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CN-122012558-A - Method for improving citrulline production of escherichia coli

CN122012558ACN 122012558 ACN122012558 ACN 122012558ACN-122012558-A

Abstract

The invention discloses a method for improving the production of citrulline by escherichia coli, which comprises the following steps of knocking out one, two or three of the following three unnecessary gene sequences in escherichia coli genome for producing L-citrulline, namely a sequence DLP12 located between genome coordinates 564755-58065, a sequence Part4 located between genome coordinates 3110645-3134692 and a sequence CP4-57 located between genome coordinates 2755941-2777971. Meanwhile, the knocking out of three gene sequences can improve the fermentation yield of L-citrulline by 27.42 percent, and has industrial application prospect.

Inventors

  • SHENG LILI
  • LIU SENSEN
  • HE MENG
  • WANG QINGPEI
  • LIU YINGMIAO
  • FAN WENCHAO

Assignees

  • 华睿生物技术(滁州)有限公司

Dates

Publication Date
20260512
Application Date
20260123

Claims (10)

  1. 1. A method for improving the production of citrulline by escherichia coli is characterized by comprising the following steps of knocking out one, two or three sequences selected from the following three sequences in escherichia coli genome for producing L-citrulline: sequence DLP12 (NCBI accession number RefSeq: NC-000913.3 version 2025) located between genomic coordinates 564755-58065, The sequence Part4 (NCBI accession number RefSeq: NC-000913.3 version 2025) located between the genomic coordinates 3110645-3134692, Sequences CP4-57 (NCBI accession number RefSeq: NC-000913.3 version 2025) located between the genomic coordinates 2755941-2777971.
  2. 2. The method of claim 1, wherein the three gene sequences DLP12, part4 and CP4-57 in the e.coli genome are knocked out simultaneously.
  3. 3. The method according to claim 1, wherein the escherichia coli is escherichia coli MG1655 derivative SH2833, and is an engineering bacterium obtained by genetic engineering of taking escherichia coli MG1655 as chassis bacterium and knocking out argG gene encoding arg-amino succinic acid synthase in genome; knocking out the gene encoding the transcription regulatory factor argR in the genome, and overexpressing the gene cluster argCJBDF and lysE gene related to the L-citrulline synthesis pathway derived from Corynebacterium glutamicum 13032.
  4. 4. The method of claim 1, wherein the knockout of the gene sequence DLP12, part4 or CP4-57 is performed by a gene editing technique.
  5. 5. The method of claim 4, wherein the gene editing technique is selected from the group consisting of homologous double crossover, red homologous recombination, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, mugent, CRISPRi, and preferably CRISPR-Cas9 gene editing system.
  6. 6. A method of constructing an L-citrulline producing bacterium comprising the steps of: (1) An integrated system of over-expressed argCJBDF gene cluster and lysE gene (2025 NCBI gene ID:1019244, NC_003450.3 version, nucleotide sequence shown as SEQ ID NO: 6) is constructed by taking Escherichia coli MG1655 as chassis bacteria and aiming at Escherichia coli MG1655 genome, CRISPR-Cas9 gene editing plasmid for constructing knockout gene is respectively designed and constructed aiming at gene argG (2025 NCBI gene ID:947590 NC_000913.3 version), gene argR (2025 NCBI gene ID:947816 NC_000913.3 version), sequence DLP12, sequence Part4 and sequence CP4-57, Wherein argCJBDF gene clusters include 5 genes argC (2025 NCBI gene ID:1019370 NC_003450.3 version, nucleotide sequence shown in SEQ ID NO: 1), argJ (2025 NCBI gene ID:1019371 NC_003450.3 version, nucleotide sequence shown in SEQ ID NO: 2), argB (2025 NCBI gene ID:1019372 NC_003450.3 version, nucleotide sequence shown in SEQ ID NO: 3), argD (2025 NCBI gene ID:1019373 NC_003450.3 version, nucleotide sequence shown in SEQ ID NO: 4), argF (2025 NCBI gene ID:1019374 NC_003450.3 version, nucleotide sequence shown in SEQ ID NO: 5); (2) Sequentially introducing the argG gene knockout system and the argR gene knockout system constructed in the step (1), the argCJBDF gene cluster overexpression vector and the lysE gene overexpression vector into escherichia coli MG1655, and obtaining a basic strain SH2833 with the argG and argR deleted genes and the argCJBDF and lysE overexpressed genes through resistance screening and genotype verification; (3) Respectively introducing CRISPR-Cas9 gene editing plasmids of the knockout sequences DLP12, the sequences Part4 or the sequences CP4-57 constructed in the step (1) into a basic strain SH2833, and respectively obtaining a single knockout strain of the knockout sequences DLP12, a single knockout strain of the knockout sequences Part4 and a single knockout strain of the knockout sequences CP4-57 through resistance screening and genotype verification; (4) And (3) taking any single knockout strain obtained in the step (3) as an initial strain, continuously knocking out the remaining two sequences, and obtaining the triple knockout recombinant escherichia coli with the simultaneous knockout sequences DLP12, part4 and CP4-57 through resistance screening and genotype verification.
  7. 7. Recombinant E.coli constructed by the method of any one of claims 1-6.
  8. 8. The recombinant E.coli according to claim 7, wherein the recombinant E.coli is constructed by knocking out three sequences DLP12, part4 and CP4-57 from the genome based on the Escherichia coli MG 1655-derived strain SH 2833/chassis.
  9. 9. Use of the recombinant E.coli according to claim 7 for the fermentative preparation of L-citrulline.
  10. 10. Use according to claim 9, characterized in that the recombinant escherichia coli according to claim 8 is cultivated in a medium under fermentation conditions and L-citrulline is extracted from the fermentation broth and/or the thallus.

