Genetically engineered bacterium for producing L-arginine as well as construction method and application of genetically engineered bacterium

Abstract

The invention relates to the technical field of genetic engineering, and discloses a genetically engineered bacterium for producing L-arginine as well as a construction method and application of the genetically engineered bacterium. Escherichia coli is taken as a starting strain, and the following treatment is performed: overexpressing genes gdhA and gdh of glutamate dehydrogenase, promoting generation of glutamic acid, overexpressing and coding arginine transporter gene argO to enhance extracellular transport of arginine, introducing exogenous argJ to replace a linear synthesis pathway in large intestines, improving the utilization rate of acetyl coenzyme A and accelerating the synthesis efficiency of arginine; a glutamine synthetase gene glnA is over-expressed, and glutamine synthesis is enhanced; the pckA is overexpressed to promote ATP synthesis; meanwhile, speA and adiA genes for coding the arginine decarboxylase are deleted, so that further synthesis of putrescine from L-arginine is inhibited, and the L-arginine accumulation capability of the strain is improved. Through the combined action of the treatment, a genetically engineered bacterium with extremely high L-arginine production yield is finally obtained, and the shake flask fermentation yield can reach 13.7 g / L.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to a gene engineering bacterium for producing L-arginine, a construction method and application thereof. Background Art

[0002] L-arginine, whose systematic name is 2-amino-5-guanidine valeric acid, is one of the semi-essential basic amino acids required by humans and animals. Its molecular weight is 174 and its chemical formula is C6H 14 N4O2 is easily soluble in water, slightly soluble in ethanol, and insoluble in ether. It is an important intermediate metabolite of the urea cycle of organisms, and has the physiological function of catalyzing the ornithine cycle, promoting the formation of urea, and reducing the concentration of blood ammonia. It has the functions of regulating blood pressure, promoting the secretion of hormones by endocrine glands, inhibiting the induction and occurrence of tumors, and promoting the healing and recovery of damaged tissues. At present, it has been widely used in the fields of medicine, industry, food, cosmetics, animal husbandry, etc., and has important economic and social value.

[0003] The production methods of L-arginine mainly include chemical synthesis, protein hydrolysis extraction and microbial fermentation. The D-type optical enantiomer is easily produced during the chemical synthesis production process, resulting in high cost of separation and purification of the subsequent products. Therefore, the chemical synthesis method is difficult to put into practical application. At present, my country's arginine production capacity is limited, and the hydrolysis method was mainly used in the early days. The hydrolysis method is to heat protein raw materials rich in L-arginine, such as pig skin, hair and animal blood, under acidic conditions to promote the cleavage of peptide bonds to obtain a variety of amino acid mixtures, and then purify L-arginine from it by ion exchange resin or barium hydroxide separation method. The hydrolysis method is low in cost and the raw materials are easy to obtain, but its production process will discharge a large amount of wastewater containing strong acid, which is easy to cause serious environmental pollution problems, poor quality stability, low recovery rate, and gradually eliminated by the market.

[0004] Microbial fermentation is a method of using cheap carbon and nitrogen sources as substrates, and screening high-yield mutants through mutagenesis or metabolic modification of model strains to achieve the purpose of directly fermenting L-arginine with cheap carbon sources. The production process of L-arginine produced by fermentation is relatively simple, has relatively little impact on the environment, is low in cost, has high yield, and has high product purity. It is suitable for large-scale industrial production. Microbial fermentation has a good development prospect in the pharmaceutical industry.

[0005] Initially, industrial strains for producing L-arginine using microbial fermentation were mainly obtained by physical radiation, chemical reagent mutagenesis, and arginine analog screening. Since the entire synthesis pathway of L-arginine is long, there are many feedback controls in the synthetic metabolic pathway, and the metabolic network involved in the precursors required for the synthesis of L-arginine is complex, the strains obtained by traditional mutagenesis breeding have poor stability and unclear metabolic networks.

[0006] The metabolic engineering transformation method has the advantages of clear goals, convenient molecular manipulation and stable fermentation process, making it a better choice for constructing industrial production strains of L-arginine. There is an urgent need for a metabolic transformation method for producing L-arginine engineered bacteria in this field to obtain a more stable high-yield L-arginine engineered strain. Summary of the invention

[0007] In order to solve the above technical problem of stably producing L-arginine, the present invention provides a genetically engineered bacterium for producing L-arginine, a construction method and application thereof.

