WO2024113418A1 - 一种厌氧高产l-精氨酸工程菌及其制备方法和应用 - Google Patents

一种厌氧高产l-精氨酸工程菌及其制备方法和应用 Download PDF

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WO2024113418A1
WO2024113418A1 PCT/CN2022/138810 CN2022138810W WO2024113418A1 WO 2024113418 A1 WO2024113418 A1 WO 2024113418A1 CN 2022138810 W CN2022138810 W CN 2022138810W WO 2024113418 A1 WO2024113418 A1 WO 2024113418A1
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arginine
arg
yield
seq
gene
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French (fr)
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蔡林涛
潘宏
唐忠亮
黄国俊
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/42—Salmonella
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Definitions

  • the invention belongs to the field of biotechnology and relates to an anaerobic high-yield L-arginine engineering bacterium and a preparation method and application thereof.
  • L-Arginine is one of the semi-essential basic amino acids required by the human body and animals, and is the final product of the arginine biosynthesis pathway.
  • L-Arg has a wide range of uses in the biomedical field. As a basic amino acid containing a guanidine group, it is an important intermediate metabolite in the urea cycle of organisms. It plays an important role in regulating the body's immune response and stimulating the immune system. At the same time, in terms of anti-tumor immune regulation, it can also inhibit tumor growth and promote the healing and recovery of damaged tissues. L-Arg metabolism is one of the important ways to regulate the functional activity of immune cells. Studies have shown that oral L-Arg has a good anti-tumor effect.
  • L-Arg can effectively improve immunity and promote the immune system to produce natural killer cells, cytotoxic T cells, phagocytes, interleukin-1 and other immune cytokines, which is beneficial to fight cancer cells and enhance immune cell metabolism.
  • high levels of L-Arg in the tumor microenvironment can increase the survival rate and anti-tumor activity of T cells, so targeting L-Arg metabolism can be used as an effective strategy to improve the body's anti-tumor immune response. Therefore, L-Arg not only has a wide range of uses in the pharmaceutical and food industries, but also shows great application potential in the field of anti-tumor immunotherapy in the future.
  • L-Arg there are two main production processes for L-Arg, namely protein hydrolysis extraction and biological fermentation.
  • Protein hydrolysis is used to produce L-Arg, but this method is time-consuming, has low yield, and has serious environmental pollution, and cannot achieve efficient targeted delivery to the lesion site.
  • the fermentation strains of L-Arg that have been studied more are mainly Corynebacterium glutamicum, Brevibacterium flavum, Corynebacterium crenatum, Escherichia coli and Bacillus subtilis, etc., but the acid production level of most fermentation production technologies is still low and is mainly limited to the food industry.
  • the construction and screening of engineered strains that can produce high L-Arg in tumors and have facultative anaerobic characteristics still face many challenges such as negative feedback from the body, lack of anaerobic promoters, and low yields.
  • the present application provides an anaerobic high-yield L-arginine engineered bacterium and a preparation method and application thereof, designs and constructs L-arginine-producing engineered bacteria, realizes efficient production of L-arginine, and can be effectively used in immunotherapy.
  • the present invention adopts the following technical solutions:
  • the present invention provides an anaerobic high-yield L-arginine engineered bacterium, wherein the engineered bacterium contains a gene encoding an N-acetylglutamate synthetase variant, wherein the gene encoding the N-acetylglutamate synthetase variant is controlled by a promoter induced by exogenous environmental conditions, and the engineered bacterium is engineered to lack functional Arg R, and the gene encoding the N-acetylglutamate synthetase variant has any one of the following DNA sequences:
  • the present invention designs a genetically engineered bacterium that can survive in a tumor and produce a high amount of L-Arg.
  • anaerobic strains such as Escherichia coli and Salmonella
  • the strains can utilize the metabolic waste generated by tumor cells as nutrients to survive, and produce L-Arg to regulate the tumor immune microenvironment through metabolism, overcome tumor immunosuppression, and make it more suitable for the proliferation and metabolism of anti-tumor effector cells.
  • immune cells are compensated for nutrients through the metabolic products of the engineered bacteria, which effectively promotes the sustainability of their anti-tumor immunity.
  • L-Arg engineered bacteria are synthesized through gene editing technology, and the repressor protein Arg R gene that inhibits the L-Arg synthesis pathway in the engineered strain is knocked out or weakened, so that the strain L-Arg yield is significantly improved, and a specific overexpression gene Arg A and an inducible promoter are integrated to achieve induction and start-up of a large amount of L-Arg, which is of great significance for improving the therapeutic efficacy of tumors.
  • a specific N-acetylglutamate synthase variant is screened, which has a lower sensitivity to the negative feedback regulation of arginine and can further increase the L-Arg production.
  • the insertion position of the gene encoding the N-acetylglutamate synthetase variant in the genome of the engineered bacteria includes between malE and malK or att ⁇ insertion site.
  • the engineering to lack functional Arg R in the present invention includes knocking out or weakening the expression of Arg R gene.
  • promoters that respond to tumor microenvironment factors are suitable for the present invention.
  • the promoter induced by exogenous environmental conditions includes any one of a hypoxia-responsive promoter, an acid-responsive promoter, a redox-responsive promoter, a light-stimulated promoter, an electrical-stimulated promoter, or a magnetic-driven promoter.
  • hypoxia-responsive promoter is selected from any one of fnr-sp, hip1, i14018, ptet-arca, ptet-fnr, r1074, ssbp1 or ysgap.
  • hypoxia-responsive promoter fnr-sp is used to construct an engineered bacterium that further responds to the tumor microenvironment and efficiently expresses L-Arg in the tumor microenvironment in a targeted manner.
  • nucleic acid sequence of the fnr-sp includes the sequence shown in SEQ ID NO.2.
  • the starting strain of the engineered bacteria is selected from any one of Corynebacterium glutamicum, Escherichia coli, Bacillus subtilis, Proteus, Enterobacter, Salmonella typhi, Salmonella or Shigella.
  • the present invention provides a method for preparing the anaerobic high-yield L-arginine engineering bacteria according to the first aspect, the method comprising:
  • the Arg R gene of the starting strain is knocked out by using the Crispr-Cas9 system, and a gene encoding a variant of N-acetylglutamate synthetase is inserted into the genome of the starting strain, wherein the gene encoding the variant of N-acetylglutamate synthetase is controlled by a promoter induced by exogenous environmental conditions, to obtain the anaerobic high-yield L-arginine engineered bacteria, wherein the gene encoding the variant of N-acetylglutamate synthetase has any one of the following DNA sequences: a) SEQ ID NO.1 or SEQ ID NO.5; b) a DNA sequence encoding a polypeptide identical to the polypeptide encoded by SEQ ID NO.1 or SEQ ID NO.5 except for redundant genetic codes; c) a DNA sequence having at least 80% homology with the DNA sequence of a) or b).
  • a specific gene modification method is designed to achieve rapid and efficient gene editing of the starting strain and rapid and stable preparation of engineered bacteria.
  • the nucleic acid sequence of the sgRNA of the Crispr-Cas9 system includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4.
  • SEQ ID NO.3 gttggtacacccagttcagc.
  • SEQ ID NO.4 tcaagttagtataaaaaagc.
  • the specific process of knocking out or inserting a gene using the Crispr-Cas9 system includes:
  • relevant primers are designed, and homology arms are constructed or integrated with the designed primers.
  • the sequencing-positive plasmid is used as a template to repair and amplify the homology arms.
  • artificially designed sgRNA is used to identify the target genome sequence and guide the Cas9 protease to cut the DNA double strands to form double-strand breaks.
  • the electroporated sgRNA plasmid is used to repair the homology arms, thereby achieving the purpose of gene knockout or knock-in. Finally, the resistance of the successfully integrated strain is eliminated.
  • the present invention provides use of the anaerobic high-yield L-arginine engineering bacteria described in the first aspect in producing L-arginine.
  • the present invention provides a method for producing L-arginine, the method comprising:
  • the anaerobic high-yield L-arginine engineering bacteria described in the first aspect are fermented to obtain L-arginine.
  • a specific culture method can be selected according to the needs and conventional means in the art.
  • the method may include: resuscitating the engineered bacteria, inoculating them into a basic culture medium without an organic nitrogen source for shaking culture, and performing anaerobic activation during the logarithmic growth phase of the bacteria, and producing L-Arg under shaking culture in a basic culture medium without an organic nitrogen source.
  • the basic culture medium containing no organic nitrogen source is a synthetic culture medium containing an inorganic nitrogen source and a carbon source, which is specially used for culturing recombinant engineered bacterial strains, including but not limited to M9 culture medium, amino acid-free culture medium, etc.
