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CN-122012453-A - Polyphosphate kinase BsPPK mutant and construction and application of production bacteria thereof

CN122012453ACN 122012453 ACN122012453 ACN 122012453ACN-122012453-A

Abstract

The invention discloses a polyphosphate kinase BsPPK mutant and construction and application of a production strain thereof, belonging to the technical field of genetic engineering. According to the invention, site-directed mutagenesis is carried out on polyphosphate kinase BsPPK from the genus Bradyrhizomes by homologous modeling, molecular docking and multiple sequence comparison equivalent methods, and the mutation of lysine at 92 th position of wild BsPPK into alanine, the mutation of threonine at 95 th position into alanine and the mutation of serine at 205 th position into arginine are carried out, so that a combined mutant BsPPK-K92A/T95A/S205R is obtained. The enzyme activity measurement result shows that the specific enzyme activity of the mutant is improved by 324.4% compared with that of a wild type, and the catalysis efficiency of the mutant on the AMP is obviously improved. When the mutant is applied to the synthesis of UDP-Gal and derivative products thereof, similar yield of the wild type which can be obtained only by 30 mM AMP can be achieved by only 15 mM AMP, and the nucleotide consumption is reduced by 50%.

Inventors

  • MAO XIANGCHAO
  • ZHAO JIAJING
  • HU YANG
  • LI WANTING
  • CHEN YIHONG

Assignees

  • 中国海洋大学

Dates

Publication Date
20260512
Application Date
20260408

Claims (10)

  1. 1. A polyphosphate kinase mutant, wherein the mutant is obtained by amino acid substitution of a polyphosphate kinase with an amino acid sequence shown in SEQ ID No.1, and the mutant comprises at least one amino acid substitution selected from the group consisting of: (a) Alanine at position 68 is mutated to glycine; (b) Lysine 92 to alanine; (c) Threonine at position 95 is mutated to alanine; (d) Serine at position 191 is mutated to glutamic acid; (e) Serine 205 is mutated to arginine.
  2. 2. The polyphosphate kinase mutant of claim 1, wherein the mutant comprises at least two of the amino acid substitutions.
  3. 3. The polyphosphate kinase mutant according to claim 1, wherein the mutant is S205R, A G/K92A, A G/S191E, A G/T95A, K A/T95A, K A/S191E, T95A/S191E、T95A/S205R、S191E/S205R、A68G/K92A/T95A、A68G/K92A/S191E、A68G/K92A/S205R、A68G/T95A/S191E、A68G/T95A/S205R、A68G/S191E/S205R、K92A/T95A/S191E、K92A/T95A/S205R、K92A/S191E/S205R Or T95A/S191E/S205R.
  4. 4. A gene encoding the polyphosphate kinase mutant according to any one of claims 1 to 3.
  5. 5. A recombinant vector carrying the gene according to claim 4.
  6. 6. A microbial cell expressing the polyphosphate kinase mutant according to any one of claims 1 to 3, or carrying the gene according to claim 4, or carrying the recombinant vector according to claim 5.
  7. 7. A method for improving the enzyme activity of polyphosphate kinase is characterized in that the polyphosphate kinase with an amino acid sequence shown as SEQ ID NO.1 is subjected to amino acid substitution, wherein the substitution is at least one of the following sites, namely, mutation of alanine at position 68 into glycine, mutation of lysine at position 92 into alanine, mutation of threonine at position 95 into alanine, mutation of serine at position 191 into glutamic acid, or mutation of serine at position 205 into arginine.
  8. 8. Use of the polyphosphate kinase mutant according to any one of claims 1 to 3 for preparing Adenosine Triphosphate (ATP).
  9. 9. Use of the polyphosphate kinase mutant according to any one of claims 1 to 3 for preparing uridine diphosphate-galactose (UDP-Gal).
  10. 10. Use of a polyphosphate kinase mutant according to any one of claims 1 to 3 for the preparation of lactosyl-N-tetraose (LNT) and/or lactosyl-N-neotetraose (LNnT).