[0008] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a genetically engineered bacterium for producing L-arginine. In the genome of the engineered bacterium, the poxB gene encoding pyruvate dehydrogenase, the ldhA gene encoding lactate dehydrogenase, the speA gene encoding arginine decarboxylase and the adiA gene are knocked out; Overexpression of the argG gene encoding sperminosuccinate synthetase, the argH gene encoding arginosuccinate lyase, the glnA gene encoding glutamine synthetase and the gdh gene encoding glutamate dehydrogenase, the pntAB gene encoding nicotinamide adenine dinucleotide transhydrogenase, the sthA gene encoding pyridine nucleotide transhydrogenase, the amtB gene encoding ammonium transporter, the pckA gene encoding phosphoenolpyruvate carboxykinase, the acnAB gene encoding aconitate hydratase, the icd gene encoding isocitrate dehydrogenase, and the gltA gene encoding citrate synthase; heterologous overexpression of the argC gene encoding N-acetyl-γ-glutamyl-phosphate reductase, the argJ gene encoding ornithine acetyltransferase, the argB gene encoding acetylglutamate kinase, the argD gene encoding acetyl-ornithine aminotransferase, and the argF gene encoding ornithine carbamoyltransferase; The engineered bacteria are obtained by transformation using Escherichia coli as a host bacterium.

[0009] The present invention uses Escherichia coli as a starting strain and performs the following treatments: overexpressing the genes gdhA and gdh of glutamate dehydrogenase to promote the generation of glutamate, overexpressing the gene argO encoding the arginine transporter to enhance the extracellular transport of arginine, introducing exogenous argJ to replace the linear synthesis pathway in the large intestine, improving the utilization rate of acetyl-CoA, and accelerating the efficiency of arginine synthesis; overexpressing the glutamine synthetase gene glnA to strengthen glutamine synthesis; overexpressing pckA to promote ATP synthesis; and simultaneously deleting the speA and adiA genes encoding the arginine decarboxylase to inhibit L-arginine from further synthesizing putrescine and improve the ability of the strain to accumulate L-arginine. Through the combined effect of the above treatments, the production performance of L-arginine of the starting strain is greatly improved, and finally a genetically engineered bacterium with extremely high L-arginine production is obtained, and the shake flask fermentation yield can reach 13.7 g / L.

[0010] As a preferred embodiment of the above-mentioned genetically engineered bacteria, the gene to be heterologously overexpressed is derived from Corynebacterium glutamicum.

[0011] In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium for producing L-arginine, characterized in that it comprises the following steps: Using Escherichia coli as the host bacteria, the CRISPR-Cas9 gene editing technology was used to knock out the poxB gene, ldhA gene, speA gene and adiA gene in the host bacteria genome, and then the gdhA gene, ppk gene, glnA gene, pckA gene, gltA gene, acnAB gene, icd gene, amtB gene, sthA gene, pntAB gene, argC gene, argJ gene, argB gene, argD gene, argF gene, argGH gene and argO gene fragments were introduced and integrated into the host bacteria genome for overexpression.

[0012] As a preferred embodiment of the above construction method, the argC gene, argJ gene, argB gene, argD gene and argF gene are derived from Corynebacterium glutamicum.

[0013] As a preferred embodiment of the above construction method, the overexpression method is: the target gene is controlled by a strong promoter Ptrc so that the target gene is overexpressed.

[0014] In a third aspect, the present invention provides the use of the above genetically engineered bacteria in the preparation of L-arginine.

[0015] In a fourth aspect, the present invention provides a method for producing L-arginine by fermentation, wherein the method is characterized in that the fermentation is carried out using the genetically engineered bacteria as described above.

[0016] Compared with the prior art, the present invention has the following technical effects: The present invention uses Escherichia coli as a starting strain and performs the following treatments: overexpressing the genes gdhA and gdh of glutamate dehydrogenase to promote the generation of glutamate, overexpressing the gene argO encoding the arginine transporter to enhance the extracellular transport of arginine, introducing exogenous argJ to replace the linear synthesis pathway in the large intestine, improving the utilization rate of acetyl-CoA, and accelerating the efficiency of arginine synthesis; overexpressing the glutamine synthetase gene glnA to strengthen glutamine synthesis; overexpressing pckA to promote ATP synthesis; and simultaneously deleting the speA and adiA genes encoding arginine decarboxylase to inhibit further synthesis of putrescine from L-arginine and improve the ability of the strain to accumulate L-arginine. Through the combined effect of the above treatments, the production performance of L-arginine of the starting strain is greatly improved, and finally a genetically engineered bacterium with extremely high L-arginine production is obtained, and the shake flask fermentation yield can reach 13.7 g / L. The present invention can provide an industrial basis for direct fermentation production of L-arginine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the pCas9 plasmid map; Figure 2 is the pEC-99A plasmid map; Figure 3 This is the pTarget plasmid map. DETAILED DESCRIPTION

[0018] The present invention uses Escherichia coli W3110 as a host bacterium for genetic engineering transformation, in order to provide a more detailed description of the present invention.