  • the shaking method and conditions include the following steps: when the revived engineered bacteria are in the logarithmic growth phase, nitrogen is added to the LB culture medium at a constant rate for 10 to 20 minutes to remove most of the oxygen in the culture medium and form an anaerobic environment; the stimulation is allowed to stand for 1 to 4 hours in an anaerobic environment at 30 to 37°C to activate the mutant gene integrated with the anaerobic promoter, 0.25 to 0.5 mL of the culture medium is taken, and the bacteria are collected by centrifugation at 2 to 8°C, 3000 to 7000 rpm, and 5 to 10 minutes.
  • the bacteria After washing the residual LB culture medium on the bacteria with PBS, the bacteria are resuspended in a basic culture medium without an organic nitrogen source (0.1 to 1 ⁇ 10 8 ), and the engineered bacteria are shaken in a shaker at 30 to 37°C and 150 to 250 rpm.
  • a basic culture medium without an organic nitrogen source 0.1 to 1 ⁇ 10 8
  • the present invention provides a pharmaceutical composition, comprising the anaerobic high-L-arginine-producing engineered bacteria described in the first aspect.
  • the present invention provides use of the anaerobic high-L-arginine-producing engineered bacteria described in the first aspect or the pharmaceutical composition described in the fifth aspect in the preparation of drugs for treating or assisting in the treatment of tumors.
  • the tumor can be selected from acute myeloid leukemia, neuroblastoma, melanoma, non-small cell lung cancer, triple-negative breast cancer, brain metastasis, colon cancer or liver cancer, etc.
  • the anaerobic high-yield L-arginine engineered bacteria designed by the present invention can significantly and stably increase the production of L-Arg in vivo and in vitro, and can effectively colonize tumor tissue and inhibit tumor growth.
  • the genetically engineered bacteria can directly produce L-Arg by absorbing nutrients such as nitrogen sources and carbon sources, and can also be used as the original strain for further improvement in order to screen out safer, more stable and efficient L-Arg production strains.
  • the present invention provides a method for producing L-Arg in a tumor for non-disease diagnosis purposes, the method comprising:
  • the anaerobic high-L-arginine-producing engineered bacteria described in the first aspect are injected into experimental animals.
  • the injection method and inoculation amount include: using nitrogen to remove most of the air in the culture medium of the engineered bacteria cultured in vitro in the logarithmic growth phase, anaerobic stimulation for 1 to 4 hours, centrifuging and collecting, washing the residual LB culture medium on the bacteria with PBS and counting, resuspending the collected bacteria with PBS to a concentration of 0.5 to 1 ⁇ 10 5 CFU/ ⁇ L, taking 100 to 200 ⁇ L of the PBS resuspension, and injecting it into the experimental animal through the tail vein, so that the engineered bacteria consume tumor metabolic waste and efficiently produce L-Arg.
  • the L-Arg content detection method includes: removing the tumor of the experimental animal 3 days after the tail vein injection of the engineered bacteria, harvesting and weighing the tumor tissue, extracting metabolites from 50-100 mg of the tumor tissue, grinding the tumor tissue in liquid nitrogen, adding 300-500 ⁇ L of cold high performance liquid chromatography (HPLC) grade 80% methanol to extract polar metabolites, and then incubating at -80%°C for 4-5 h, and then concentrating the supernatant of the sample at 4°C, 10,000-16,000 ⁇ g, 10-20 min high-speed vacuum, and using LC-MS to detect the intratumoral L-Arg content.
  • HPLC cold high performance liquid chromatography
  • anaerobic high-yield L-arginine engineered bacteria enter the body by intravenous injection or the like, they can automatically target and aggregate in the tumor based on their anaerobic targeted colonization characteristics, and utilize nutrients such as nitrogen sources and carbon sources in the tumor microenvironment to consume tumor metabolic waste to efficiently express and produce L-Arg, which can be effectively applied to the study of basic tumor behavior for non-therapeutic purposes, etc.
  • the present invention designs a genetically engineered bacterium that can survive in a tumor and produce a high amount of L-Arg.
  • the L-Arg engineered bacterium is synthesized by gene editing technology, and the repressor protein Arg R gene that inhibits the L-Arg synthesis pathway in the engineered strain is knocked out or weakened, so that the L-Arg yield of the strain is significantly improved, and the overexpression of a specific Arg A gene and an inducible promoter are integrated to achieve induction and start of large-scale production of L-Arg, and effectively overcome the microenvironmental inhibition of the tumor through metabolic immune regulation, which is of great significance for improving the therapeutic efficacy of tumors.
  • Figure 1 is a design diagram for strain engineering transformation
  • Figure 2 is an electrophoresis diagram for ArgR knockout verification
  • FIG3 is an electrophoretic diagram for verification of Fnr-ArgA215 insertion
  • FIG4 is a growth curve diagram of wild bacteria ECN and genetically engineered bacteria L-Arg Bac. strain
  • FIG5 is a graph showing the L-Arg production of wild bacteria ECN and genetically engineered bacteria L-Arg Bac.
  • FIG6 is a graph showing the colony results of wild-type ECN and genetically engineered L-Arg Bac. in tumors;
  • FIG7 is a graph showing the tumor growth curves of wild ECN and genetically engineered L-Arg Bac.
  • FIG8 is a graph showing the production rate of arginine by different genetically engineered bacteria.
  • the present invention provides an engineered bacterium that can produce high L-Arg and regulate the tumor microenvironment.
  • the core of the invention is to transform the engineered strain by using CRISPR gene editing means, knock out the L-Arg production inhibitory gene ArgR, and insert the negative feedback regulation mutant gene Fnr-Arg A215 of the integrated hypoxia response promoter at a specific position by integrating the overexpression gene Arg A and the hypoxia response promoter, so that the transformed strain reduces the negative feedback regulation restriction, thereby obtaining a genetically engineered anaerobic expression strain L-Arg Bac. that is stable and produces high L-Arg.
  • the culture conditions and standard production methods for the engineered strain to stably and efficiently produce L-Arg under anaerobic conditions are explored and screened.
  • the genetically engineered bacterium L-Arg Bac utilizes the facultative anaerobic respiration characteristics to target and colonize the inside of the tumor.
  • the transformed engineered bacteria regulate the abnormal metabolism of tumor cells through L-arginine, improve the immunosuppression of the tumor microenvironment, increase the infiltration of immune cells in the tumor microenvironment, promote the proliferation and metabolism of immune cells, and comprehensively improve the effect of tumor immunotherapy.
  • the present invention adopts a genetic engineering design scheme as shown in the figure (Figure 1), which mainly includes the following steps:
  • Competent bacterial cells can be obtained by centrifugation of wild-type strains in the logarithmic growth phase, and engineered bacteria Bac. pCas9 containing pCas9 plasmid is formed by electroporation. The culture is restored with LB medium and plated on a resistant plate containing antibiotics. At the same time, Bac. pCas9 is prepared into a competent state by electroporation, and the Arg R inhibitory gene of L-Arg and its homologous arm are amplified and repaired using the designed primers. Finally, the engineered bacteria are transformed with sgRNA and repair homologous arms. The resistance of the successfully transformed strain is eliminated through temperature sensitivity, and the strain without resistance after knocking out the ArgR gene of L-Arg synthesis pathway repressor protein is named Bac. ⁇ ArgR;
  • the competent cells were added with sgRNA plasmid and repair homology arms and electrotransformed under 2000 ⁇ 3000 KV conditions.
  • the cloned products were spread and cultured on plates containing resistance.
  • the cloned products were inoculated in LB medium without resistance. After dilution, they were cultured on LB (no resistance), LB (spectinomycin), and LB (kanamycin) spots.
  • the cloned strains with little growth on the resistance plate were selected and inoculated in LB culture without resistance and maintained.
  • the non-resistant strain that successfully integrated the hypoxia-inducible promoter and inserted the mutant ArgA215 gene on the basis of knocking out the Arg R gene was named L-Arg Bac.;
  • Intratumoral expression of L-Arg the engineered bacteria activated by anaerobic stimulation in 1 were collected by centrifugation at 1000-10000 rpm, 2-20 min, and 4°C, and the cells were counted. The cells were injected into the experimental animals through the tail vein at an inoculum of 0.1-1 ⁇ 108 , and the concentration of L-Arg in the tumor tissue was detected by HPLC-MS at consecutive time points of 12-72 h. Due to the anaerobic tumor-targeting characteristics of the bacteria, tumor metabolism in the tumor microenvironment produces a large amount of inorganic salt NH4 + , and the engineered bacteria use this as a nutrient to produce a large amount of L-Arg.
  • This example involves the construction of genetically engineered bacteria L-Arg Bac. and gene sequencing identification.
  • ECN Escherichia coli Nissle1917
  • Knockout of the Arg_R gene, a repressor protein of the L-Arg synthesis pathway Knockout of the Arg R gene, a repressor protein of the arginine synthesis pathway, in the E. coli ECN genome to obtain the gene knockout strain ECN ⁇ ArgR; the first step is the amplification and construction of the homology arm; first extract the E.
