Genetically engineered bacterium with high yield of L-cysteine as well as construction method and application of genetically engineered bacterium

Abstract

The invention relates to the technical field of L-cysteine production, and discloses a genetically engineered bacterium with high yield of L-cysteine as well as a construction method and application of the genetically engineered bacterium. Through knockout of a tcyP gene, overexpression of a PqqU gene, a galP gene, a zwf gene, a talA gene and a pta gene and introduction and overexpression of an fxpK gene, a carbon metabolic flux is reasonably distributed between an important carbon metabolic node 3-phosphoglycerate and acetyl coenzyme A necessary for an SAT catalytic reaction, the SAT can efficiently catalyze L-serine to synthesize OAS, and overflow of the L-serine is reduced. Finally, through the above strategy, more carbon metabolism flows to synthesis of L-cysteine in the strain fermentation process, and efficient production of L-cysteine is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of L-cysteine production, and particularly relates to a genetically engineered bacterium for high-yield production of L-cysteine and a construction method and application thereof. BACKGROUND

[0002] L-cysteine (L-Cysteine) is an amino acid containing sulfhydryl (-SH), which has physiological functions such as antioxidant, detoxification and anti-inflammatory, and is widely used in medicine, food (such as bread improver), cosmetics (such as hair care products) and feed industry, and is also a precursor of many important derivatives such as L-methionine and glutathione.

[0003] For the synthesis of L-cysteine, the traditional chemical synthesis method needs complex reaction steps and a large amount of toxic reagents, which not only has high production cost, but also produces harmful by-products, causing threat to the environment and human health. The animal hair extraction method has problems such as unstable raw material supply, low purity and ethical controversy, especially in Europe and other regions with strict requirements on animal welfare, and such method has been gradually restricted or eliminated. In contrast, the microbial fermentation method has significant advantages by constructing microbial cell factories and directly synthesizing L-cysteine by genetically engineered microorganisms, and the research and development of microbial fermentation method can fundamentally solve the defects of traditional production technology and provide technical support for large-scale industrial production of L-cysteine.

[0004] At present, many researchers have conducted in-depth research on the synthesis pathway and mechanism of L-cysteine in Corynebacterium glutamicum and Escherichia coli, and applied metabolic engineering strategies and synthetic biology means to the construction of L-cysteine high-yield strains. The early construction of L-cysteine synthesis strains is basically the same, that is, first, strengthen the supply of precursor L-serine, knock out L-serine degradation genes sdaA, sdaB and tdcG, and overexpress L-serine synthesis genes serA, serB and serC. Then, overexpress the SAT mutant that relieves feedback inhibition, and some SAT mutants such as Escherichia coli SATMet256Ile, SATMet256Ala, SATThr167Ala, SATVal95Arg and SATAsp96Pro have been used to construct L-cysteine high-yield strains. Then, overexpress cysM, nrdH and glpE for sulfur module strengthening, and knock out tnaA, yciW and cyuA for weakening L-cysteine degradation. Finally, overexpress L-cysteine efflux genes eamA, eamB and tolC. Thus, the basic L-cysteine production strain is finally constructed.

[0005] In the prior art, the research on the microbial fermentation method of L-cysteine mainly focuses on the regulation and balance of carbon-sulfur metabolism, dynamic regulation, auxiliary factor supply and redox balance regulation. However, the yield of the L-cysteine production strain constructed by the foregoing method does not meet the current demand for L-cysteine, and some key problems still limit its industrialization, for example, the topological structure of the carbon-sulfur metabolic network is not complete, and in particular, the dynamic coupling mechanism of the sulfur assimilation module and the central carbon metabolism is not clear, which leads to the unclear dynamic flux distribution rule of the key metabolic nodes, and restricts the design efficiency of the precise metabolic regulation strategy. More importantly, the intracellular accumulation of L-cysteine can increase the intracellular ROS level through the hydroxyl radical (·OH) generated by the Fenton reaction, which leads to DNA damage and irreversible inactivation of essential enzymes. SUMMARY

[0006] In order to solve the technical problem of low yield of the L-cysteine production strain, the present application provides a genetically engineered bacterium for high-yield L-cysteine and a construction method and application thereof.

[0007] The specific technical scheme of the present application is as follows: In the first aspect, the present application provides a genetically engineered bacterium for high-yield L-cysteine, which is obtained by knocking out the tcyP gene in the chassis bacterium, overexpressing the PqqU gene, the galP gene, the zwf gene, the talA gene, the pta gene, and introducing the fxpK gene and overexpressing.

[0008] The genetically engineered bacterium for high-yield L-cysteine is constructed by knocking out the tcyP gene in the chassis bacterium, overexpressing the PqqU gene, the zwf gene, the talA gene, the pta gene, and introducing the fxpK gene and overexpressing. The genetically engineered bacterium can make more carbon metabolic flow to the synthesis of L-cysteine during the fermentation process of the strain, and realize the efficient production of L-cysteine.

