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CN-122012444-A - Aminotransferase mutants and their use in leucine production

CN122012444ACN 122012444 ACN122012444 ACN 122012444ACN-122012444-A

Abstract

The invention discloses an aminotransferase mutant from thermophilic thermobacteria (Tepidiphilus thermophilus) and application thereof in L-leucine production, wherein the aminotransferase mutant has high aminotransferase activity, so that the yield of L-leucine is improved, and meanwhile, the accumulation of byproduct valine is effectively reduced.

Inventors

  • HAO YI
  • ZHANG ZHIYUAN
  • ZHANG JIARONG
  • LI HONGXUAN
  • JIN XIANG

Assignees

  • 北京量维生物科技研究院有限公司
  • 陕西量维生物工程有限公司
  • 甘肃量维生物工程有限公司

Dates

Publication Date
20260512
Application Date
20260407

Claims (6)

  1. 1. An aminotransferase mutant, characterized by being obtained by mutating the amino acid sequence shown in SEQ ID NO. 1 as a basis at a site selected from the group consisting of: 1) Mutating amino acid 169 from A to D; 2) Mutation of amino acid 233 from H to R; 3) Mutation of amino acid 234 from H to A; 4) The 236 th amino acid is mutated from L to A.
  2. 2. A nucleic acid molecule encoding the mutant of claim 1.
  3. 3. A biological material comprising the nucleic acid molecule of claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector or a viral vector.
  4. 4. A recombinant microorganism comprising the aminotransferase mutant of claim 1 or the nucleic acid molecule of claim 2.
  5. 5. Use of an aminotransferase mutant according to claim 1, a nucleic acid molecule according to claim 2, a biological material according to claim 3 or a recombinant microorganism according to claim 4 for the production of L-leucine.
  6. 6. A method for producing L-leucine, characterized in that the aminotransferase mutant according to claim 1, the nucleic acid molecule according to claim 2, the biological material according to claim 3 or the recombinant microorganism according to claim 4 is used.

Description

Aminotransferase mutants and their use in leucine production Technical Field The invention belongs to the technical fields of genetic engineering and fermentation engineering, and particularly relates to an amino transferase mutant and application thereof in leucine production. Background L-leucine is one of eight essential amino acids, and has wide application and market demands in the fields of medicine, food, feed, cosmetics and the like. The global L-leucine market scale is continuously increased, and compared with the traditional hair hydrolysis method for producing L-leucine, the microbial fermentation method has the characteristics of low cost, green, high efficiency and the like. However, the technology for producing L-leucine by microbial fermentation has the defects of long synthesis path and strict feedback regulation mechanism of the L-leucine in the microorganism, and has the problems of low yield, low conversion rate, long fermentation period, more accumulation of branched-chain amino acid byproducts represented by L-valine and the like, thus limiting the economic benefit. Aminotransferase (aminotransferase, also known as aminotransferase) in bacteria is a large and functionally diverse enzyme family that catalyzes the transfer of the amino group of one amino acid to another alpha-keto acid to produce a new keto acid and a new amino acid, playing a key role in nitrogen metabolism, amino acid synthesis and degradation, and cell wall synthesis, among other core vital activities. The last step in L-leucine biosynthesis in bacteria such as E.coli is the key rate limiting step, and the branched chain amino acid aminotransferase encoded by ilvE catalyzes the precursor alpha-keto isohexide (alpha-KIC) to produce L-leucine by transamination. However, the enzyme has low catalytic efficiency and weak substrate specificity, and is involved in the synthesis of other branched-chain amino acids, thus being extremely easy to accumulate L-valine and L-isoleucine (the metabolic pathway is shown in figure 1). In order to specifically enhance L-leucine synthesis, a common strategy is to select an enzyme with stronger affinity for L-leucine precursor alpha-KIC and higher catalytic efficiency to replace endogenous branched-chain amino acid aminotransferase. The strategy adopted by CN116355818A is to introduce a leucine dehydrogenase (Leucine dehydrogenase, NCBI protein database number and EC number are NP_390288.1 and EC:1.4.1.9 respectively) derived from bacillus subtilis into escherichia coli, the encoding genes UniProt are named leuDH, NCBI are named bcd, and an asymmetric reductive amination reaction catalyzed by the enzyme is utilized to replace one oxygen atom on a keto acid molecule with an amino group (-NH 2) with the help of coenzyme NADH, so that the L-amino acid is finally generated. The L-leucine yield of the constructed engineering bacteria in a fermentation tank reaches 85.6 g/L, the conversion rate is 0.36 g/g, and the engineering bacteria become the highest production performance strain reported in the prior art. However, the Bacillus LeuDH has high affinity for L-leucine precursor alpha-ketoisohexide (alpha-KIC) and also has affinity for L-valine precursor alpha-ketoisovalerate (alpha-KIV), and accumulation of L-valine is easy to cause during high expression, so that the production performance of leucine is reduced, and the separation and purification cost is increased. CN110607268B is prepared by using the characteristic of LeuDH, and when constructing high-yield L-valine engineering bacteria, a leucine dehydrogenase gene of bacillus subtilis is also introduced to replace a branched-chain amino acid aminotransferase gene ilvE of escherichia coli to catalyze alpha-KIV to produce L-valine. In addition, it was found that an Aromatic amino acid aminotransferase (coding gene tyrB) mainly responsible for Aromatic amino acid synthesis such as E.coli also has a function of specifically catalyzing synthesis of L-leucine by alpha-KIC, and that the affinity of the Aromatic amino acid aminotransferase tyrB encoded by tyrB for leucine substrate alpha-KIC is significantly higher than that of valine substrate alpha-KIV, compared with the leucine dehydrogenase encoded by leuDH. However, there are few studies on the direct catalysis of alpha-KIC to L-leucine by TyrB, and there is little in-depth excavation and optimization of this function of TyrB. Although the strain for producing L-leucine is constructed by substituting tyrB for ilvE gene, the yield of leucine is not ideal and is difficult to meet the requirement of industrial production. Therefore, the method for excavating and constructing the aminotransferase with high enzyme activity and strong substrate specificity is an important direction for improving the synthesis quality and the synthesis efficiency of the L-leucine. In recent years, artificial intelligence technology has shown great potential in enzyme mining and protein design modification, and inte