  • coli ECN DNA was used as a template, and the primers ArgR-UP-F (nucleic acid sequence: ctccgctgccgcgaccttaatcc) and ArgR-up-R (nucleic acid sequence: cgacggggcagagaaagtcacccgatatggtggttgatac); ArgR-down-F (nucleic acid sequence: tctgccccgtcgcttctggcggggggaa) and ArgR-down-R (nucleic acid sequence: cacaccacttacggatacggtcc) were used to amplify the ArgR inhibitory gene and its homologous arms of L-Arg.
  • ArgR-UP-F nucleic acid sequence: ctccgctgccgcgaccttaatcc
  • ArgR-up-R nucleic acid sequence: cgacggggca
  • the primers ArgR-up-F/ArgR-down-R were used to prepare the PCR system to repair the overlapping fusion amplification of the homologous arms and connect to the T vector.
  • the product was added with A-linked pUX-T vector, transformed into Ecoli DH5alpha competent cells, and evenly spread on the plate containing ampicillin (Amp) resistance. Single clones were picked and mixed in 10 ⁇ L sterile water, and 0.5 ⁇ L was used as a template for amplification according to the corresponding system.
  • the second step is Crispr-Cas9 gene knockout.
  • electroporate ECN to become competent cells Inoculate wild-type E. coli ECN into 5 mL LB liquid culture medium, activate and culture overnight at 37°C, transfer 1% to 50 mL LB liquid culture medium, and collect the cells by centrifugation when the OD600 value is 0.6-0.8. Wash them three times with 10% glycerol, and finally resuspend the cells with 2 mL 10% glycerol to obtain the prepared competent cells. After electroporation of the plasmid containing pCas9, 10 ⁇ L of the plasmid was added to the prepared E.
  • coli ECN competent cells and placed on ice for 5 min, electroporated at 2500 KV, and 1 mL of LB medium was added. After culturing at 37°C for 1 h, the cells were plated and cultured on kanamycin (Kan) resistant plates.
  • the pCas9 ECN was prepared in the same way to become competent cells, and then sgRNA (SEQ ID NO. 3) and the homology arms were repaired.
  • the primers ArgR-up -F/ArgR-down-R were used for amplification to identify whether the ArgR gene, a repressor protein of the arginine synthesis pathway, was knocked out (Figure 2);
  • the third step is to edit and eliminate the resistance of the strain, eliminate the resistance through temperature sensitivity, obtain the strain with successful knockout, and name the non-resistant strain ECN ⁇ ArgR;
  • the first step is the construction of homology arms.
  • the primers are designed and the primer fnr-ArgA215 is amplified.
  • the designed primers ArgA-att ⁇ -F (tgaagcctgctttttttaaaaacgccgcaaagtttgagcga)/argA-att ⁇ -R (gctcaagttagtatttaccctaaatccgccatcaacactt) are used to amplify the fnr-ArgA215 fragment according to the set conditions with the gene synthesis plasmid as the template.
  • primers att ⁇ ArgA215-F (tggcggatttagggtaaatactaacttgagcgaacggga)/att ⁇ Arg A 215-R (actttgcggcgttttttaaaaaagcaggcttcaacggattc) were used to reversely amplify the att ⁇ homology arm using the ECN att ⁇ -puxT plasmid as a template.
  • the recovered product was seamlessly cloned and connected according to the set conditions, and allowed to stand on ice for 30 min.
  • the product was transformed into Ecoli DH5alpha competent cells and evenly spread on a plate containing ampicillin (Amp) resistance and cultured overnight at 37°C. Finally, the clones grown were identified by colony PCR. Single clones were picked and mixed in 10 ⁇ L sterile water. 0.5 ⁇ L was used as a template for amplification under the set system conditions. Primers M13-F (pUC): CCCAGTCACGACGTTGTAAAACG and M13-R (pUC): AGCGGATAACAATTTCACACAGG were used for identification. Positive clones were sequenced and compared.
  • the att ⁇ -ArgA215 pux-T plasmid with positive sequencing was used as a template to amplify and repair homology arms according to the set system conditions.
  • the sequencing result of positive clones was 99.70%, and the integrated homology arms were successfully obtained.
  • the second step is Crispr-Cas9 gene insertion.
  • the ECN ⁇ ArgR Cas9 strain was electroporated into competent cells, and 100 ⁇ L of the prepared electroporated competent cells was added with sgRNA (SEQ ID NO. 4) plasmid and repair homology arms. The cells were electroporated at 2500KV, and then plated on kanamycin/spectinomycin (Kan/spc) resistant plates. Finally, the primers att ⁇ -up-F/att ⁇ -down-R were used for integration identification (Figure 3).
  • the third step is to eliminate resistance. Inoculate the successfully inserted strain into LB liquid medium (no resistance) at 37°C overnight, dilute it and plate it on LB (no resistance) plates, incubate it at 37°C overnight, pick a single clone and plate it on LB (no resistance), LB (spectinomycin), and LB (kanamycin), select the clone with little resistance plate and inoculate it on LB no resistance medium to preserve the strain, and name it ECN ⁇ ArgR att ⁇ :fnr-argA215.
  • ArgA213, ArgA214 and ArgA216 genes were used to replace the ArgA215 gene to prepare engineered bacteria, and the arginine production rate of each engineered bacteria was detected.
  • the specific method includes: incubating the engineered bacteria containing the engineering plasmid with glutamine and ornithine for 6 hours, and using LC-MS to determine the total production of free arginine in cells containing plasmids expressing mutant argA (ArgA213, ArgA214, ArgA215, ArgA216), and obtaining the synthesis rate of free arginine per dry weight per hour in the cells.
  • the results are shown in Figure 8. It can be seen that the use of the ArgA215 gene can further increase the arginine production rate.
  • the ArgA213 gene sequence is:
  • the ArgA214 gene sequence is (SEQ ID NO.5):
  • the ArgA216 gene sequence is:
  • This example detects the activity of L-Argbac. in genetically engineered bacteria and the production of L-Arg in vitro.
  • the activity of the engineered bacteria was tested under aerobic and anaerobic conditions respectively.
  • the ECN and L-ArgBac. strains were revived from -80°C glycerol bacteria and divided into two groups: aerobic and anaerobic variable groups.
  • the four groups of strains were inoculated into shaking tubes of LB culture medium and cultured at 37°C and 100r/min for 12h.
  • the OD600 value of the strain was detected by ultraviolet spectrophotometer.
  • the bacterial solution with OD600 between 0.6 and 0.8 was obtained and nitrogen was added to eliminate oxygen in the bacterial solution of LB culture medium of the anaerobic group.
  • the anaerobic group was placed in an anaerobic environment at 37°C for 2h for pathway induction.
  • glycerol bacteria frozen at -80°C were revived and divided into two groups, with the same marking groups as in Example 21. They were inoculated into shaking tubes of LB culture medium, and cultured at 37°C and 100 r/min for 12 h. The OD600 value of the bacterial solution was monitored. The bacterial solution with an OD600 value of 0.6-0.8 was centrifuged to collect the bacteria, washed 3 times with PBS and counted. 5 ⁇ 107 bacteria were resuspended in PBS and injected into tumor-bearing mice through the tail vein.
  • mice were killed by cervical dislocation, the tumor tissue was taken and weighed, and the tumor tissue was digested and resuspended into a single cell suspension. 20 ⁇ L of the diluted single cell suspension samples were evenly spread on a bacterial culture plate, cultured at 37°C for 12 h, and the colonies were counted to calculate the number of colonies contained in the unit tumor tissue (Figure 6), indicating that the tumor targeting ability of the strain modified by the present invention after tail vein injection was not much different from that of the original strain.
  • the B16 animal tumor model was established using C57/BL6J mice with normal immunity. Each mouse was subcutaneously injected with 5 ⁇ 10 6 murine melanoma B16F10 cells. After 5 days, the tumor volume reached 50 mm 3 .
  • the tumor-bearing mice were randomly divided into two groups: ECN group and L-Argbac. group.
  • the ECN and L-ArgBac. glycerol bacteria frozen at -80°C were revived and labeled as groups in Example 21. They were inoculated into shaking tubes of LB culture medium respectively, and cultured overnight at 37°C and 100 r/min. The bacterial liquid with OD 600 value of 0.6-0.8 was centrifuged to collect the bacteria.
  • the bacteria were collected by centrifugation, washed 3 times with PBS and counted. 5 ⁇ 10 7 CFU of the strain was resuspended with PBS and injected into the tumor-bearing mice through the tail vein. The tumor of the mice was recorded regularly ( FIG. 7 ). As shown in FIG. 7 , the L-Arg produced in the tumor of the L-ArgBac. group made the tumor grow slower than that of the ECN group, and had a certain inhibitory effect on the growth of the tumor.