[0009] Specifically: After overexpressing only the pqqU gene in the starting strain, the yield of L-cysteine is only increased from 2.39 g / L to 2.50 g / L. However, after knocking out the tcyP gene in the starting strain and then overexpressing the PqqU gene, the yield of L-cysteine is only increased from 2.39 g / L to 3.43 g / L. Therefore, the simultaneous knockout of the tcyP gene and the overexpression of the PqqU gene form a cascade amplification effect to drive the synthesis of L-cysteine, which significantly improves the yield of cysteine.

[0010] Overexpression of galP establishes a new carbon metabolic driving force, which can effectively improve the flow direction of carbon metabolic flow, and significantly increase the yield of cysteine to 3.74 g / L.

[0011] By overexpressing zwf, talA, and introducing the exogenous phosphoketolase encoding gene fxpK, the carbon metabolic flow can be reasonably distributed between 3-phosphoglycerate and acetyl-CoA, which are necessary for the catalytic reactions of SAT, so that SAT can efficiently catalyze the synthesis of OAS from L-serine.

[0012] Through the above strategy, more carbon metabolic flow is directed to the synthesis of L-cysteine during fermentation of the strain, so that efficient production of L-cysteine is achieved.

[0013] As a preferred embodiment of the genetically engineered strain, the chassis strain is Escherichia coli (E. coli).

[0014] As a preferred embodiment of the genetically engineered strain, the fxpK gene is derived from Bifidobacterium adolescentis.

[0015] As a preferred embodiment of the genetically engineered strain, the sequence of the tcyP gene is shown in SEQ ID No. 1, the sequence of the pqqU outer membrane protein is shown in SEQ ID No. 2, the sequence of the galP gene is shown in SEQ ID No. 3, the sequence of the zwf gene is shown in SEQ ID No. 4, the sequence of the talA gene is shown in SEQ ID No. 5, the sequence of the fxpK gene is shown in SEQ ID No. 6, and the sequence of the pta gene is shown in SEQ ID No. 7.

[0016] In a second aspect, the present application provides a method for constructing a genetically engineered strain for high-yield production of L-cysteine, which comprises the following steps in a chassis strain: knocking out the tcyP gene; overexpressing the PqqU gene; overexpressing the galP gene; overexpressing the zwf gene; overexpressing the talA gene; overexpressing the pta gene; overexpressing the fxpK gene derived from Bifidobacterium adolescentis.

[0017] As a preferred embodiment of the method for constructing the genetically engineered strain, the chassis strain is E. coli W3110ΔtdcG::Ptrc-serA mut ΔsdaA::Ptrc-serBΔsdaB::Ptrc-serCΔtnaAΔcyuA::Ptrc-cysE T167A ΔyciW::Ptrc-cysM-nrdHΔycjV::Ptrc-eamB mutAyncI::Ptrc-eamA AcydDC.

[0018] As the preferred construction method, the sequence of the tcyp gene is shown as SEQ ID No. 1, the sequence of the pqqU outer membrane protein is shown as SEQ ID No. 2, the sequence of the galP gene is shown as SEQ ID No. 3, the sequence of the zwf gene is shown as SEQ ID No. 4, the sequence of the talA gene is shown as SEQ ID No. 5, the sequence derived from the fxpK gene is shown as SEQ ID No. 6, and the sequence of the pta gene is shown as SEQ ID No. 7.

[0019] In a third aspect, the application provides the use of the genetically engineered bacteria in the production of L-cysteine.

[0020] As the preferred use, the genetically engineered bacteria are inoculated into a fermentation medium, and fermentation culture is carried out at 30-37℃, 180rpm-200rpm, and after the fermentation is completed, the fermentation broth is separated and purified to obtain L-cysteine.

[0021] Compared with the prior art, the application has the following technical effects: By knocking out the tcyp gene, the L-cysteine / L-cystine shuttle system is destroyed; by overexpressing the outer membrane protein PqqU with L-cystine efflux capacity, L-cystine in the periplasmic space can be efficiently transported out of the cell; by overexpressing the galP gene, the flow direction of the carbon metabolic stream is significantly improved, so as to flow to the production of L-cysteine; by overexpressing the zwf and talA genes, the metabolic stream of glycolysis and the PP pathway is connected, and the exogenous phosphoketolase encoding gene fxpK is introduced, and pta is overexpressed, so that the carbon metabolic stream is reasonably distributed between 3-phosphoglycerate and acetyl coenzyme A necessary for SAT catalytic reaction, so that SAT can efficiently catalyze the synthesis of OAS from L-serine, and the overflow of L-serine is reduced. Ultimately, through the above strategies, more carbon metabolic stream flows to the synthesis of L-cysteine during the fermentation of the strain, and efficient production of L-cysteine is realized. DETAILED DESCRIPTION

[0022] The application will be further described below in conjunction with examples. Those skilled in the art will be able to implement the application based on these descriptions. In addition, the examples of the application involved in the following descriptions are generally only a part of the examples of the application, not all examples. Therefore, based on the examples in the application, all other examples obtained by those skilled in the art without creative labor shall fall within the scope of protection of the application.