Description

Method for improving citrulline production of escherichia coli Technical Field The invention belongs to the technical field of genetic engineering, and relates to a method for improving the production of L-citrulline by escherichia coli. Background Citrulline is an important non-protein amino acid, and is widely applied to the fields of foods, medicines, health care products and the like by virtue of unique physiological functions and chemical properties, so that the market demand is continuously rising. In the food industry, citrulline can be used as a natural flavor enhancer to be added into meat products, beverages and baked foods, so that the fresh flavor and the mellow feeling of the products can be improved, the necessary nitrogen source nutrition of human bodies can be supplemented, the nutritional value and the edible taste of the foods can be improved, in the medicine field, the citrulline can be used as a key intermediate product of urea circulation, can effectively regulate in-vivo nitrogen metabolism balance, promote detoxification and excretion of ammonia, can be used for preparing medicines for treating hepatic encephalopathy, liver injury, hypertension and cardiovascular diseases, and simultaneously has good application potential in the aspects of wound repair, immunoregulation and the like, and in the health care product field, the citrulline can promote nitric oxide synthesis by vascular endothelial cells, expand blood vessels, improve blood circulation, further promote exercise endurance and relieve fatigue after exercise, and is one of core components of high-end exercise nutrition supplements. Along with the rapid development of the global health industry, the annual average growth rate of the market on the high-purity citrulline is kept at 8% -12%, and the development of efficient, low-cost and environment-friendly citrulline production technology has become the key for guaranteeing the sustainable development of related industries and has important economic value and social significance. At present, the production method of citrulline mainly comprises a chemical synthesis method, an enzyme method and a microbial fermentation method. The chemical synthesis method has the defects of harsh reaction conditions, more byproducts, serious environmental pollution, low product purity and the like, is gradually eliminated by the market, the enzyme method for producing the citrulline has the advantages of high reaction specificity and high product purity, but the enzyme preparation cost is high, the stability is poor, the large-scale industrial production is difficult to realize, and the microbial fermentation method has the advantages of wide raw material sources, mild reaction conditions, environmental friendliness, easiness in large-scale amplification and the like, so the method becomes the main flow direction of the current citrulline industrial production. In the research of citrulline production by a microbial fermentation method, the selection and transformation of strains are the core of improving the yield. As a classical mode microorganism, the escherichia coli has the unique advantages of clear genetic background (about 4.6Mb of whole genome, 4405 coding genes are contained, the sequence is completely resolved), high growth and propagation speed (the doubling time of the log phase is only 20-30 minutes), clear metabolic regulation mechanism, easy precise transformation by genetic engineering means (such as CRISPR-Cas9, red homologous recombination and the like) and the like, and is widely applied to fermentation production of various amino acids such as glutamic acid, lysine, threonine and the like and biological products such as organic acid, enzyme preparations and the like. The escherichia coli MG1655 is a typical representative of a wild escherichia coli K-12 strain, has the characteristics of no plasmid, no resistance mark, stable metabolism and the like, has complete analysis of genome sequences in 1997, has perfect related gene operation tools and metabolism regulation databases, is an ideal starting strain for constructing high-yield engineering strains, and has an irreplaceable application foundation in the field of constructing amino acid high-yield strains. In the research of producing citrulline by using escherichia coli fermentation, the prior art mainly focuses on local genetic modification of citrulline synthesis pathways and branch metabolic pathways, and the core thought is to enhance citrulline accumulation by strengthening expression of key enzymes of the synthesis pathways, blocking degradation of products or branch metabolic pathways. For example, the distribution of carbon and nitrogen metabolism to citrulline is enhanced by over-expressing key genes such as N-acetylglutamate synthase gene (argA), N-acetylornithine aminotransferase gene (argD) and ornithine carbamoyltransferase gene (argF) in the arginine synthesis pathway, or the conversion of citrulline to argi