  • the present invention synthesizes L-Arg engineered bacteria through gene editing technology, knocks out or weakens the repressor protein ArgR gene that inhibits the L-Arg synthesis pathway in the engineered strain, so that the strain L-Arg production is significantly improved, and integrates the specific overexpression gene ArgA and the inducible promoter, thereby achieving induction and starting to produce a large amount of L-Arg, and successfully constructs anaerobic high-yield L-arginine engineered bacteria.
  • the L-arginine production is nearly 1000 times higher than that of wild bacteria, and the engineered bacteria can be targeted and colonized in the tumor microenvironment, using the metabolic waste generated by tumor cells as nutrients to survive, and producing L-Arg to regulate the tumor immune microenvironment through metabolism, which is of great significance for improving the efficacy of tumor treatment.
  • the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

一种厌氧高产L-精氨酸工程菌及其制备方法和应用。所述工程菌中含有编码N-乙酰谷氨酸合成酶变体的基因，所述编码N-乙酰谷氨酸合成酶变体的基因由被外源环境条件诱导的启动子控制，所述工程菌被工程化为缺乏功能性ArgR。通过基因编辑技术合成L-Arg工程菌，实现诱导启动大量产生L-Arg，L-精氨酸产量与野生菌相比提高近1000倍，且所述工程菌能够在肿瘤微环境中靶向定殖，利用肿瘤细胞生成的代谢废物作为营养物质生存，并产生L-Arg通过代谢来调控肿瘤免疫微环境，对提高肿瘤治疗疗效有着重要的意义。

Description

一种厌氧高产L-精氨酸工程菌及其制备方法和应用 技术领域
本发明属于生物技术领域，涉及一种厌氧高产L-精氨酸工程菌及其制备方法和应用。
 背景技术
L-精氨酸（L-Arginine，简称L-Arg）是人体和动物体内所需半必需碱性氨基酸之一，是精氨酸生物合成途径的最终产物。L-Arg在生物医学领域具有广泛用途，它作为一种含有胍基的碱性氨基酸，是生物体尿素循环的一种重要中间代谢物，对于调节机体免疫反应，激励免疫系统具有重要作用。同时，在抗肿瘤免疫调控方面，它还可抑制肿瘤生长、促进损伤组织愈合康复等作用。L-Arg代谢是调节免疫细胞功能活性的重要途径之一。研究表明，口服L-Arg具有良好的抗肿瘤效果，L-Arg可有效提高免疫力、促进免疫系统产生自然杀伤细胞、细胞毒性T细胞、吞噬细胞和白介素-1（Interleukin-1）等免疫细胞因子，有利于对抗癌细胞并增强免疫细胞新陈代谢，尤其是肿瘤微环境中高含量的L-Arg可以提高T细胞的存活率和抗肿瘤活性，从而靶向L-Arg代谢可作为提高机体抗肿瘤免疫应答的有效策略。因此，L-Arg不仅在在医药和食品工业中具有广泛用途，而且在未来抗肿瘤免疫治疗领域展现出巨大的应用潜力。
目前L-Arg主要有两种生产工艺，即蛋白质水解提取和生物发酵。蛋白质水解法来生产L-Arg，但是该方法操作费时、收率低，且环境污染严重，不能实现高效的靶向递送至病灶部位。随着生物发酵技术的兴起与大力发展，利用发酵菌种生产L-Arg逐渐兴起，L-Arg的发酵菌种研究的较多的主要有谷氨酸棒杆菌(Corynebacterium glutamicum)、黄色短杆菌(Brevibacterium flavum)、钝齿棒杆菌(Corynebacteriumcrenatum)、大肠杆菌(Escherichia coli)和枯草芽孢杆菌(Bacillus subtilis)等，但大多数发酵生产技术的产酸水平仍然较低，并且主要局限于食品工业。近年来，美国宾夕法尼亚州立大学的Roger Geiger教授研究发现基因工程菌能调控肿瘤微环境，有效改善微环境的免疫抑制。然而，目前用来生产L-Arg的微生物菌株基本都是在有氧环境下生存，缺少能肿瘤乏氧环境下利用肿瘤代谢产物合成L-Arg的工程菌株。基因工程技术对于L-Arg高产菌株选育具有重要的推动作用，利用基因工程构建L-Arg高产菌株是一种高效率、理性化的育种手段。尽管如此，如何构建和筛选出能瘤内高产L-Arg和含有兼性厌氧特性的工程菌株，仍然面临着机体负反馈、缺乏厌氧启动子等诸多挑战。
综上所述，构建产L-精氨酸工程菌，实现高效生产L-精氨酸且能有效应用于免疫治疗，具有重要意义。
 技术问题
目前构建和筛选出能瘤内高产L-Arg和含有兼性厌氧特性的工程菌株，仍然面临着机体负反馈、缺乏厌氧启动子、产量低等诸多挑战，针对上述问题，本申请提供一种厌氧高产L-精氨酸工程菌及其制备方法和应用，设计构建产L-精氨酸工程菌，实现高效生产L-精氨酸且能有效应用于免疫治疗。
 技术解决方案
为达上述目的，本发明采用以下技术方案：
第一方面，本发明提供一种厌氧高产L-精氨酸工程菌，所述工程菌中含有编码N-乙酰谷氨酸合成酶变体的基因，所述编码N-乙酰谷氨酸合成酶变体的基因由被外源环境条件诱导的启动子控制，所述工程菌被工程化为缺乏功能性Arg R，所述编码N-乙酰谷氨酸合成酶变体的基因具有选自以下的DNA序列中任意一种：
a)SEQ ID NO.1或SEQ ID NO.5；
b)除了冗余的遗传密码以外，编码与由SEQ ID NO.1或SEQ ID NO.5编码的多肽相同的多肽的DNA序列；
c)与a)或b)的DNA序列具有至少80%同源性的DNA序列。
本发明设计一种能在肿瘤内生存并高产L-Arg工程菌株的基因工程菌，通过对大肠杆菌、沙门氏菌等厌氧菌株的基因工程改造，使其能利用肿瘤细胞生成的代谢废物作为营养物质生存，并产生L-Arg通过代谢来调控肿瘤免疫微环境，克服肿瘤免疫抑制，使之更适合抗肿瘤效应细胞的增殖代谢，尤其是免疫细胞通过工程菌的代谢产物进行营养补偿，有效促进其抗肿瘤免疫的持续性，为了克服现有L-Arg生产菌株在厌氧环境中表达水平低的难题，通过基因编辑技术合成L-Arg工程菌，敲除或弱化工程菌株中抑制L-Arg合成途径的阻遏蛋白Arg R基因，使得菌株L-Arg产量获得显著的提升，并整合特定过表达基因Arg A和诱导型启动子，从而实现诱导启动大量产生L-Arg，对提高肿瘤治疗疗效有着重要的意义。
本发明中，筛选特定的N-乙酰谷氨酸合酶变体，其对精氨酸的负反馈调节的敏感性更低，能够进一步提高L-Arg产量。
SEQ ID NO.1：
atggtaaaggaacgtaaaaccgagttggtcgagggattccgccattcggttccctgtatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccgtctggtggtggtctatggcgcacgtccgcagatcgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcggtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggctaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaa。
优选地，所述编码N-乙酰谷氨酸合成酶变体的基因在工程菌基因组上的插入位置包括malE和malK之间或attλ插入位点。
本发明中，发现编码N-乙酰谷氨酸合成酶变体的基因插入的位置的差异性，会产生不同的基因表达效果，插入特定的位置具有高度选择性与特异性，在attλ位点插入，能使得基因更加的稳定的表达。
可以理解，本发明所述工程化为缺乏功能性Arg R包括敲除或弱化Arg R基因表达量。
可以理解，响应肿瘤微环境因素的启动子均适用于本发明。
可选地，所述被外源环境条件诱导的启动子包括乏氧响应启动子、酸响应启动子、氧化还原响应启动子、光刺激启动子、电刺激启动子或磁驱动启动子中任意一种。
优选地，所述乏氧响应启动子选自fnr-sp、hip1、i14018、ptet-arca、ptet-fnr、r1074、ssbp1或ysgap中任意一种。
本发明中采用乏氧响应启动子fnr-sp，即可构建进一步响应肿瘤微环境的工程菌，靶向性地在肿瘤微环境中高效表达L-Arg。
可选地，所述fnr-sp的核酸序列包括SEQ ID NO.2所示的序列。
SEQ ID NO.2：
aaaaacgccgcaaagtttgagcgaagtcaataaactctctacccattcagggcaatatctctcttggatccaaagtgaactctagaaataattttgtttaactttaagaaggagatatacat。
可选地，所述工程菌的出发菌株选自谷氨酸棒杆菌、大肠杆菌、枯草芽孢杆菌、变形杆菌、肠杆菌、伤寒杆菌、沙门氏菌或志贺氏菌中任意一种。
第二方面，本发明提供一种第一方面所述的厌氧高产L-精氨酸工程菌的制备方法，所述方法包括：