[0023] In the embodiments of the present application, the chassis strain is Escherichia coli W3110. In order to better embody the present application, in the following embodiments, the Escherichia coli with the following genotype is used as the starting strain, and the genotype of the starting strain is E. coli W3110ΔtdcG::Ptrc-serA mut ΔsdaA::Ptrc-serBΔsdaB::Ptrc-serCΔtnaAΔcyuA::Ptrc-cysE T167A ΔyciW::Ptrc-cysM-nrdHΔycjV::Ptrc-eamB mut ΔyncI::Ptrc-eamAΔcydDC. The starting strain is recorded as strain CYS-0. The sequence information of the genes involved in the genotype is shown in Table 1. The gene accession number is the accession number in the GenBank gene (nucleic acid sequence database) of NCBI. The starting strain can be obtained by technical means in the field of biological engineering, which is prior art.

[0024] The starting strain can also be obtained by other prior art, for example, “Construction of Escherichia coli strain with high yield of l-cysteine based on carbon-sulfur module balance strategy” and “CydDC functions as a cytoplasmic cystine reductase to sensitize Escherichia coli to oxidative stress and aminoglycosides”.

[0025] Table 1 Gene name Gene source Gene accession number tdcG Escherichia coli 2847724 serA Escherichia coli 945258 sdaA Escherichia coli 946331 serB Escherichia coli 948913 sdaB Escherichia coli 947262 serC Escherichia coli 945527 tnaA Escherichia coli 948221 cyuA Escherichia coli 2847723 cysE Escherichia coli 948126 yciW Escherichia coli 945859 cysM Escherichia coli 946888 nrdH Escherichia coli 947161 ycjV Escherichia coli 945890 eamB Escherichia coli 947065 yncI Escherichia coli 945121 eamA Escherichia coli 946081 cydC Escherichia coli 945504 cydD Escherichia coli 949052 In the present application, the strain CYS-0 / pE is used as the chassis strain, the tcyP gene is knocked out to destroy the L-cysteine / L-cystine shuttle system, the outer membrane protein PqqU with L-cystine efflux capacity is overexpressed to make L-cystine in the periplasmic space be efficiently transported out of the cell; the galP gene is overexpressed to significantly improve the flow direction of carbon metabolic flow, so that the production of L-cysteine is flowed; and the zwf and talA genes are overexpressed to connect the metabolic flow of glycolysis and PP pathway, and the exogenous phosphoketolase encoding gene fxpK is introduced, and pta is overexpressed to make the carbon metabolic flow be reasonably distributed between 3-phosphoglycerate and acetyl-CoA necessary for SAT catalytic reaction, so that SAT can efficiently catalyze L-serine to synthesize OAS, and the overflow of L-serine is reduced. Ultimately, through the above strategies, L-cystine is reduced to enter the cell to consume reducing power GSH and NADPH, and more carbon metabolic flow is directed to the synthesis of L-cysteine, so that the efficient production of L-cysteine is realized. In an embodiment, the yield of the strain constructed in this way reaches 28.03 g / L in a 5L fermenter.

[0026] In the embodiments of the present application, the gene source, sequence information and functions involved are shown in Table 2.

[0027] Table 2 Gene name Gene source Nucleotide sequence Involved function tcyP Escherichia coli SEQ ID No. 1 Importin PqqU Escherichia coli SEQ ID No. 2 Efflux protein galP Escherichia coli SEQ ID No. 3 Initiating glucose, galactose and arabinose metabolism zwf Escherichia coli SEQ ID No. 4 Expressing phosphopentose pathway talA Escherichia coli SEQ ID No. 5 Encoding transaldolase A fxpK B. adolescentis SEQ ID No. 6 Encoding phosphoketolase pta Escherichia coli SEQ ID No. 7 Promoting AcP synthesis into acetyl-CoA In the embodiments of the present application, the primer names and sequences involved are shown in Table 3.

[0028] Table 3 In the embodiments of the present application, the formula of LB medium is as follows: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder; the LB solid medium additionally contains 4% agar powder.

[0029] In the embodiments of the present application, the formula of SM1 fermentation medium is as follows: 30 g / L glucose, 4.5 g / L KH2PO4, 9.5 g / L Na2HPO4, 8 g / L NH4Cl, 5 g / L (NH4)2SO4, 5 g / L Na2S2O3, 5 g / L yeast powder, 1 g / L peptone, 5 mg / L vitamin B1 and 1 mL / L salt solution; the salt solution is: 5 g / L MnSO4·8H2O, 500 g / L MgSO4·7H2O, 5 g / L FeSO4·7H2O, 5 g / L ZnSO4.

[0030] The formula of SM2 medium is as follows: 30 g / L glucose, 1 g / L KH2PO4, 1 g / L Na2HPO4, 10 g / L (NH4)2SO4, 10 g / L Na2S2O3, 5 g / L yeast powder, 10 g / L peptone, 2 g / L betaine, 5 mg / L vitamin B1, 1 g / L methionine, 1 g / L isoleucine, 1 g / L threonine and 1 mL / L salt solution; the salt solution is: 5 g / L MnSO4·8H2O, 500 g / L MgSO4·7H2O, 5 g / L FeSO4·7H2O, 5 g / L ZnSO4.