[0019] The invention uses Escherichia coli W3110 as a host bacterium, over-expresses the gdhA gene encoding glutamate dehydrogenase, the glnA gene encoding glutamine synthetase, the pckA gene encoding phosphoenolpyruvate carboxykinase, the pntAB gene encoding nicotinamide adenine dinucleotide transhydrogenase (NAD(P) transhydrogenase), the sthA gene encoding pyridine nucleotide transhydrogenase and the amtB gene encoding ammonium transporter in Escherichia coli W3110; and over-expresses the gene encoding glutamate dehydrogenase in Corynebacterium glutamicum ATCC 13032. The gdh gene encoding N-acetyl-γ-glutamyl-phosphate reductase, argC gene encoding N-acetyl-γ-glutamyl-phosphate reductase, argJ gene encoding ornithine acetyltransferase, argB gene encoding acetylglutamate kinase, argD gene encoding acetylornithine aminotransferase, and argF gene encoding ornithine carbamoyltransferase were overexpressed; the argG gene encoding argininosuccinate synthetase and the argH gene encoding argininosuccinate lyase were overexpressed, and at the same time, the genes poxB encoding pyruvate dehydrogenase, ldhA encoding lactate dehydrogenase, and arginine decarboxylase were overexpressed. The speA and adiA genes of the decarboxylase were knocked out. After the above gene editing, a genetically recombinant strain was obtained, and the fermentation production of L-arginine was studied.

[0020] Specifically, the host bacteria are firstly made to lack arginine decarboxylase activity to block the further degradation of L-arginine into spermidine; and the target genes controlled by the strong promoter Ptrc are inserted into the genome to overexpress the genes encoding glutamate dehydrogenase gdhA, glutamine synthetase glnA, phosphoenolpyruvate carboxykinase pckA, nicotinamide adenine dinucleotide transhydrogenase pntAB, pyridine nucleotide transhydrogenase sthA, ammonium transporter amtB, aconitate hydratase acnAB, isocitrate dehydrogenase icd and citrate synthase gltA, so as to enhance the transport of arginine and the synthesis expression of its precursor glutamate.

[0021] In the present invention, the citrulline produced by the host bacteria is further degraded into arginine by knocking out the host's speA and adiA genes encoding arginine decarboxylase.

[0022] In the present invention, the host bacteria are enhanced to synthesize glutamate, a precursor of arginine, mainly by inserting glutamate dehydrogenase gene gdh, aconitate hydratase gene acnAB, isocitrate dehydrogenase gene icd and citrate synthase gene gltA controlled by a strong promoter Ptrc into the genome.

[0023] In the present invention, the host bacteria are enhanced in arginine synthesis mainly by inserting a gene cluster overexpressing a gene encoding N-acetyl-γ-glutamyl-phosphate reductase gene argC, an ornithine acetyltransferase gene argJ, an acetylglutamate kinase gene argB, an acetyl-ornithine aminotransferase gene argD, an ornithine carbamoyltransferase gene argF, a spermine succinate synthetase gene argG and an arginine succinate lyase gene argH from Corynebacterium glutamicum controlled by a strong promoter Ptrc into the genome.

[0024] In the present invention, the host bacteria are enhanced to transport arginine mainly by inserting a target gene controlled by a strong promoter Ptrc into the genome to overexpress the arginine transporter gene argO.

[0025] In the present invention, the genetically engineered bacteria adopts CRISPR-Cas9 gene editing technology to perform site-specific modification on the host genome.

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the following embodiments, the genes and corresponding pathways involved in gene editing are shown in Table 1. Among them, the accession number is an identifier used to uniquely identify a gene in the Entrez Gene database of the National Center for Biotechnology Information (NCBI).

[0027] Table 1 Genes involved in gene editing and corresponding pathways Gene name Accession Number Involved pathways argJ 69621908 L-Arginine Synthesis argC 69621907 L-Arginine Synthesis argB 69621909 L-Arginine Synthesis argD 69621910 L-Arginine Synthesis argF 69621911 L-Arginine Synthesis argG 69621913 L-Arginine Synthesis argH 69621914 L-Arginine Synthesis poxB 946132 Acetic acid synthesis oeLh 946315 Lactic acid synthesis speA 947432 Agmatine Synthesis adiA 948638 Agmatine Synthesis gLn 948370 Glutamine synthesis gd Cg2280 Glutamate synthesis gD 946802 Glutamate synthesis pntA 946628 Transhydrogenation between NADH and NADP and respiration pntB 946144 Transhydrogenation between NADH and NADP and respiration sthA 948461 Peripheral catabolic pathways B 945084 Ammonia transport pckA 945667 Phosphoenolpyruvate synthesis acn 946724 Isocitrate synthesis acnB 944864 Isocitrate synthesis icd 945702 α-Ketoglutarate synthesis gltA 945323 TCA cycle argO 947418 Arginine transport yx ECK2993 Pseudogene yncI ECK1452 Pseudogene Y ECK2674 Pseudogene Yj ECK4333 Pseudogene Yj Y ECK0308 Pseudogene Yj ECK4329 Pseudogene argR 947861 Amino acid synthesis yeeP ECK1991 Pseudogene Yj Y ECK1313 Pseudogene r ECK3633 Pseudogene pd 944827 TCA cycle HkDJ ECK1157 Pseudogene In the following examples, the primer sequences involved in gene editing are shown in Table 2.