利用Crispr-Cas9系统敲除出发菌株的Arg R基因，并在出发菌株的基因组中插入编码N-乙酰谷氨酸合成酶变体的基因，所述编码N-乙酰谷氨酸合成酶变体的基因由被外源环境条件诱导的启动子控制，得到所述厌氧高产L-精氨酸工程菌，所述编码N-乙酰谷氨酸合成酶变体的基因具有选自以下的DNA序列中任意一种：a)SEQ ID NO.1或SEQ ID NO.5；b)除了冗余的遗传密码以外，编码与由SEQ ID NO.1或SEQ ID NO.5编码的多肽相同的多肽的DNA序列；c)与a)或b)的DNA序列具有至少80%同源性的DNA序列。
本发明中，设计特定的基因修饰方法，能够实现对出发菌株快速、高效地基因编辑，快速、稳定地制备工程菌。
优选地，所述Crispr-Cas9系统的sgRNA的核酸序列包括SEQ ID NO.3和SEQ ID NO.4所示的序列。
SEQ ID NO.3：gttggtacacccagttcagc。
SEQ ID NO.4：tcaagttagtataaaaaagc。
本发明中，利用Crispr-Cas9系统敲除或插入基因的具体过程包括：
首先，设计相关引物，用设计的引物构建或整合同源臂，以测序阳性的质粒为模板进行同源臂的修复扩增；其次，用人工设计的sgRNA识别目的基因组序列，并引导Cas9蛋白酶进行DNA双链的切割，形成双链断裂，电转化的sgRNA质粒进行同源臂修复，从而达到基因的敲除或敲入的目的；最后，将整合成功的菌株进行抗性消除。
第三方面，本发明提供第一方面所述的厌氧高产L-精氨酸工程菌在生产L-精氨酸中的应用。
第四方面，本发明提供一种生产L-精氨酸的方法，所述方法包括：
对第一方面所述的厌氧高产L-精氨酸工程菌进行发酵培养，获得L-精氨酸。
可以理解，本发明中可根据需求及本领域常规手段选择具体的培养方法，例如所述方法可包括：复苏工程菌，接种到含无有机氮源基础培养基中进行摇菌培养，并在该菌对数生长期时进行无氧激活，在无有机氮源基础培养基的摇菌下产生L-Arg。
可选地，所述含无有机氮源基础培养基是一种合成含无机氮源和碳源的培养基，专门用于培养重组工程菌菌株的培养，包括但不限于M9培养基、无氨基酸培养基等。
可选地，所述摇菌的方法及条件包括以下步骤：将复苏的工程菌在对数生长期时，持续10~20 min往LB培养基菌液中充入恒定速率的氮气，排除培养基中的绝大部分氧气，形成无氧环境；在30~37℃、无氧环境中静置刺激1~4 h启动，使得整合无氧启动子的突变体基因被激活，取0.25~0.5  mL培养基菌液，在2~8℃、3000~7000 rpm、5~10min的条件下离心收集菌体，用PBS洗去菌体上残留的LB培养基后，用无有机氮源基础培养基菌体重悬（0.1~1×10 8），在30~37℃、150~250 rpm条件下的摇床中进行工程菌的摇菌培养。
第五方面，本发明提供一种药物组合物，所述药物组合物包括第一方面所述的厌氧高产L-精氨酸工程菌。
第六方面，本发明提供第一方面所述的厌氧高产L-精氨酸工程菌或第五方面所述的药物组合物在制备治疗或辅助治疗肿瘤的药物中的应用。
优选地，所述肿瘤可选自急性髓系白血病、神经母细胞瘤、黑色素瘤、非小细胞肺癌、三阴性乳腺癌、脑转移瘤、结肠癌或肝癌等等。
本发明设计的厌氧高产L-精氨酸工程菌能够显著稳定提高体内、外L-Arg的产量，同时能有效定植肿瘤组织并抑制肿瘤的生长。该基因工程菌既可以直接通过吸收氮源、碳源等营养物质来生产L-Arg，也可以作为原始菌株做进一步改良以期筛选出更加安全、稳定、高效L-Arg生产菌株。
第七方面，本发明提供一种以非疾病诊断为目的的瘤内产生L-Arg的方法，所述方法包括：
将第一方面所述的厌氧高产L-精氨酸工程菌注射到实验动物体内。
所述注射的方法及接种量包括：将体外培养在对数生长期的工程菌用氮气排除培养基内大部分空气，无氧刺激1~4 h后离心收集，并用PBS洗去菌体上残留的LB培养基后计数，用PBS将收集的菌体重悬至0.5~1×10 5 CFU/μL浓度，并取100~200 μL的PBS重悬液，通过尾静脉注射到实验动物体内，工程化细菌消耗肿瘤代谢废物高效产生L-Arg。
所述L-Arg含量检测方法包括：将尾静脉注射工程菌后3天后的实验动物肿瘤取下，收获并称重肿瘤组织，在50~100 mg肿瘤组织中提取代谢物，在液氮中研磨处理肿瘤组织，加入300~500 μL冷的高效液相色谱(HPLC)级80%甲醇提取极性代谢物，随后在-80%℃孵育4~5 h，然后将样品在4℃、10,000~16,000×g、10~20 min高速真空浓缩上清液，并使用LC-MS检测瘤内L-Arg含量。
本发明中，所述厌氧高产L-精氨酸工程菌通过静脉注射等方式进入体内后，可基于厌氧靶向定殖特性，自动靶向聚集到肿瘤内，并利用肿瘤微环境中的氮源和碳源等营养物质，消耗肿瘤代谢废物高效表达产生L-Arg，可有效应用于非治疗为目的的肿瘤基础行为研究等。
 有益效果
本发明设计一种能在肿瘤内生存并高产L-Arg工程菌株的基因工程菌，通过基因编辑技术合成L-Arg工程菌，敲除或弱化工程菌株中抑制L-Arg合成途径的阻遏蛋白Arg R基因，使得菌株L-Arg产量获得显著的提升，并整合过表达特定Arg A基因和诱导型启动子，从而实现诱导启动大量产生L-Arg，并通过代谢免疫调控有效克服肿瘤的微环境抑制，对提高肿瘤治疗疗效有着重要的意义。
 附图说明
图1为菌株工程化改造设计图；
图2为ArgR敲除验证电泳图；
图3为Fnr-ArgA215插入验证电泳图；
图4为野生菌ECN与基因工程菌L-Arg Bac.菌株的生长曲线图；
图5为野生菌ECN与基因工程菌L-Arg Bac.的L-Arg产量图；
图6为野生菌ECN与基因工程菌L-Arg Bac.瘤内菌落结果图；
图7为野生菌ECN与基因工程菌L-Arg Bac.肿瘤生长曲线图；
图8为不同基因工程菌精氨酸的生成速率图。
 本发明的实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道购买获得的常规产品。
本发明提供了一种能高产L-Arg并调节肿瘤微环境的工程菌，其核心为利用CRISPR基因编辑手段来改造工程菌株，敲除L-Arg产生抑制基因ArgR，并通过整合过表达基因Arg A和乏氧响应启动子，在特定位置插入整合乏氧响应启动子的负反馈调节突变体基因Fnr-Arg A215，使得改造后的菌株降低负反馈调节限制，从而获得稳定并高产L-Arg的基因工程厌氧表达菌株L-Arg Bac.。同时，探索并筛选了工程菌株在厌氧条件下稳定、高效生产L-Arg的培养条件与标准生产方法。该基因工程菌L-Arg Bac.其利用兼性厌氧呼吸特性能靶向定殖肿瘤内部，改造后的工程菌通过L-精氨酸调节肿瘤细胞的异常代谢，改善肿瘤微环境免疫抑制，提高免疫细胞在肿瘤微环境中的浸润，促进免疫细胞的增殖代谢，全面提高肿瘤免疫治疗的效果。
为了使得工程菌达到上述目的，本发明采用如图所示的基因工程设计方案（图1），主要包括以下步骤：
（1）L-Arg合成途径阻遏蛋白Arg R抑制基因的敲除：野生型菌株在对数生长期时经离心可得到感受态菌细胞，通过电转化形成含pCas9质粒的工程菌Bac. pCas9，用LB培养基恢复培养，在含有抗生素的抗性平板上进行涂布培养，同时以电转化的方法将Bac. pCas9制备成感受态，并使用设计的引物对L-Arg的Arg R抑制基因及其同源臂进行扩增和修复，最后对工程细菌进行sgRNA及修复同源臂的转化，转化成功的菌株通过温度敏感型将抗性消除，并将敲除L-Arg合成途径阻遏蛋白ArgR基因无抗性菌株命名为Bac. △ArgR；
（2）L-Arg负反馈突变体Arg A215基因和乏氧诱导型启动子的整合并插入：将工程菌Bac. △ArgR Cas9细胞电转化成感受态，并引物以基因合成质粒为模板扩增突变体基因fnr-ArgA215片段，同时以attλ-puxT质粒为模板进行反向扩增attλ同源臂，产物经PCR产物纯化后，将回收的产物进行无缝克隆连接，阳性产物以attλ-ArgA215 pux-T质粒为模板扩增获得修复同源臂。最后取感受态细胞加入sgRNA质粒和修复同源臂在2000~3000 KV条件下电转化，在有含有抗性的平板进行涂布培养，克隆产物接种于无抗的LB培养基，稀释后分别在LB（无抗）、LB（壮观霉素）、LB（卡那霉素）点板培养，选择抗性板不长的克隆菌株接种于LB无抗培养并保菌，在敲除Arg R基因基础上成功整合乏氧诱导型启动子并插入突变体ArgA215基因的无抗性菌株命名为L-Arg Bac.；
（3）L-Arg体内外表达方法：①L-Arg的体外表达，工程菌在37℃、150~250 rpm条件下LB培养基中进行活化，在培养基中持续充入氮气10~20 min后，无氧刺激1~4 h激活乏氧诱导型启动子，并用无有机氮源培养基以活化细菌的LB培养基条件进行扩大培养12~24 h，该工程菌即可在体外高效、稳定分泌表达L-Arg；②L-Arg的瘤内表达，取上述①中无氧刺激激活的工程菌，在1000~10000 rpm、2~20 min、4℃条件下离心收集菌体并计数，以0.1~1×10 8的接种量通过尾静脉注射到实验动物体内，分布在12 ~72 h连续时间点内HPLC-MS检测L-Arg在肿瘤组织中的浓度。由于该菌厌氧靶向肿瘤特性，在肿瘤微环境中肿瘤代谢产生大量无机盐NH4 +，工程菌以此为营养物质产生大量的L-Arg。
实施例1
本实施例进行基因工程菌L-Arg Bac.构建与基因测序鉴定。
使用大肠杆菌Nissle1917（ECN）为例进行基因工程改造，基因工程菌L-Arg Bac.的构建方法主要分为以下两个步骤：