[0031] Example 1 In this embodiment, CYS-0 is used as the starting strain, and CRISPR-Cas9 gene editing technology is used to achieve the knockout of the tcyP gene with the nucleotide sequence shown in SEQ ID No. 1 on the genome.

[0032] The specific operation is as follows: 1-1: Construction of plasmid backbone pTarget Using the pTarget plasmid as a template, use the primers tcyP-PAM F and tcyP-PAM R listed in Table 3 to mutate the pTarget plasmid. After PCR amplification, perform electrophoresis analysis and analyze the PCR product band size using a gel imager. If successful, purify and recover the PCR product. Using the purified product as a template, linearize it using the primers pT-Line-F / pT-Line-R listed in Table 3. Detect the amplified linearized product by agarose gel electrophoresis and purify to obtain the linearized pTarget backbone.

[0033] 1-2: Obtaining DNA fragments Using the genome of E. coli W3110 strain as a template, PCR amplification was performed using tcyP-UP-F / tcyP-UP-R and tcyP-DOWN-F / tcyP-DOWN-R in Table 3 as primers to obtain the fragment tcyP-up and the fragment tcyP-down; the PCR products were detected by electrophoresis and purified and recovered.

[0034] 1-3: Construction of plasmid pT-ΔtcyP for knocking out tcyP The pTarget backbone linearized in step 1-1, the tcyP-up fragment and the tcyP-down fragment in step 1-2 were obtained by Nanjing Novozymes Recombinant seamless cloning was performed using the MultiS One-Step Cloning Kit. The ligated product was transformed into competent DH5α culture medium and incubated at 37°C for 12 hours. A single colony was selected from the plate as a template and verified by colony PCR using primers pTarget-YZ-F and pTarget-YZ-R (Table 3). The PCR amplification product was verified by agarose gel electrophoresis to confirm the band size. PCR products consistent with the target band size were sent to Qingke Biotechnology (Hangzhou) for sequencing and further verification. After sequencing verification, a single colony with the correct sequence was selected and incubated in a LB tube containing spectinomycin resistance at 37°C on a shaker for 14 hours. The plasmid was then extracted to obtain the plasmid pT-ΔtcyP for knocking out tcyP.

[0035] 1-4: Prepare CYS-0 competent cells and transform pCas plasmid A small amount of CYS-0 bacterial solution was taken from the glycerol cryovial with an inoculating loop and streaked onto antibiotic-free LB solid medium. The culture was incubated overnight at 37°C for 14 hours. A single colony was picked from the plate and inoculated into a test tube containing 5 mL of LB medium. The culture was incubated overnight at 37°C at 200 rpm in a shaker incubator for 14 hours. 1 mL of bacterial solution was transferred to 50 mL of antibiotic-free LB medium (the amount of bacterial solution was 2%) and incubated at 37°C at 200 rpm in a shaker incubator until the OD 600between 0.4-0.6, the shake flask was taken out and placed on ice for 12 min; under sterile conditions, the bacterial solution was poured into a 50 mL centrifuge tube, which was centrifuged at 4°C, 4000 rpm for 10 min to collect the bacterial cells. 20 mL of 0.1 M CaCl2 was added to resuspend the bacterial cells in ice, and the ice bath was performed for 30 min. The bacterial cells were collected by centrifugation at 4°C, 4000 rpm for 10 min, and 1 mL of pre-cooled sterile solution containing 0.1 M CaCl2 and 15% glycerol was added. After resuspension, it was divided into 1.5 mL sterile ep tubes (100 μL per tube), which was CYS-0 competent cells. 3 μL of pCas plasmid (Addgene Plasmid #62225) was placed in a centrifuge tube containing 100 μL of CYS-0 competent cells, and the ice bath was performed for 30 min. The 42°C heat shock was performed for 90 s (quickly placed, timing), and the ice bath was performed for 3 min. Then 700 μL of LB culture solution was added in the clean bench, and the 30°C incubator was cultured for 2.5-3 h. The centrifugation was performed at 5000 rpm for 3 min, and 200 μL of supernatant was taken. After resuspension, it was coated on a plate containing 50 mg / L kanamycin, and the 30°C culture was performed for 15 h. CYS-0 / pCas strain was obtained.