[0028] Table 2 Primer sequence list Example 1 Construction of genetically engineered bacteria W3110-R 1. Knockout of poxB, ldhA, speA and adiA genes The poxB, ldhA, speA and adiA genes in Escherichia coli W3110 were knocked out using CRISPR-Cas9 gene editing technology to obtain the W3110-ΔpoxB-ΔldhA-ΔspeA-ΔadiA strain. There are two tool plasmids in this editing system. The plasmid map is shown in Figure 1 and 2 , where pCas9 carries the elimination system of the sgRNA expression plasmid pTarget, the Red recombination system and the Cas9 protein expression system, kanamycin resistance (working concentration: 50 mg / L), and is cultured at 32°C; pTarget contains the J23119 (SpeI) promoter, the sgRNA and Cas9 protein binding region sequence, and spectinomycin resistance (working concentration: 50 mg / L). Figure 3 The pTarget plasmid map is shown.

[0029] The specific operations of this step are as follows: 1. Construction of pTarget plasmid (1) Construction of plasmid The purpose of constructing the pTarget plasmid is to transcribe the corresponding sgRNA in the bacteria, combine with the Cas9 protein to form a complex, recognize the target gene through base complementary pairing, and cut it, causing the DNA double strand of the target gene to break. The target sequence was designed using chopchop, and the pTarget plasmid was constructed using PCR.

[0030] The pTarget-poxB, pTarget-ldhA, pTarget-speA and pTarget-adiA plasmids containing the target sequence were amplified by reverse PCR using primers poxB-PAM-F, poxB-PAM-R, ldhA-PAM-F, ldhA-PAM-R, speA-PAM-F, speA-PAM-R and adiA-PAM-F, adiA-PAM-R, respectively. The template plasmid was removed by digestion with DpnⅠ at 37℃ for 40min.

[0031] (2) Plasmid transformation Take 5uL PCR reaction solution, add it to 30uL DH5α transfection competent medium, mix well, place on ice for 15min, heat shock at 42℃ for 45-90s, immediately ice bath for 3min, add 800uL LB medium, and recover at 37℃ for 60min. Centrifuge at 5000rpm for 2min, discard the supernatant, keep 100uL to resuspend the bacteria and apply it to a plate containing 50mg / L spectinomycin, turn the plate upside down, and culture at 37℃ overnight. After the colonies grow on the plate, select the positive recombinants.

[0032] The selected positive recombinants were inoculated into a culture medium containing spectinomycin resistance and cultured overnight to extract the plasmid for later use.

[0033] 2. Preparation of recombinant DNA fragments The recombinant DNA fragment consists of the upstream and downstream homologous arms of the knocked-out gene. The software snapgene was used to design primers. The upstream and downstream sequences of the gene to be knocked out were used as templates to design upstream and downstream homologous arm primers (amplification length is about 500bp); the amplification primers of the integrated gene sequence were designed using the gene to be integrated as a template. The upstream and downstream homologous arms were amplified separately by PCR, and the recombinant fragments were prepared by overlapping PCR.

[0034] Taking poxB gene knockout as an example, the preparation of the recombinant DNA fragment of poxB gene knockout is described. Similarly, recombinant DNA fragments of ldhA, speA and adiA knockout are prepared. The preparation of the recombinant DNA fragment of poxB gene knockout is as follows: First, the genome of Escherichia coli W3110 is used as a template, and the upstream and downstream homology arm fragments required for knocking out the poxB gene are amplified using primers poxB-U-1 / poxB-U-2 and primers poxB-D-1 / poxB-D-2, and then the two fragments are purified and recovered respectively. The two fragments are connected by fusion PCR using primers poxB-U-1 / poxB-D-2 to make a recombinant DNA fragment, which is purified and recovered for use. The primer sequences are shown in Table 2.