①L-Arg合成途径阻遏蛋白Arg_R基因的敲除：敲除大肠杆菌ECN基因组中的敲除精氨酸合成途径阻遏蛋白Arg R基因，获得基因敲除菌株ECN ΔArgR；第一步，同源臂的扩增及构建；首先提取大肠杆菌ECN DNA为模板，使用序列为ArgR-UP-F（核酸序列为：ctccgctgccgcgaccttaatcc）和ArgR-up-R（核酸序列为：cgacggggcagagaaagtcacccgatatggtggttgatac）；ArgR-down-F（核酸序列为：tctgccccgtcgcttctggcggcggggaa）和ArgR-down-R（核酸序列为：cacaccacttacggatacggtcc）的引物对L-Arg的ArgR抑制基因及其同源臂进行扩增，用引物ArgR-up-F/ArgR-down-R进行PCR体系配制修复同源臂overlapping融合扩增及T载体连接，产在物加入A连pUX-T载体，转化 Ecoli DH5alpha 感受态细胞到含氨苄青霉素（Amp）抗性的平板均匀涂布。并挑取单克隆至10 μL无菌水中混匀后取，0.5 μL为模板按照相应体系进行扩增，用引物M13-F(pUC)：CCCAGTCACGACGTTGTAAAACG和M13-R(pUC)：AGCGGATAACAATTTCACACAGG进行菌落PCR鉴定，并挑取阳性克隆产物进行测序比对，得到阳性克隆测序结果为98.1%，成功构建上下游同源臂；
ArgR敲除菌测序结果（argR-up-F单向）：
cggttttcatggaccacatatcaagaatgtgtagtcacgcaagtttagcgtttatgcatttaattgccgtaatcaggaacctaacttacgtaattaacagtcacttatgcattagcgcaacattccaacaggtggtgacaatataccctaccgctcagccaaaacaatatcaatttgataacaattaatttacttttaagcagaatttgcaggccgtgacgcaggcatgtttctcaataacgaaatttgataaaatcccgctctttcataacattatttcagccttcttcagggctgactgtttgcataaaaattcatctgtatgcacaataatgttgtatcaaccaccatatcgggtgactttctctgccccgtcgcttctggcggcggggaaaatgttgcttttgcctatcaaccccctgctttcccctgcgattcatttaacgaatagtgcgttttactgcgacatatcattcacacaatgaatacatatagtaaaaaagcacattatgcaaaattcattatctaattgaaaatactataattaacaataaatgatcgtgtttttaattcttttttgttattaaaattcacatttttaacacttagtatcaactgaaacagttagcgtggtattaattagttcaataattagtgtatacttgattttgtgatatgggtcacgaaacaaaggcccagctaaaagattatgtcgaggtaaaaatcatgaaaatcaaaaccactgttgctgcattaagtgtgctttctgttctctctttcggtgcattcgctgccgactccattgatgctgcacaagcacaaaatcgtgaagcaatcgggacgtc；
比对结果：（与预计敲除后序列比对）
Fast alignment of DNA sequences DNAMAN2 and DNAMAN1
Ktuple=2  Gap_penalty=7
Upper line: DNAMAN2, from 3 to 843
Lower line: DNAMAN1, from 35 to 875
DNAMAN2:DNAMAN1 identity= 99.17%(838/845) gap=3.65%(32/877)；
第二步，Crispr-Cas9基因敲除，首先电转化ECN成为感受态细胞，将野生型大肠杆菌ECN接种至5 mL LB液体培养基中，在37℃时活化培养过夜，将1%转接至50 mL LB液体培养基中，测OD 600值为0.6~0.8时离心收集菌体，使用10%甘油洗三次，最后用2 mL 10%甘油重悬菌体即可得到制备好的感受态细胞。将含pCas9的质粒电转化后，取10 μL质粒加到制备好的大肠杆菌ECN感受态细胞在冰上放置5 min，2500 KV电转化，加入1 mL LB培养基，在37℃恢复培养1 h后，在卡那霉素(Kan)抗性平板上进行涂布培养，以同样的方法制备pCas9 ECN成为感受态后转化sgRNA（SEQ ID NO.3）及修复同源臂，并用引物ArgR-up -F/ArgR-down-R进行扩增鉴定精氨酸合成途径阻遏蛋白ArgR基因是否敲除（图2）；
第三步，编辑消除菌株的抗性，通过温度敏感型消除抗性，得到敲除成功的菌株，并把无抗性菌株命名为ECN △ArgR；
②L-Arg负反馈调节N-乙酰谷氨酸合酶ArgA215突变体基因的插入：对①中ArgR基因敲除菌ECN ΔArgR的基因组中插入负反馈调节N-乙酰谷氨酸合酶ArgA的突变体基因ArgA215，获得L-精氨酸生产能力更高的基因工程重组菌株L-Arg bac.(ECN ΔArgR attλ:fnr-ArgA215)，首先获得整合乏氧诱导型启动子的突变体Fnr-ArgA215基因核苷酸序列，以及该基因的插入位点核苷酸序列信息；
Fnr-ArgA215核苷酸序列：
aaaaacgccgcaaagtttgagcgaagtcaataaactctctacccattcagggcaatatctctcttggatccaaagtgaactctagaaataattttgtttaactttaagaaggagatatacatatggtaaaggaacgtaaaaccgagttggtcgagggattccgccattcggttccctgtatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccgtctggtggtggtctatggcgcacgtccgcagatcgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcggtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggctaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaa；
attλ插入位点附近核苷酸序列信息：
atttgccgcgcgggttgacgtcgtgacgccagtcggcagggctcatgccaccatcaagggaaagcccaatcttcacatcaatcggtttttcacccgtaccgtacagagtaattccacccggagcggcagggacatacaccgttccctgatactcaccaggcatcacggcaatatactggcgcttgttggtacgcttgataattgccgcatctaccgccgcctgaatcgtggtatgcgttacaccttgagtacccgccgggccgacaacaaagtcaggttgcgcaggcagggtaatcggggaaggattccacgctgccgcacctggtgtcagggatgcaaaatagtgttgagcatcgaaattctgcgcttcttttgccgacagaatcgggcgcgaagaggtaccaggcgcggtttgatcagaaggacgttgatcgggcggtgttgagctacaggcggtcagcgtcacgccaaaagccaatgccagcgccagacgggaaactgaaaatgtgttcacaggttgctccgggctatgaaatagaaaaatgaatccgttgaagcctgcttttttatactaacttgagcgaaacgggaaggtaaaaagacaaaaagttgtttttaatacctttaagtgataccagatggcattgcgccatctggcagagtgattaactaaacatcgcagtaatcgaggcactcgccagagagtgaaaatgaacgttaaacccgaccatcgcgccgctggcaccttcatcgacatcaatacgttctacatccagcgcgtgaacggtaaaaatgtagcgatgggtttcgcctttcggcggcgctgcgccatcgtacccggttttaccaaagtcggtacgcgtctgcaaaacgccgtctggcatagctaccagaccagagccaaacccttgcggtaatacgcgggtatcagcgggtaaattaacaactacccagtgccaccagccggagccggttggcgcatccgggtcat；
插入Fnr-ArgA215后，整个表达盒序列信息如下：