[0036] 1-5: Preparation of CYS-0 / pCas competent cells and transformation of pT-ΔtcyP plasmid to complete tcyP gene knockout A single colony was picked from the CYS-0 / pCas plate of step 1-4, inoculated into 10 mL of LB test tube containing 50 mg / L kanamycin and 100 μL of 1 M arabinose, and cultured at 30°C for 15 h. 200 μL was inoculated into an LB small shake flask, and the same final concentration of kanamycin and arabinose was also added. The 30°C culture was performed for 3-4 h, and the OD 600The OD600 of the bacteria was between 0.4 and 0.6. The pre-cooled bacterial solution was centrifuged at 4°C and 4000 rpm for 2 min. The supernatant was discarded, and an equal volume of pre-cooled sterile ultrapure water was added. After resuspension, it was centrifuged at 4°C and 4500 rpm for 2 min. This step was repeated twice, and pre-cooled 10% glycerol was added. The resuspension was aliquoted into 1.5 mL ep tubes (100 μL per tube) in an ice water bath. This was CYS-0 / pCas competent cells. 10 μL of plasmid pT-tcyP from steps 1-3 was added to the ultraclean table, mixed, and placed in an ice water bath for 1 min. Then it was transferred to a pre-cooled 2 mm electroporation cup, placed in an electroporation instrument, and quickly completed using ECO2. After the electroporation was completed, 1 mL of pre-cooled LB medium was added to the electroporation cup groove, and the entire bacterial solution was aspirated into a 2 mL sterile ep tube by tilting the electroporation cup. It was recovered at 30°C and 180 rpm for 3 h. It was centrifuged at 5000 rpm for 3 min, and 200 μL of supernatant was taken. After resuspension, it was plated on a plate containing spectinomycin and kanamycin, and incubated at 30°C for about 24 h. The primers tcyP-V F and tcyP-V R in Table 3 were designed about 100 bp outside the DNA. Single colonies on the plate were picked as templates for colony PCR verification. After agarose gel electrophoresis detection, the PCR product was sent to Genescript (Hangzhou) Co., Ltd. for sequencing for further verification, and the tcyP gene was knocked out.

[0037] 1-6: Elimination of pT-ΔtcyP plasmid and pCas plasmid The positive clones were inoculated in 10 mL LB tubes containing kanamycin, and 10 μL of 1 M IPTG stock solution was added. They were placed in a 30°C shaker at 180 rpm and cultured for 8-16 h. They were streaked on LB solid medium containing kanamycin and incubated at 30°C overnight. Single colonies were numbered, and some single colonies were picked with a small gun head and streaked on LB solid medium containing kanamycin and spectinomycin, respectively, and incubated at 30°C and 37°C overnight, respectively. Single colonies that could not grow in the region corresponding to LB solid medium containing spectinomycin were pT-ΔtcyP successfully eliminated clones, i.e., CYS-0 / pCas. The CYS-0 / pCas clone successfully eliminated pT-ΔtcyP was inoculated in 10 mL LB tubes without antibiotics and incubated at 37°C in a shaker at 180 rpm for 10 h. They were streaked on LB solid medium without antibiotics and incubated at 37°C overnight. Single colonies were numbered, and some single colonies were picked with a small gun head and streaked on LB solid medium containing kanamycin and without antibiotics, respectively, and incubated at 30°C and 37°C overnight, respectively. Single colonies that could not grow in the region corresponding to LB solid medium without antibiotics were pCas successfully eliminated clones, i.e., strain CYS-0ΔtcyP, which was designated as strain CYS-1.

[0038] 1-7: Shake flask fermentation of recombinant strain and yield detection To verify the ability of the strain CYS-0ΔtcyP obtained in step 1-6 to produce L-cysteine, single colonies of CYS-0ΔtcyP and the starting strain CYS-0 were inoculated into 10 LB test tubes, and cultured overnight at 37°C on a shaker at 180 rpm. Then, according to a 5% inoculation volume, the strains were transferred into sterilized SM1 fermentation medium, and 0.2 g of calcium carbonate, 10 μL of 0.1 M IPTG (final concentration: 0.1 mM), 20 μL of 50 g / L Kan (final concentration: 50 mg / L), and 20 μL of 0.2 g / L VB1 (final concentration: 0.2 mg / L) were added. Each strain was prepared in triplicate, and the cultures were incubated at 30°C on a shaker at 180 rpm for 48 h. After the fermentation, 1 mL of the fermentation broth was centrifuged at 12,000 rpm for 2 min to obtain the bacterial cells and supernatant A, and the supernatant was stored for subsequent detection. The precipitate in the flask contained both CaCO3 and L-cysteine. The precipitate was first washed twice with ddH2O, then dissolved in 1 M acetic acid to remove CaCO3, washed with PBS, and then dissolved in 500 μL of 0.5 M sulfuric acid to remove L-cysteine. The supernatant B was obtained by centrifugation and stored for subsequent detection. Finally, the precipitate was washed with PBS to obtain the bacterial cells. 600 The supernatants A and B were mixed, and 100 μL of the mixture was used for detection of the content of L-cysteine by high performance liquid chromatography. The results are shown in Table 1.

[0039] Table 4 Strain L-cysteine production (g / L) OD 600 ]] CYS-0 2.39 8.87 CYS-1 2.37 10.65 CYS-V2 2.50 10.91 CYS-2 3.43 14.79 CYS-3 3.74 15.42 CYS-4 4.62 16.07 CYS-5 4.98 18.12 CYS-6 6.51 19.60 CYS-7 8.74 19.98 As shown in Table 4, the starting strain CYS-0 of the present embodiment produced L-cysteine, and the yield of L-cysteine was 2.39 g / L. After the gene tcyP was knocked out on the genome, the strain did not show poor growth after the addition of antibiotics and shake flask fermentation, and the OD 600 reached 10.65.