[0035] 3. Transformation of plasmids and recombinant fragments (1) Transformation of pCas plasmid The plasmid was transformed into the prepared W3110 competent cells by chemical transformation, and the cells were revived and cultured at 30°C and then plated on LB plates containing kanamycin for overnight culture. Single colonies grown on the resistant plates were identified to screen out the correct recombinants.

[0036] (2) Preparation of electroporation competent cells of W3110 strain containing pCas9 plasmid The W3110 strain containing the pCas9 plasmid was cultured at 30°C, arabinose was added to induce the expression of the pCas9 plasmid recombinase system, and the competent state was prepared when the OD was 0.5.

[0037] (3) Electroporation of pTarget and recombinant DNA fragments.

[0038] pTarget and the recombinant DNA fragment were simultaneously electro-transformed into the electro-transformation competent medium of the W3110 strain containing the pCas9 plasmid, and then revived for 3 hours, plated on a plate containing kanamycin and spectinomycin, and cultured overnight at 30°C. Colony PCR verification was performed using the upstream primer of the upper homology arm and the downstream primer of the lower homology arm to screen out positive recombinants.

[0039] 4. Elimination of tool plasmids (1) Elimination of pTarget plasmid.

[0040] The positive recombinants were placed in LB medium containing IPTG and kanamycin resistance and cultured at 30°C for 16 hours, then streaked on a plate containing kanamycin and cultured at 30°C. The single colonies that grew on the kanamycin plate but not on the spectinomycin plate were selected for preservation.

[0041] (2) Elimination of pCas9 plasmid The positive recombinants that eliminated the pTarget plasmid were inoculated into LB medium without antibiotics, cultured at 37°C, streaked on LB plates, and cultured at 42°C. The single colonies that grew on the LB plate without antibiotics but did not grow on the kanamycin plate were selected and preserved. Finally, the electrophoresis diagram verified that the poxB, ldhA, speA, and adiA genes were successfully knocked out.

[0042] 2. Construction of knock-in plasmids for overexpression of gdhA, gdh, glnA, pckA, gltA, acnAB, icd, amtB, sthA, pntAB, argCJBDF, argGH and argO genes 1. The construction of knock-in plasmids for overexpression of gdhA, gdh, glnA, pckA, gltA, acnAB, icd, amtB, sthA, pntAB, argCJBDF, argGH and argO genes is as follows: The knock-in plasmid pT-yncI::P trc Construction of gdhA plasmid: The Escherichia coli W3110 genome and plasmid pEC-99A were used as templates (the plasmid map used is shown in Figure 2), using 99A-gdhA-1, 99A-gdhA-2, 99A-line-F and 99A-line-R as primers to amplify the gdhA gene fragment and the pEC-99A plasmid fragment, and then using the gene recombination method to construct a new plasmid 99A-gdhA containing the gdhA gene; using the Escherichia coli (Escherichiacoli) W3110 genome, plasmid 99A-gdhA and plasmid pTarget as templates, using yncI-U-1, yncI-U-2, yncI-D-1, yncI-D-2, TRC-gdhA-1, TRC-gdhA-2, pT-line-1 and pT-line-2 as primers to amplify the yncI homology arm, the pEC-99A plasmid fragment, and the pEC-99A plasmid fragment. trc The gdhA gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc gdhA-inserted pT-yncI::P trc gdhA new plasmid.

[0043] The knock-in plasmid pT-yeeP::P of gdh gene trc Construction of gdh plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-gdh-1, 99A-gdh-2, 99A-line-F and 99A-line-R were used as primers to amplify the gdh gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-gdh containing the gdhA gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-gdh and plasmid pTarget as templates, yeeP-U-1, yeeP-U-2, yeeP-D-1, yeeP-D-2, TRC-gdh-1, TRC-gdh-2, pT-line-1 and pT-line-2 were used as primers to amplify the yeeP homology arm, the P trc The gdh gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc gdhA-inserted pT-yeeP::P trc gdh new plasmid.

[0044] The knock-in plasmid pT-yjiT::P trcConstruction of glnA plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-glnA-1, 99A-glnA-2, 99A-line-F and 99A-line-R were used as primers to amplify the glnA gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-glnA containing the glnA gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-glnA and plasmid pTarget as templates, yjiT-U-1, yjiT-U-2, yjiT-D-1, yjiT-D-2, TRC-glnA-1, TRC-glnA-2, pT-line-1 and pT-line-2 were used as primers to amplify the yjiT homology arm, the P trc The promoter glnA gene fragment and the pTarget plasmid fragment were then used to construct P trc pT-yjiT::P trc glnA new plasmid.