cagtcggcagggctcatgccaccatcaagggaaagcccaatcttcacatcaatcggtttttcacccgtaccgtacagagtaattccacccggagcggcagggacatacaccgttccctgatactcaccaggcatcacggcaatatactggcgcttgttggtacgcttgataattgccgcatctaccgccgcctgaatcgtggtatgcgttacaccttgagtacccgccgggccgacaacaaagtcaggttgcgcaggcagggtaatcggggaaggattccacgctgccgcacctggtgtcagggatgcaaaatagtgttgagcatcgaaattctgcgcttcttttgccgacagaatcgggcgcgaagaggtaccaggcgcggtttgatcagaaggacgttgatcgggcggtgttgagctacaggcggtcagcgtcacgccaaaagccaatgccagcgccagacgggaaactgaaaatgtgttcacaggttgctccgggctatgaaatagaaaaatgaatccgttgaagcctgcttttttaaaaacgccgcaaagtttgagcgaagtcaataaactctctacccattcagggcaatatctctcttggatccaaagtgaactctagaaataattttgtttaactttaagaaggagatatacatatggtaaaggaacgtaaaaccgagttggtcgagggattccgccattcggttccctgtatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccgtctggtggtggtctatggcgcacgtccgcagatcgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcggtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggctaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaaatactaacttgagcgaaacgggaaggtaaaaagacaaaaagttgtttttaatacctttaagtgataccagatggcattgcgccatctggcagagtgattaactaaacatcgcagtaatcgaggcactcgccagagagtgaaaatgaacgttaaacccgaccatcgcgccgctggcaccttcatcgacatcaatacgttctacatccagcgcgtgaacggtaaaaatgtagcgatgggtttcgcctttcggcggcgctgcgccatcgtacccggttttaccaaagtcggtacgcgtctgcaaaacgccgtctggcatagctaccagaccagagccaaacc。
第一步，同源臂的构建，首先进行引物的设计，并对引物fnr-ArgA215扩增，用设计的引物ArgA-attλ-F（tgaagcctgcttttttaaaaacgccgcaaagtttgagcga）/ argA-attλ-R（gctcaagttagtatttaccctaaatccgccatcaacactt），以基因合成质粒为模板按照设定条件扩增fnr-ArgA215的片段。其次用引物attλ ArgA215-F（tggcggatttagggtaaatactaacttgagcgaaacggga）/attλArg A 215-R（actttgcggcgtttttaaaaaagcaggcttcaacggattc）通过ECN attλ-puxT质粒为模板进行反向扩增attλ同源臂，得到产物经PCR纯化后，按照设定条件将回收的产物进行无缝克隆连接，并在冰上静置30 min，转化 Ecoli DH5alpha成感受态细胞到含氨苄青霉素(Amp)抗性的平板均匀涂布，在37℃培养过夜。最后对长出的克隆进行菌落PCR鉴定，挑取单克隆至10 μL无菌水中混匀后取，0.5μL为模板在设定体系条件体系下进行扩增，用引物M13-F(pUC)：CCCAGTCACGACGTTGTAAAACG和M13-R(pUC)：AGCGGATAACAATTTCACACAGG进行鉴定，得到阳性的克隆进行测序比对，以测序阳性的attλ-ArgA215 pux-T质粒为模板按照设定体系条件进行扩增修复同源臂扩增，得到阳性克隆测序结果为99.70%，成功获得整合的同源臂；
Nissle1917△ArgR attλ-argA插入测序及比对测序拼接结果：（测序引物1917 attλ up-F、1917 attλcx-F、1917 attλcx-R）
atcggtttttccccgtaccgtacagagtaattccacccggagcggcagggacatacaccgttccctgatactcaccaggcatcacggcaatatactggcgcttgttggtacgcttgataattgccgcatctaccgccgcctgaatcgtggtatgcgttacaccttgagtacccgccgggccgacaacaaagtcaggttgcgcaggcagggtaatcggggaaggattccacgctgccgcacctggtgtcagggatgcaaaatagtgttgagcatcgaaattctgcgcttcttttgccgacagaatcgggcgcgaagaggtaccaggcgcggtttgatcagaaggacgttgatcgggcggtgttgagctacaggcggtcagcgtcacgccaaaagccaatgccagcgccagacgggaaactgaaaatgtgttcacaggttgctccgggctatgaaatagaaaaatgaatccgttgaagcctgcttttttaaaaacgccgcaaagtttgagcgaagtcaataaactctctacccattcagggcaatatctctcttggatccaaagtgaactctagaaataattttgtttaactttaagaaggagatatacatatggtaaaggaacgtaaaaccgagttggtcgagggattccgccattcggttccctgtatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccgtctggtggtggtctatggcgcacgtccgcagatcgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcggtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggctaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaaatactaacttgagcgaaacgggaaggtaaaaagacaaaaagttgtttttaatacctttaagtgataccagatggcattgcgccat；
与预期插入后序列比对：
Fast alignment of DNA sequences DNAMAN2 and DNAMAN1
Ktuple=2  Gap_penalty=7
Upper line: DNAMAN2, from 58 to 2078
Lower line: DNAMAN1, from 6 to 2026
DNAMAN2:DNAMAN1 identity= 99.70%(2020/2026) gap=2.50%(52/2078)；
第二步，Crispr-Cas9基因插入，将ECN △ArgR Cas9菌株电转成感受态制备，并取制备好的电转化感受态100 μL加入sgRNA（SEQ ID NO.4）质粒和修复同源臂，在2500KV条件下电转化，其次在卡那霉素/壮观霉素（Kan/spc）抗性平板上进行涂布培养，最后用引物attλ-up-F/attλ-down-R进行整合鉴定（图3）；
第三步，消除抗性，接种插入成功菌株至LB液体培养基（无抗），在37℃过夜，稀释倍数进行涂板LB（无抗）平板，37℃过夜，挑取单克隆分别点板在LB（无抗）、LB（壮观霉素）、LB（卡那霉素），选择抗性板子不长的克隆接种于LB无抗培养基保存菌株，并命名为ECN △ArgR attλ：fnr-argA215。
参照上述方法，分别使用ArgA213、ArgA214和ArgA216基因替换ArgA215基因制备工程菌，并检测各工程菌精氨酸的生成速率，具体方法包括：将含有工程质粒的工程菌与谷氨酰胺和鸟氨酸共同孵育6 h，并使用LC-MS测定含有表达突变的argA（ArgA213、ArgA214、ArgA215、ArgA216）质粒的细胞游离精氨酸的总产量，获得细胞每小时每干重游离精氨酸的合成速率，结果如图8所示，可知使用ArgA215基因能够进一步提高精氨酸的生成速率。
ArgA213基因序列为：
atggtaaaggaacgtaaaaccgagttggtcgagggattccgccattcggttccctatatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccatctggtggtggtctatggcgcacgtccgcagattgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcggtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggcgaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaa。
ArgA214基因序列为（SEQ ID NO.5）：
atggtaaaggaacgtaaaaccgagttggtcgagggattccgctattcggttccctatatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccgtctggtggtggtctatggcgcacgtccgcagatcgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcggtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggctaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaa。
ArgA216基因序列为：