[0040] Example 2 In this example, the outer membrane protein coding gene PqqU with the nucleotide sequence shown in SEQ ID No. 2 was overexpressed in the strain CYS-1, and the strain CYS-0ΔtcyP / pE-pqqU and shake flask fermentation were constructed.

[0041] The specific operation is as follows: 2-1: Construction of pE-pqqU plasmid The plasmid pTrc-99A-cysE was extracted A237V(denoted as pE) as a template, PCR amplification was performed using the linearized primers 99A-Line-F and 99A-Line-R in Table 3. The amplified PCR product was detected by agarose gel electrophoresis, and the band size was observed using a gel imager. After the band was correct, the PCR product was recovered to obtain the linearized pTrc-99A-cysE. A237V Using the wild-type W3110 genome as a template, primers pqqU-F and pqqU-R were designed to amplify the complete pqqU fragment; The MultiS One Step Cloning Kit was used for seamless recombination cloning. The product was transformed into DH5α competent cells. After single colonies were verified by colony PCR and sequencing, the plasmid pTrc99A-pqqU was extracted. This plasmid was used as a template and RBS-pqqU-F and RBS-pqqU-R in Table 3 were used as primers for PCR amplification to obtain a pqqU fragment connected to the RBS on the pTrc99A plasmid, namely RBS-pqqU. The linearized pTrc-99A-cysE obtained above was cloned into the pTrc99A-pqqU fragment. A237V The fragment and RBS-pqqU fragment were seamlessly cloned and transformed into DH5α competent cells. After single colonies were verified by colony PCR using primers 99A-VF and 99A-VR in Table 3 and sequencing was correct, the plasmid was extracted, which was the pE-pqqU plasmid.

[0042] Among them, pTrc-99A-cysE was obtained by inserting the cysE gene sequence (nucleotide sequence with gene ID 948126) into the multiple cloning site of plasmid pTrc-99A through genetic engineering.

[0043] 2-2: Obtaining the CYS-0ΔtcyP / pE-pqqU strain Take 3 μL of the plasmid pE-pqqU obtained in step 2-1 and put it into the ep tube containing 100 μL of CYS-0 competent cells and the ep tube of CYS-1 competent cells obtained in Example 1, respectively, ice bath for 30 min, heat shock at 42 ° C for 90 s (quickly put it in and time it), ice bath for 3 min, add 700 μL of LB culture medium in the clean bench, incubate at 37 ° C for 1-1.5 h, centrifuge at 5000 rpm for 3 min, take 200 μL of the supernatant, resuspend and spread on a plate containing 50 mg / L kanamycin, and culture at 37 ° C for 15 h to obtain CYS-0 / pE-pqqU strain and CYS-0ΔtcyP / pE-pqqU strain, which were recorded as CYS-V2 and CYS-2, respectively.

[0044] The production strain CYS-0ΔtcyP / pE-pqqU constructed in step 2-2 was fermented and tested in shake flasks according to steps 1-7 of Example 1. 600The results of L-cysteine content in the fermentation supernatant are shown in Table 4.

[0045] As shown in Table 4, after overexpression of the pqqU gene in the strain CYS-0, the yield of L-cysteine was only increased to 2.50 g / L; but in the strain CYS-1, i.e. in the strain CYS-0ΔtcyP, after overexpression of the pqqU gene by a plasmid, the yield of L-cysteine was increased from 2.37 g / L to 3.43 g / L, which indicated that overexpression of the gene could significantly enhance the yield of cysteine. It was inferred that there was some kind of cooperation between knocking out tcyP and overexpression of the pqqU gene, which could significantly enhance the yield of cysteine.

[0046] Example 3 In this example, the galP gene (nucleotide sequence as shown in SEQ ID No. 3) was overexpressed in the strain CYS-2, and the CYS-0ΔtcyP::Ptrc-galP / pE-pqqU was constructed and subjected to shake flask fermentation.

[0047] The specific operation is as follows: 3-1: Construction of pT-ΔtcyP::Ptrc-galP plasmid The plasmid pT-ΔtcyP was extracted as a template, and PCR amplification was carried out by using the linearized primers pT-Line-F and pT-Line-R in Table 3. The amplified PCR product was subjected to agarose gel electrophoresis detection, and the band size was observed by a gel imaging instrument. After the band was correct, the PCR product was recovered, i.e. the linearized pT-ΔtcyP was obtained. The wild type W3110 genome was used as a template, and the complete galP fragment was amplified by using the primers galP-F and galP-R in Table 3. The MultiSOne Step Cloning Kit was used for recombination seamless cloning. The product was transformed into DH5α competent cells, and single colony colony PCR verification and sequencing were carried out. After the correct plasmid pTrc99A-galP was extracted, the plasmid was used as a template, and the primers trc-galP-F and trc-galP-R in Table 3 were used for PCR amplification to obtain the galP fragment connected with the trc promoter on the pTrc99A plasmid, i.e. trc-galP. The linearized pT-ΔtcyP fragment and the trc-galP fragment obtained above were subjected to seamless cloning, and were transformed into DH5α competent cells. Single colony colony PCR verification and sequencing were carried out by using the primers pTarget-YZ-F and pTarget-YZ-R in Table 3. After the correct plasmid was extracted, it was the pT-ΔtcyP::Ptrc-galP plasmid. 3-2: Obtain CYS-0ΔtcyP::Ptrc-galP strain