[0045] The knock-in plasmid pT-ygaY::P trc Construction of pckA plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-pckA-1, 99A-pckA-2, 99A-line-F and 99A-line-R were used as primers to amplify the pckA gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-pckA containing the pckA gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-pckA and plasmid pTarget as templates, ygaY-U-1, ygaY-U-2, ygaY-D-1, ygaY-D-2, TRC-pckA-1, TRC-pckA-2, pT-line-1 and pT-line-2 were used as primers to amplify the ygaY homology arm, the P trc The pckA gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc pckA-inserted pT-ygaY::P trc pckA new plasmid.

[0046] The knock-in plasmid of gltA gene pT-pdhR::P trcConstruction of gltA plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-gltA-1, 99A-gltA-2, 99A-line-F and 99A-line-R were used as primers to amplify the gltA gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-gltA containing the gltA gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-gltA and plasmid pTarget as templates, pdhR-U-1, pdhR-U-2, pdhR-D-1, pdhR-D-2, TRC-gltA-1, TRC-gltA-2, pT-line-1 and pT-line-2 were used as primers to amplify the pdhR homology arms, the pD-1 containing P trc The gltA gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc gltA-inserted pT-pdhR::P trc gltA new plasmid.

[0047] acnAB gene knock-in plasmid pT-rph::P trc Construction of acnAB plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-acnAB-1, 99A-acnAB-2, 99A-line-F and 99A-line-R were used as primers to amplify the acnAB gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-acnAB containing the acnAB gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-acnAB and plasmid pTarget as templates, rph-U-1, rph-U-2, rph-D-1, rph-D-2, TRC-acnAB-1, TRC-acnAB-2, pT-line-1 and pT-line-2 were used as primers to amplify the rph homology arms, the P trc The promoter acnAB gene fragment and pTarget plasmid fragment were then used to construct P trc acnAB inserted into pT-rph::P trc acnAB new plasmid.

[0048] icd gene knock-in plasmid pT-ycjV::P trcConstruction of icd plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-icd-1, 99A-icd-2, 99A-line-F and 99A-line-R as primers were used to amplify the icd gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-icd containing the icd gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-icd and plasmid pTarget as templates, ycjV-U-1, ycjV-U-2, ycjV-D-1, ycjV-D-2, TRC-icd-1, TRC-icd-2, pT-line-1 and pT-line-2 were used as primers to amplify the ycjV homology arms, the P trc The icd gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc icd-inserted pT-ycjV::P trc icd new plasmid.

[0049] The knock-in plasmid pT-yjiP::P of amtB gene trc Construction of amtB plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-amtB-1, 99A-amtB-2, 99A-line-F and 99A-line-R were used as primers to amplify the amtB gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-amtB containing the amtB gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-amtB and plasmid pTarget as templates, yjiP-U-1, yjiP-U-2, yjiP-D-1, yjiP-D-2, TRC-amtB-1, TRC-amtB-2, pT-line-1 and pT-line-2 were used as primers to amplify the yjiP homology arm, the P trc The amtB gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc pT-yjiP::P with amtB inserted trc amtB new plasmid.

[0050] sthA gene knock-in plasmid pT-ykgH::P trcConstruction of sthA plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-sthA-1, 99A-sthA-2, 99A-line-F and 99A-line-R were used as primers to amplify the sthA gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-sthA containing the sthA gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-sthA and plasmid pTarget as templates, ykgH-U-1, ykgH-U-2, ykgH-D-1, ykgH-D-2, TRC-sthA-1, TRC-sthA-2, pT-line-1 and pT-line-2 were used as primers to amplify the ykgH homology arms, the P trc The promoter sthA gene fragment and pTarget plasmid fragment were then used to construct P trc pT-ykgH::P trc sthA new plasmid.

[0051] The knock-in plasmid pT-yghX::P trc Construction of pntAB plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-pntAB-1, 99A-pntAB-2, 99A-line-F and 99A-line-R were used as primers to amplify the pntAB gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-pntAB containing the pntAB gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-pntAB and plasmid pTarget as templates, yghX-U-1, yghX-U-2, yghX-D-1, yghX-D-2, TRC-pntAB-1, TRC-pntAB-2, pT-line-1 and pT-line-2 were used as primers to amplify the yghX homology arm, the P trc The pntAB gene fragment of the promoter and the pTarget plasmid fragment were then constructed by gene recombination. trc pntAB inserted into pT-yghX::P trc pntAB new plasmid.

[0052] argCJBDFGH gene knock-in plasmid pT-argR::P trcConstruction of argCJBDFGH plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-argCJBDFGH-1, 99A-argCJBDFGH-2, 99A-line-F and 99A-line-R were used as primers to amplify the argCJBDFGH gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-argCJBDFGH containing the argCJBDFGH gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-argCJBDFGH and plasmid pTarget as templates, argR homology arms, argR containing P trc The promoter argCJBDFGH gene fragment and pTarget plasmid fragment were then used to construct P trc pT-argR::P trc argCJBDFGH new plasmid.