Description

Polyphosphate kinase BsPPK mutant and construction and application of production bacteria thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to a polyphosphate kinase BsPPK mutant and construction and application of a production strain thereof. Background The disclosure of this background section is only intended to increase some understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. Adenosine triphosphate (Adenosine' -triphosphate, ATP) is a high-energy phosphate compound that plays an important role in metabolic processes, enzymatic, biosynthesis, cofactor synthesis and cell-free protein synthesis. In industrial production, the use of in vitro ATP-dependent enzyme cascades is limited by the addition of the expensive substrate ATP and the accumulation of the by-product Adenosine Diphosphate (ADP). Therefore, the development of an ATP regeneration system is of great importance for the design of an efficient and economical cascade reaction. PPK enzymes reported at present are mainly divided into two families, PPK1 and PPK2, PPK1 preferentially catalyzes the transfer of phosphate groups from ATP to PolyP n to synthesize PolyP n+1, while PPK2 preferentially synthesizes ATP using AMP or ADP. Achbergerov a et al analysis compared sequence homologs of PPK1 from E.coli (ESCHERICHIA COLI) and PPK2 from P.aeruginosa (Pseudomonas aeruginosa), confirming low amino acid sequence similarity of PPK1 and PPK 2. Polyphosphate (polyP)/polyphosphate kinase (PPK) based ATP regeneration systems are of great interest. There are many reports in the prior art, for example, rpPPK from Rujie's bacteria (Ruegeria pomeroyi) is used for the production of LacNAc and DrPPK from Pediococcus radiodurans (Deinococcus radiodurans) is used for the production of D-enone. Among them, three kinds of PPK2 family enzyme members (PPK 2-3) can use cheap and stable sodium hexametaphosphate (PolyP 6) as phosphate donor to catalyze the synthesis of ATP by the phosphoric acid of adenosine diphosphate (Adenosine-5 '-diphosphate, ADP) and adenosine monophosphate (Adenosine-5' -monophosphate, AMP), and are considered to have more practical application prospect. Polyphosphate kinase (BsPPK) derived from the genus braytosis (Bulleidia sp.) has been disclosed in the prior art which is capable of using sodium hexametaphosphate as a phosphate donor and catalyzing both AMP and ADP phosphorylation to ATP. The discovery of this enzyme provides a new element choice for the construction of ATP regeneration systems. In practical use, when AMP is used as a phosphate acceptor, AMP is first synthesized into ADP and then further phosphorylated to ATP. In the two-step reaction process, the catalytic efficiency of the second step is relatively low, and the second step becomes the speed limiting step of ATP synthesis of the AMP, and is expressed as a certain gap between k cat/KmAMP and k cat/KmADP, which influences the application efficiency of BsPPK in the reaction actually taking the AMP as the initial nucleotide to a certain extent. Therefore, the development of a polyphosphate kinase mutant with further improved AMP catalytic activity to meet the requirement of efficient ATP regeneration has important practical significance. Disclosure of Invention Aiming at the prior art, the invention aims to provide a polyphosphate kinase mutant and construction and application of a production strain thereof. The inventor screens out a polyphosphate kinase from the genus Bradybacterium (Bulleidia sp.) through database mining comparison, clones the polyphosphate kinase into ESCHERICHIA COLIBL (DE 3) to realize high-efficiency expression, and verifies the function of recombinant enzyme. In order to further improve the catalytic activity of the enzyme on the AMP, the amino acid site of the polyphosphate kinase is mutated by a homologous modeling, molecular docking and multiple sequence comparison equivalent method, so that the problem that k cat/KmAMP is 1 order of magnitude lower than k cat/KmADP is solved, and the mutant with obviously improved AMP catalytic efficiency is obtained. The technical scheme adopted by the invention is as follows: in a first aspect, the present invention provides a polyphosphate kinase mutant obtained by amino acid substitution of a polyphosphate kinase having an amino acid sequence shown in SEQ ID No.1, wherein the mutant comprises at least one amino acid substitution selected from the group consisting of: (a) The alanine at position 68 is mutated into glycine, and the amino acid sequence is an A68G mutant shown as SEQ ID NO. 2; (b) The 92 th lysine is mutated into alanine, and the amino acid sequence is shown as K92A mutant shown as SEQ ID NO. 3; (c) The threonine at position 95 is mutated into alanine, and the amino acid se