[0048] ​Take 3 μL of the pT-ΔtcyP::Ptrc-galP plasmid constructed in step 3-1, add it to the ep tube containing 100 μL of CYS-0 / pCas competent cells obtained in Example 1, ice bath for 30 min, 42°C heat shock for 90 s (quickly put in, timing), add 700 μL of LB culture solution in a clean bench after ice bath for 3 min, 37°C incubator for 1 h, 5000 rpm centrifugation for 3 min, take 200 μL of supernatant, resuspend and spread on plates containing 50 mg / L kanamycin, 37°C culture for 15 h, and CYS-0ΔtcyP::Ptrc-galP strain is obtained.

[0049] 3-3: Preparation of CYS-0ΔtcyP::Ptrc-galP competent cell transformation pE plasmid According to the method of step 2-2, the plasmid pE-pqqU is transformed into CYS-0ΔtcyP::Ptrc-galP competent cells to obtain CYS-0ΔtcyP::Ptrc-galP / pE-pqqU strain, which is denoted as CYS-3.

[0050] 3-4: The production strain CYS-0ΔtcyP::Ptrc-galP / pE-pqqU constructed in step 3-3 is subjected to shake flask fermentation and detection according to step 1-7 of Example 1, with CYS-0 as the control group, OD 600 The detection results of L-cysteine content in the fermentation supernatant are shown in Table 4.

[0051] As can be seen from Table 4, overexpression of galP on the plasmid, through Ptrc-galP gene to establish a new carbon metabolic driving force, to promote the synthesis rate of 3-phosphoglycerate, and then form a cascade amplification effect to drive L-cysteine synthesis, which can effectively improve the flow direction of carbon metabolic flow, so that the yield of cysteine is significantly increased, and the yield is increased to 3.74 g / L.

[0052] Example 4 In this example, the zwf gene (nucleotide sequence as shown in SEQ ID No. 4), talA gene (nucleotide sequence as shown in SEQ ID No. 5), exogenous fxpK gene (nucleotide sequence as shown in SEQ ID No. 6), and pta gene (nucleotide sequence as shown in SEQ ID No. 7) are overexpressed in the strain CYS-3 to construct the CYS-7 strain, and shake flask fermentation is carried out.

[0053] The specific operation is as follows: 4-1: The same as the method of overexpressing gene galP in strain CYS-2 in Example 3, overexpressing zwf gene with nucleotide sequence as shown in SEQ ID No. 4 in strain CYS-3, to obtain CYS-0ΔtcyP::Ptrc-galP::Ptrc-zwf / pE-pqqU strain, recorded as CYS-4.

[0054] 4-2: The same as the method of overexpressing gene galP in strain CYS-2 in Example 3, overexpressing talA gene with nucleotide sequence as shown in SEQ ID No. 5 in strain CYS-4, to obtain CYS-0ΔtcyP::Ptrc-galP::Ptrc-zwf::Ptrc-talA / pE-pqqU strain, recorded as CYS-5.

[0055] 4-3: The same as the method of overexpressing gene galP in strain CYS-2 in Example 3, overexpressing fxpK gene with nucleotide sequence as shown in SEQ ID No. 6 in strain CYS-5, to obtain CYS-0ΔtcyP::Ptrc-galP::Ptrc-zwf::Ptrc-talA::Ptrc-fxpK / pE-pqqU strain, recorded as CYS-6.

[0056] 4-4: The same as the method of overexpressing gene PqqU in strain CYS-1 in Example 2, overexpressing pta gene with nucleotide sequence as shown in SEQ ID No. 7 in strain CYS-6, to obtain CYS-0ΔtcyP::Ptrc-galP::Ptrc-zwf::Ptrc-talA::Ptrc-fxpK / pE-pqqU-pta strain, recorded as CYS-7.

[0057] 4-5: The production strains CYS-4, CYS-5, CYS-6, CYS-7 constructed in steps 4-1 to 4-4 were subjected to shake flask fermentation and detection according to step 1-7 in Example 1, with CYS-0 as the control group, OD 600 The detection results of L-cysteine content in fermentation supernatant are shown in Table 4.

[0058] As shown in Table 4, by overexpressing zwf, talA, and introducing the exogenous phosphoketolase gene fxpK, and overexpressing pta, the final yield of L-cysteine can reach 8.74 g / L. It is speculated that the principle lies in that overexpression of zwf, talA, fxpK, and pta can reasonably distribute the carbon metabolic flow between 3-phosphoglycerate and acetyl-CoA necessary for SAT catalytic reaction, so that SAT can efficiently catalyze L-serine to synthesize OAS, and more carbon metabolic flow can be directed to the synthesis of L-cysteine.