[0053] argO gene knock-in plasmid pT-ycgH::P trc Construction of argO plasmid: Using the Escherichia coli W3110 genome and plasmid pEC-99A as templates, 99A-argO-1, 99A-argO-2, 99A-line-F and 99A-line-R were used as primers to amplify the argO gene fragment and the pEC-99A plasmid fragment, and then a new plasmid 99A-argO containing the argO gene was constructed by gene recombination; using the Escherichia coli genome, plasmid 99A-argO and plasmid pTarget as templates, ycgH-U-1, ycgH-U-2, ycgH-D-1, ycgH-D-2, TRC-argO-1, TRC-argO-2, pT-line-1 and pT-line-2 were used as primers to amplify the ycgH homology arms, the P trc The promoter argO gene fragment and pTarget plasmid fragment were then used to construct P trc pT-ycgH::P with argO inserted trc argO new plasmid.

[0054] The above gene insertion was successfully verified by electrophoresis.

[0055] 2. The recombinant plasmids were sequentially transformed into W3110-ΔpoxB-ΔldhA-ΔspeA-ΔadiA competent cells to obtain the genetically engineered bacteria W3110-G, with the genotype of W3110-ΔpoxB-ΔldhA-ΔspeA-ΔadiA-yncI::P trc gdhA,yeeP::P trc gdh,yjiT::P trc glnA,ygaY::Ptrc pckA,pdhR::P trc gltA,rph::P trc acnAB,ycjV::P trc icd,yjiP::P trc amtB,ykgH::P trc sthA,yghX::P trc pntAB,argR::P trc argCJBDFGH,ycgH::P trc argO.

[0056] Example 2 Genetically engineered bacteria W3110-R shake flask fermentation The wild-type Escherichia coli W3110, the strain obtained by the gene editing process in Example 1, and the genetically engineered bacteria W3110-R were taken out and fermented according to the following method: Take the strain stored at -80℃, streak it on the slant, culture it at 37℃ for 12h, subculture it twice, and restore the vitality of the strain. Use a sterile inoculation loop to take the strain and inoculate it into the seed culture medium, and culture it at 37℃ for 8h. Inoculate the seed culture medium into the fermentation medium at an inoculum of 10% (volume ratio), and the fermentation medium is filled with 10% (volume ratio) of the shake flask, and culture it at 37℃ and 200r / min. Manually adjust the fermentation liquid every 4h, add dilute ammonia and glucose solution, keep the fermentation liquid sugar content at 2-5g / L, maintain the fermentation pH at 6.5-7.5, and the fermentation cycle is 60h.

[0057] The slant culture medium is: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 20g / L, and the rest is water, pH 6.9-7.1.

[0058] The seed culture medium is: 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, and the rest is water, pH 6.9-7.1.

[0059] The fermentation medium is: glucose: 20g / L, yeast powder 5g / L, peptone 5g / L, magnesium sulfate 2g / L, potassium dihydrogen phosphate 3.5g / L, ferrous sulfate and manganese sulfate 20mg / L. The rest is water, pH 6.8-7.2.

[0060] The fermentation results of this embodiment are as follows: (1) The arginine production of the starting strain wild-type Escherichia coli W3110 in a 60-hour fermentation shake flask was 0.04 g / L. Furthermore, the arginine production of the engineered strain W3110-ΔpoxB-ΔldhA-ΔspeA-ΔadiA after poxB, ldhA, speA and adiA were knocked out in the starting strain was 0.12 g / L; (2) Further, based on the engineered bacteria W3110-ΔpoxB-ΔldhA-ΔspeA-ΔadiA, a strong promoter P was inserted into the genome. trc The controlled argCJBDFGH gene cluster produced 4.2 g / L arginine in shake flasks; (3) Further, P was inserted into the engineered bacteria W3110-ΔpoxB-ΔldhA-ΔspeA-ΔadiA trc After the argCJBDFGH gene cluster controlled by the strong promoter P trc Controlled argO enhanced arginine efflux, and the arginine production in the shake flask was 5.1 g / L; (4) Further, the strain genome overexpressing the synthetic gene cluster argCJBDFGH and the efflux protein argO with an arginine production of 5.1 g / L in the shake flask was inserted with gltA, acnAB and icd in the TCA cycle, and the arginine production in the shake flask was 6.2 g / L; (5) sthA and pntAB that promote cofactor synthesis were inserted into the strain with a yield of 6.2 g / L, and the arginine yield in the shake flask was 6.7 g / L; (6) Based on the above strain with a yield of 6.7 g / L, glnA was inserted into its genome, and the shake flask arginine production was 7.8 g / L; pckA was further inserted into its genome, and the shake flask arginine production was 8.5 g / L; amtB was further inserted into its genome, and the shake flask arginine production was 9.3 g / L. Further, after inserting gdhA of Escherichia coli itself and gdh from Corynebacterium glutamicum, the shake flask arginine production was 13.7 g / L.