atggtaaaggaacgtaaaaccgagttggtcgagggattccgccattcggttccctatatcaatacccaccggggaaaaacgtttgtcatcatgctcggcggtgaagccattgagcatgagaatttctccagtatcgttaatgatatcgggttgttgcacagcctcggcatccgtctggtggtggtctatggcgcacgtccgcagatcgacgcaaatctggctgcgcatcaccacgaaccgctgtatcacaagaatatacgtgtgaccgacgccaaaacactggaactggtgaagcaggctgcgggaacattgcaactggatattactgctcgcctgtcgatgagtctcaataacacgccgctgcagggcgcgcatatcaacgtcgtcagtggcaattttattattgcccagccgctgggcgtcgatgacggcgtggattactgccatagcgggcgtatccggcggattgatgaagacgcgatccatcgtcaactggacagcggtgcaatagtgctaatggggccggtcgctgtttcagtcactggcgagagctttaacctgacctcggaagagattgccactcaactggccatcaaactgaaagctgaaaagatgattggtttttgctcttcccagggcgtcactaatgacgacggtgatattgtctccgaacttttccctaacgaagcgcaagcgcgggtagaagcccaggaagagaaaggcgattacaactccggtacggtgcgctttttgcgtggcgcagtgaaagcctgccgcagcggcgtgcgtcgctgtcatttaatcagttatcaggaagatggcgcgctgttgcaagagttgttctcacgcgacggtatcagtacgcagattgtgatggaaagcgccgagcagattcgtcgcgcaacaatcaacgatattggcggtattctggagttgattcgcccactggagcagcaaggtattctggtacgccgttctcgcgagcagctggagatggaaatcgacaaattcaccattattcagcgcgataacacgactattgcctgcgccgcgctctatccgttcccggaagagaagattggggaaatggcctgtgtggcagttcacccggattaccgcagttcatcaaggggtgaagttctgctggaacgcattgccgctcaggcgaagcagagcggcttaagcaaattgtttgtgctgaccacgcgcagtattcactggttccaggaacgtggatttaccccagtggatattgatttactgcccgagagcaaaaagcagttgtacaactaccagcgtaaatccaaagtgttgatggcggatttagggtaa。
实施例2
本实施例进行基因工程菌L-Argbac.活性的检测与体外培养L-Arg产量检测。
①活性检测。
分别在有氧和无氧条件下测试工程菌活性，将ECN和L-ArgBac.菌株从-80℃的甘油菌复苏，并各分成两组：有氧和无氧变量组，四组菌株分别接种到LB培养基的摇菌管中，在37℃、100r/min条件下震荡培养12h，利用紫外分光光度计检测菌株OD 600值，取OD 600在0.6~0.8之间得菌液，充入氮气排除无氧组LB培养基菌液中的氧气，无氧组在无氧环境中，37℃静置2h进行通路诱导。各组取0.25mLLB培养基菌液用离心法收集菌体，并用PBS清洗一次后稀释20倍在M9培养基中，用摇菌管在37℃、100rpm环境中摇瓶培养，采集各组0h时的代表性样本，同时震荡培养，分别取3h、9h和24h等不同时间节点的样品，检测OD 600值（图4），由OD 600值数据变化可知，本发明所得工程菌株L-ArgBac.活性较野生菌减弱，但是总体上菌株活性改变不大，且活性趋势向上；
②L-Arg产量检测。
在①中取无氧组3h、9h和24h取有代表性的样本LC-MS检测改造后的菌株L-Arg产量，由检测结果可得L-ArgBac.经基因工程改造后L-Arg产量是野生大肠杆菌ECN菌的1000倍（图5）。
实施例3
本实施例进行基因工程菌L-ArgBac.的肿瘤靶向定殖。
取-80℃冻存的ECN和L-ArgBac.甘油菌复苏，各分成两组，同实施例2①中标记组别，分别接种到LB培养基的摇菌管中，37℃、100r/min，震荡培养12h，监测菌液OD 600值，取OD 600值0.6~0.8菌液离心收集菌体，用PBS洗涤3次并计数，取5×10 7个菌体用PBS重悬后尾静脉注入荷瘤的老鼠体内，24h后，断颈处死小鼠，取肿瘤组织并称重，将肿瘤组织消化重悬成单细胞悬液，稀释的单细胞悬液样品各20μL，在细菌培养板上进行均匀涂板，37℃培养12h，进行菌落计数，计算单位肿瘤组织内所含的菌落数（图6），表明本发明改造所得菌株通过尾静脉注射后肿瘤靶向能力与原始菌株靶向能力改变不大。
实施例4
本实施例进行基因工程菌L-ArgBac.瘤内产L-Arg与抑瘤效果分析。
使用免疫正常的C57/BL6J小鼠构建B16动物肿瘤模型，每只小鼠皮下注射5×10 6鼠源黑色素瘤B16F10细胞，5天后肿瘤体积达到50mm 3，并将荷瘤小鼠随机分成两组：ECN组和L-Argbac.组，取-80℃冻存的ECN和L-ArgBac.甘油菌复苏，同实施例2①中标记组别，分别接种到LB培养基的摇菌管中，37℃、100r/min，震荡培养过夜，取OD 600值0.6~0.8菌液离心收集菌体，离心收集菌体，用PBS洗涤3次并计数，将5×10 7CFU菌株用PBS重悬后尾静脉注射到荷瘤的小鼠体内，定期记录小鼠肿瘤定期记录（图7），由图7可知，L-ArgBac.组瘤内产生的L-Arg使得肿瘤较ECN组生长缓慢，对肿瘤的生长有一定的抑制效果。
综上所述，本发明通过基因编辑技术合成L-Arg工程菌，敲除或弱化工程菌株中抑制L-Arg合成途径的阻遏蛋白ArgR基因，使得菌株L-Arg产量获得显著的提升，并整合特定过表达基因ArgA和诱导型启动子，从而实现诱导启动大量产生L-Arg，成功构建厌氧高产L-精氨酸工程菌，L-精氨酸产量与野生菌相比提高近1000倍，且所述工程菌能够在肿瘤微环境中靶向定殖，利用肿瘤细胞生成的代谢废物作为营养物质生存，并产生L-Arg通过代谢来调控肿瘤免疫微环境，对提高肿瘤治疗疗效有着重要的意义。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种厌氧高产L-精氨酸工程菌，其特征在于，所述工程菌中含有编码N-乙酰谷氨酸合成酶变体的基因，所述编码N-乙酰谷氨酸合成酶变体的基因由被外源环境条件诱导的启动子控制；
    所述工程菌被工程化为缺乏功能性Arg R；
    所述编码N-乙酰谷氨酸合成酶变体的基因具有选自以下的DNA序列中任意一种：
    a)SEQ ID NO.1或SEQ ID NO.5；
    b)除了冗余的遗传密码以外，编码与由SEQ ID NO.1或SEQ ID NO.5编码的多肽相同的多肽的DNA序列；
    c)与a)或b)的DNA序列具有至少80%同源性的DNA序列。
  2. 根据权利要求1所述的厌氧高产L-精氨酸工程菌，其特征在于，所述编码N-乙酰谷氨酸合成酶变体的基因在工程菌基因组上的插入位置包括malE和malK之间或attλ插入位点；
    优选地，所述工程化为缺乏功能性Arg R包括敲除Arg R基因或弱化Arg R基因表达量。
  3. 根据权利要求1所述的厌氧高产L-精氨酸工程菌，其特征在于，所述被外源环境条件诱导的启动子包括乏氧响应启动子、酸响应启动子、氧化还原响应启动子、光刺激启动子、电刺激启动子或磁驱动启动子中任意一种；
    优选地，所述乏氧响应启动子选自fnr-sp、hip1、i14018、ptet-arca、ptet-fnr、r1074、ssbp1或ysgap中任意一种。
  4. 根据权利要求1所述的厌氧高产L-精氨酸工程菌，其特征在于，所述工程菌的出发菌株选自谷氨酸棒杆菌、大肠杆菌、枯草芽孢杆菌、变形杆菌、肠杆菌、伤寒杆菌、沙门氏菌或志贺氏菌中任意一种。
  5. 一种权利要求1-4任一项所述的厌氧高产L-精氨酸工程菌的制备方法，其特征在于，所述方法包括：
    利用Crispr-Cas9系统敲除出发菌株的Arg R基因，并在出发菌株的基因组中插入编码N-乙酰谷氨酸合成酶变体的基因，所述编码N-乙酰谷氨酸合成酶变体的基因由被外源环境条件诱导的启动子控制，得到所述厌氧高产L-精氨酸工程菌；
    所述编码N-乙酰谷氨酸合成酶变体的基因具有选自以下的DNA序列中任意一种：
    a)SEQ ID NO.1或SEQ ID NO.5；
    b)除了冗余的遗传密码以外，编码与由SEQ ID NO.1或SEQ ID NO.5编码的多肽相同的多肽的DNA序列；
    c)与a)或b)的DNA序列具有至少80%同源性的DNA序列。
  6. 根据权利要求5所述的厌氧高产L-精氨酸工程菌的制备方法，其特征在于，其特征在于，所述Crispr-Cas9系统的sgRNA的核酸序列包括SEQ ID NO.3和SEQ ID NO.4所示的序列。
  7. 权利要求1-4任一项所述的厌氧高产L-精氨酸工程菌在生产L-精氨酸中的应用。
  8. 一种生产L-精氨酸的方法，其特征在于，所述方法包括：
    对权利要求1-4任一项所述的厌氧高产L-精氨酸工程菌进行发酵培养，获得L-精氨酸。
  9. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-4任一项所述的厌氧高产L-精氨酸工程菌。
  10. 权利要求1-4任一项所述的厌氧高产L-精氨酸工程菌或权利要求9所述的药物组合物在制备治疗或辅助治疗肿瘤的药物中的应用；
    优选地，所述肿瘤包括急性髓系白血病、神经母细胞瘤、黑色素瘤、非小细胞肺癌、三阴性乳腺癌、脑转移瘤、结肠癌或肝癌。
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