[0059] Example 5 As shown in Table 4, the recombinant strain CYS-0ΔtcyP::Ptrc-galP::Ptrc-zwf::Ptrc-talA::Ptrc-fxpK / pE-pqqU-pta (strain CYS-7) is the optimal strain, and through the modification of Examples 1 to 5, the carbon metabolic flow can be further enhanced to promote the production of more L-cysteine, and the final yield of L-cysteine can reach 8.74 g / L.

[0060] In this example, strain CYS-7 was inoculated and fermented in a 5L fermenter, and the fermentation method was as follows: The strain CYS-7 was streaked onto a plate resistant to Kan, and after overnight culture in a 37°C constant temperature incubator, a single colony was picked and inoculated in 10 mL of LB test tube to complete the culture of the primary seed liquid. The primary seed liquid was inoculated in two bottles containing 100 mL of SM1 medium at a 5% inoculation ratio, and the bottles were placed in a 30°C, 180 rpm shaker for 12 h to obtain the secondary seed liquid. 2L of SM2 medium was added to the 5L fermenter after air blowing, sterilized, and then connected to the pipeline and connected to the alkali supplement bottle. The pH was adjusted to 7.0, the temperature was adjusted to 30°C, and then the secondary seed liquid was inoculated into the fermenter at a 10% inoculation amount. The aeration amount was 8vvm (i.e. the aeration volume:liquid volume was 4:1), the rotation speed was linked with the dissolved oxygen, and the dissolved oxygen control was set to 30%. After the initial sugar was consumed, IPTG with a final concentration of 0.9 mM was added and the feed bottle was connected for feeding. The feeding strategy was adjusted to pH linkage feeding, i.e. when the pH was lower than 7.0, ammonia water was added to raise the pH, and when the pH was higher than 7.10, glucose was added to lower the pH, and the fermentation temperature was adjusted to 24°C. Then samples were taken every 4 h for detection.

[0061] After fermentation, the content of L-cysteine in the supernatant was detected by high performance liquid chromatography, and the results showed that the fermentation yield of L-cysteine was 28.03 g / L. It can be seen that strain CYS-7 can also achieve efficient production of L-cysteine in a 5L fermenter.

[0062] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0063] The above is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent transformation of the above embodiment based on the technical essence of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A genetically engineered bacterium for high L-cysteine ​​production, characterized by: It is obtained by knocking out the tcyP gene in the chassis bacteria genome, overexpressing the PqqU gene, galP gene, zwf gene, talA gene, and pta gene, and introducing and overexpressing the fxpK gene.

2. The genetically engineered bacterium for high L-cysteine ​​production according to claim 1, wherein: The bottom plate bacteria is Escherichia coli.

3. The genetically engineered bacteria for high L-cysteine ​​production according to claim 1, wherein: The fxpK gene comes from Bifidobacterium adolescentis.

4. The genetically engineered bacterium for high L-cysteine ​​production according to claim 1, wherein: The sequence of the tcyP gene is shown in SEQ ID No. 1, the sequence of the pqqU efflux protein is shown in SEQ ID No. 2, the sequence of the galP gene is shown in SEQ ID No. 3, the sequence of the zwf gene is shown in SEQ ID No. 4, the sequence of the talA gene is shown in SEQ ID No. 5, the sequence of the fxpK gene is shown in SEQ ID No. 6, and the sequence of the pta gene is shown in SEQ ID No.

7.

5. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 4, wherein: The following steps are performed in the bottom plate: Knockout of the tcyP gene; Overexpression of the PqqU gene; Overexpression of the galP gene; Overexpression of the zwf gene; Overexpression of the talA gene; Overexpression of the pta gene; Overexpression of the fxpK gene from Bifidobacterium adolescentis.

6. The construction method according to claim 5, wherein: The base bacteria is E. coli W3110ΔtdcG::Ptrc-serA mut ΔsdaA::Ptrc-serBΔsdaB::Ptrc-serCΔtnaAΔcyuA::Ptrc-cysE T167A ΔyciW::Ptrc-cysM-nrdHΔycjV::Ptrc-eamB mut ΔyncI::Ptrc-eamAΔcydDC.

7. The construction method according to claim 5, wherein: The sequence of the tcyP gene is shown in SEQ ID No. 1, the sequence of the pqqU efflux protein is shown in SEQ ID No. 2, the sequence of the galP gene is shown in SEQ ID No. 3, the sequence of the zwf gene is shown in SEQ ID No. 4, the sequence of the talA gene is shown in SEQ ID No. 5, the sequence derived from the fxpK gene is shown in SEQ ID No. 6, and the sequence of the pta gene is shown in SEQ ID No.

7.

8. Use of the genetically engineered bacterium according to any one of claims 1 to 4 or the genetically engineered bacterium constructed by the construction method according to any one of claims 5 to 7 in the production of L-cysteine.

9. The use according to claim 8, characterized in that: The application method comprises the following steps: inoculating the genetically engineered bacteria into a fermentation medium, and fermenting and culturing the culture medium at 30-37° C. and 180-200 rpm to obtain L-cysteine.

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