoserine producing bacterium of claim 1, wherein the overexpression plasmid containing gene metA is pTrc99A-metA.
6. A method for constructing the genetically engineered O-succinyl-L-homoserine producing bacterium according to any one of claims 1 to 5, comprising the steps of:
(1) The CRISPR-Cas9 gene editing technology is utilized to replace the promoters of the gene metB and the gene thrB in the genome of the Chaetoceros E.coli W3110 with M 12 The promoter is used for obtaining engineering bacteria E.coli W3110P M12 -metB P M12 -thrB；
(2) Using CRISPR-Cas9 gene editing technology to obtain engineering bacteria E.coli W3110P from step (1) M12 -metBP M12 The promoters of thrB gene aspA, aspC, metL, thrA and lysC are replaced by trc promoter to obtain engineering bacteria E.coli W3110P M12 -metB P M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrAP trc -lysC；
(3) Integrating the gene pyc into engineering bacteria E.coli W3110P obtained in the step (2) M12 -metB P M12 -thrB P trc -aspAP trc -aspC P trc -met P trc -thrA P trc In the genome of lysC, engineering bacteria E.coli W3110P were obtained M12 -metBP M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA P trc -ompT::pyc cg ；
(4) Constructing plasmid pTrc99A-metA and introducing into engineering bacterium E.coli W3110P obtained in step (3) M12 -metBP M12 -thrB P trc -aspA P trc -aspCP trc -met P trc -thrA(Trc)P trc -ompT::pyc cg In the step (a), the genetically engineered bacterium E.coli W3110P for producing O-succinyl-L-homoserine is obtained M12 -metB P M12 -thrB P trc -aspA P trc -aspC P trc -metP trc -thrA(Trc)P trc -ompT::pyc cg /pTrc99A-metA。
7. The method for constructing a genetically engineered bacterium producing O-succinyl-L-homoserine according to claim 6, wherein the nucleotide sequence of the gene metB is shown in SEQ ID NO.1, the nucleotide sequence of the gene thrB is shown in SEQ ID NO.2, the nucleotide sequence of the gene aspA is shown in SEQ ID NO.3, the nucleotide sequence of the gene aspC is shown in SEQ ID NO.4, the nucleotide sequence of the gene metL is shown in SEQ ID NO.5, the nucleotide sequence of the gene thrA is shown in SEQ ID NO.6, the nucleotide sequence of the gene lysC is shown in SEQ ID NO.7, and the nucleotide sequence of the gene metA is shown in SEQ ID NO. 8.
8. The method for constructing a genetically engineered bacterium that produces O-succinyl-L-homoserine according to claim 6, wherein the nucleotide sequence of the gene pyc is shown as SEQ ID NO. 9.
9. The use of the genetically engineered bacterium for producing O-succinyl-L-homoserine according to any one of claims 1 to 5 or the genetically engineered bacterium for producing O-succinyl-L-homoserine constructed by the method according to claim 7 or 8 in the production of O-succinyl-L-homoserine by microbial fermentation.
10. The application of claim 9, wherein the application is: inoculating the genetically engineered bacteria producing O-succinyl-L-homoserine into a fermentation culture medium, fermenting and culturing for 70-90 h at 28-37 ℃ and 100-500 rpm, and separating and purifying the supernatant of the fermentation broth after fermentation is finished to obtain O-succinyl-L-homoserine.
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