[0061] From the above results, it can be seen that after 60h of shake flask culture, the final L-arginine accumulation in the fermentation broth of the genetically engineered bacteria W3110-G reached 13.7g / L, which is much higher than that of the wild-type chassis bacteria. And it can be seen from this that the gene editing of Example 1: overexpression of the genes gdhA and gdh of glutamate dehydrogenase promotes the generation of glutamate, overexpression of the gene argO encoding the arginine transporter enhances the extracellular transport of arginine, and the introduction of exogenous argJ replaces the linear synthesis pathway in Escherichia coli, improves the utilization rate of acetyl-CoA, and accelerates the efficiency of arginine synthesis; overexpression of the glutamine synthetase gene glnA strengthens glutamine synthesis; overexpression of pckA promotes ATP synthesis; at the same time, the speA and adiA genes encoding arginine decarboxylase are deleted to inhibit the further synthesis of putrescine from L-arginine and improve the ability of the strain to accumulate L-arginine. Through the above-mentioned combined effects of gene editing, the L-arginine production performance of the starting strain can be greatly improved, and finally a genetically engineered strain with extremely high L-arginine production is obtained, and the shake flask fermentation yield can reach 13.7g / L. It is further shown that enhancing the synthesis expression of L-arginine precursors and blocking the degradation pathway of L-arginine can increase the accumulation of L-arginine.

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

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

Claims

1. A genetically engineered bacterium for producing L-arginine, characterized in that: The genome of the engineered bacteria includes: Knock out the poxB gene encoding pyruvate dehydrogenase, the ldhA gene encoding lactate dehydrogenase, the speA gene encoding arginine decarboxylase, and the adiA gene; Overexpression of the argG gene encoding sperminosuccinate synthetase, the argH gene encoding arginosuccinate lyase, the glnA gene encoding glutamine synthetase and the gdh gene encoding glutamate dehydrogenase, the pntAB gene encoding nicotinamide adenine dinucleotide transhydrogenase, the sthA gene encoding pyridine nucleotide transhydrogenase, the amtB gene encoding ammonium transporter, the pckA gene encoding phosphoenolpyruvate carboxykinase, the acnAB gene encoding aconitate hydratase, the icd gene encoding isocitrate dehydrogenase, and the gltA gene encoding citrate synthase; heterologous overexpression of the argC gene encoding N-acetyl-γ-glutamyl-phosphate reductase, the argJ gene encoding ornithine acetyltransferase, the argB gene encoding acetylglutamate kinase, the argD gene encoding acetyl-ornithine aminotransferase, and the argF gene encoding ornithine carbamoyltransferase; The engineered bacteria are obtained by transformation using Escherichia coli as a host bacterium.

2. A genetically engineered bacterium for producing L-arginine as claimed in claim 1, characterized in that: The heterologous overexpressed gene is derived from Corynebacterium glutamicum.

3. A method for constructing a genetically engineered bacterium for producing L-arginine, characterized in that: The following steps are involved: Using Escherichia coli as the host bacteria, the CRISPR-Cas9 gene editing technology was used to knock out the poxB gene, ldhA gene, speA gene and adiA gene in the host bacteria genome, and then the gdhA gene, ppk gene, glnA gene, pckA gene, gltA gene, acnAB gene, icd gene, amtB gene, sthA gene, pntAB gene, argC gene, argJ gene, argB gene, argD gene, argF gene, argGH gene and argO gene fragments were introduced and integrated into the host bacteria genome for overexpression.

4. The construction method according to claim 3, characterized in that: The argC gene, argJ gene, argB gene, argD gene, and argF gene are derived from Corynebacterium glutamicum.

5. The construction method according to claim 3, characterized in that: The overexpression method is: the target gene is controlled by a strong promoter Ptrc so that the target gene is overexpressed.

6. Use of the genetically engineered bacterium according to claim 1, or the genetically engineered bacterium constructed by the construction method according to any one of claims 3 to 5, in the preparation of L-arginine.

7. A method for producing L-arginine by fermentation, characterized in that: The fermentation production is carried out using the genetically engineered bacteria as claimed in claim 1 or the genetically engineered bacteria constructed by the construction method as claimed in any one of claims 3 to 5.

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