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KR-20260066190-A - ESCHERICHIA COLI-BASED RECOMBINANT STRAIN, CONSTRUCTION METHOD THEREFOR AND USE THEREOF

KR20260066190AKR 20260066190 AKR20260066190 AKR 20260066190AKR-20260066190-A

Abstract

The present invention provides a recombinant strain after site-specific mutation based on Escherichia coli strain K12 or a derivative strain thereof, a method for constructing the same, and an application thereof. The recombinant strain has undergone site-specific mutation for the kdtA gene, spoT gene, or yebN gene and can produce a higher concentration of L-threonine compared to a non-mutated wild-type strain.

Inventors

  • 웨이, 에잉
  • 멩, 강
  • 지아, 휴이핑
  • 가오, 시아오항
  • 마, 펭용
  • 저우, 시아오군
  • 자오, 청구앙
  • 양, 리펭
  • 수, 호유보

Assignees

  • 헤이룽지앙 에펜 바이오테크 컴퍼니 리미티드

Dates

Publication Date
20260512
Application Date
20200827
Priority Date
20190828

Claims (9)

  1. A polynucleotide comprising a sequence having a mutation in which guanine (G) is mutated to adenine (A) at the 82nd base of sequence number 1.
  2. In paragraph 1, The mutated nucleotide sequence is a polynucleotide, which is the sequence denoted by SEQ ID NO. 2.
  3. A recombinant protein containing the amino acid sequence represented by SEQ ID NO. 4.
  4. A recombinant vector comprising a polynucleotide encoding a recombinant protein according to paragraph 3.
  5. A recombinant strain comprising a polynucleotide encoding a recombinant protein according to paragraph 3.
  6. In paragraph 5, A recombinant strain is formed by introducing a recombinant vector according to claim 4 into a host strain; and the host strain is a recombinant strain selected from Escherichia coli .
  7. A method for constructing a recombinant strain according to claim 5, comprising the step of obtaining a recombinant strain by introducing a polynucleotide encoding a recombinant protein according to claim 3 into a host strain.
  8. Method of using polynucleotide according to claim 1 in the fermentation production of L-threonine.
  9. Method of using a recombinant protein according to paragraph 3 in the fermentation production of L-threonine.

Description

Escherichia coli-based recombinant strain, construction method therefor, and application therefor Cross-reference of related applications The present invention claims priority of a prior application with patent application number 2019109262958 filed with the National Intellectual Property Administration of China on September 27, 2019, priority of a prior application with patent application number 2019108046792 filed with the National Intellectual Property Administration of China on August 28, 2019, and priority of a prior application with patent application number 2019108046881 filed with the National Intellectual Property Administration of China on August 28, 2019, wherein the full text of the said prior applications is incorporated into the present invention by reference. The present invention belongs to the field of genetic engineering and microbial technology, and specifically relates to a recombinant strain modified by the kdtA gene, a method for constructing the same, and its applications. L-threonine is one of the eight essential amino acids that humans and animals cannot synthesize on their own. L-threonine can enhance the absorption of grains, regulate metabolic balance in the body, and promote physical growth and development, and is widely used in the feed, pharmaceutical, and food industries. Currently, L-threonine production is mainly carried out through chemical synthesis, protein hydrolysis, and microbial fermentation. Among these, microbial fermentation is the most widely used method for the industrial production of L-threonine due to its low production cost, high production intensity, and minimal environmental pollution. Various bacteria can be used for the microbial fermentation production of L-threonine; for example, mutant strains obtained through wild-type induction, such as Escherichia coli, Corynebacterium , and Serratia , can be used as production strains. Specific embodiments include anti-amino acid analog mutant strains or strains with various nutritional requirements, such as methionine, threonine, and isoleucine. However, traditional mutant breeding makes it difficult to obtain strains with high yields because random mutations result in slow strain growth and the generation of more byproducts. Therefore, constructing recombinant Escherichia coli using metabolic engineering methods is an effective pathway for producing L-threonine. Currently, the overexpression or attenuation of key enzyme genes in amino acid synthesis pathways and competitive pathways mediated by expression plasmids is a major means for genetic modification of E. coli. However, the development of methods to produce L-threonine more economically with high yield is still necessary. Escherichia coli is highly regarded by genetic engineering experts as a host for exogenous gene expression due to its clear genetic background, simple technical manipulation and culture conditions, and the economic feasibility of large-scale fermentation. The genomic DNA of Escherichia coli consists of cyclic molecules within the nucleoid, and multiple cyclic plasmid DNAs may exist simultaneously. The nucleoid of an E. coli cell contains one DNA molecule with a length of approximately 4,700,000 base pairs; about 4,400 genes are distributed within this DNA molecule, with an average length of about 1,000 base pairs per gene. With a few exceptions, the E. coli strains commonly used in molecular biology, and the majority of strains used in DNA recombination experiments, are E. coli strain K12 and its derivatives. The present invention provides a recombinant strain based on Escherichia coli strain K12 or a derivative thereof, a method for constructing the recombinant strain thereof, and an application in the fermentation and production of amino acids. The present invention is based on the wild-type kdtA gene (ORF sequence as indicated by sequences 73556-74833 in Genbank accession number CP032667.1), the wild-type spoT gene (ORF sequence as indicated by sequences 3815907-3818015 in Genbank accession number AP009048.1), and the wild-type yebN gene (ORF sequence as indicated by sequences 1907402-1907968 in Genbank accession number AP009048.1) in E. coli K12 strain and derivative strains thereof (e.g., MG1655, W3110, etc.), wherein a mutant gene obtained after site-specific mutation of said genes and a recombinant strain containing said genes can be used for the production of L-threonine, and the obtained strain can significantly increase the yield of L-threonine compared to a non-mutated wild-type strain. It was discovered that the strain has excellent stability, allowing for reduced production costs as an L-threonine producing strain. Based on the above invention, the present invention provides technical solutions in three parts as follows. The first part is a nucleotide sequence, said nucleotide sequence includes a sequence formed by a mutation in the 82nd base of the wild-type kdtA gene coding sequence represented by SEQ ID NO. 1. According to