CN-121495901-B - Polyphosphate kinase and its application in increasing ATP production

Abstract

The invention discloses polyphosphate kinase and application thereof in improving ATP production, belonging to the technical field of genetic engineering. The polyphosphate kinase disclosed by the invention is PePPK, psPPK, rbPPK or RrPPK, and the amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO. 5-8. The polyphosphate kinase disclosed by the invention can increase the ATP production amount and synthesize glutathione and 3 '-adenosine phosphate-5' -phosphosulfate.

Inventors

• TAN TIANWEI
• JI FAN
• YANG JIGANG
• LI XIAOJING

Assignees

• 北京化工大学

Dates

Publication Date

20260508

Application Date

20260113

Claims (2)

1. 1. The application of polyphosphate kinase PePPK in improving ATP production is characterized in that the amino acid sequence of polyphosphate kinase PePPK is shown in SEQ ID NO. 6.
2. 2. The application of the polyphosphate kinase PePPK in extracellular synthesis of glutathione or intracellular synthesis of 3 '-adenosine-5' -phosphosulfate is characterized in that the amino acid sequence of the polyphosphate kinase PePPK is shown as SEQ ID NO. 6.

Description

Polyphosphate kinase and its application in increasing ATP production Technical Field The invention relates to the technical field of genetic engineering, in particular to polyphosphate kinase and application thereof in improving ATP production. Background In vitro bioconversion is an innovative bio-fabrication platform, but energy supply remains a critical challenge. However, direct supplementation of stoichiometric ATP is extremely costly and may cause problems such as enzyme inhibition. Efficient ATP regeneration process involving PPK (polyphosphate kinase) is an important way to solve the in vitro bioconversion energy supply. PPK can catalyze inorganic polyphosphate (Poly P) and ADP to be converted into ATP, so that a high-efficiency ATP regeneration module is constructed, dependence on a large amount of exogenous ATP is avoided, and continuous energy support is provided for in-vitro enzymatic reaction. Second, poly P has the significant advantage of low cost and strong stability compared to higher phosphate compounds such as phosphopyruvic acid used in substrate level phosphorylation. The PPK uses Poly P as a phosphate donor to regenerate ATP, so that the economic cost of in-vitro bioconversion is greatly reduced, and the practical obstacle caused by overhigh phosphate donor cost in the traditional substrate horizontal phosphorylation is solved. PPK2B cg from Corynebacteriumglutamicum was studied to catalyze the conversion of ADP to ATP and to introduce it into Streptomyces albulus, increasing intracellular ATP levels and ultimately increasing epsilon-polylysine production. PPK enzyme derived from Meiothermus ruber was also studied to provide ATP production in glutathione synthesis, resulting in 80% conversion of synthetic glutathione. Also, the activities of various PPK enzymes on ADP and polyphosphate substrates with different polymerization degrees were analyzed, and the PPK2C pa derived from Pseudomonas aeruginosa was found to show the highest specific activity on polyP 6, thus realizing the continuous supply of ATP in 3 '-adenosine-5' -phosphosulfate. Accordingly, providing polyphosphate kinase and its use in increasing ATP production is a problem that those skilled in the art are highly aware of. Disclosure of Invention In view of this, the present invention provides polyphosphate kinase and its use in increasing ATP production. The invention innovatively provides PePPK, psPPK, rbPPK and RrPPK polyphosphate kinases which have 30.45%, 31.65%, 30.74% and 32.81% homology with PPK2C pa, respectively, which has good ATP regeneration effect. PePPK, psPPK, rbPPK and RrPPK achieve significantly better effects than the above-described p.aeroginosa-derived control PPK2C pa in the extracellular and intracellular synthesis of glutathione and 3 '-phosphoadenosine-5' -phosphosulfuric acid, respectively, with advanced and practical value. In order to achieve the above purpose, the present invention adopts the following technical scheme: the polyphosphate kinase is PePPK, psPPK, rbPPK or RrPPK, and the amino acid sequence of the polyphosphate kinase is shown as SEQ ID NO. 6-9. Further, the use of the polyphosphate kinase in increasing ATP production. Further, the use of the polyphosphate kinase in the synthesis of intracellular and extracellular products. Further, the use of the polyphosphate kinase in extracellular synthesis of glutathione or intracellular synthesis of 3 '-phosphoadenosine-5' -phosphosulfate. Recombinant vectors comprising the genes of the four wild-type polyphosphate kinases, respectively. Recombinant strains respectively comprising the genes of the four wild-type polyphosphate kinases. The preparation method of the polyphosphate kinase comprises the following steps of connecting the encoding gene of the polyphosphate kinase with an original expression vector to obtain a recombinant vector, and transforming host cells by the recombinant vector to obtain PePPK, psPPK, rbPPK and RrPPK recombinant strains respectively. The original expression vector is pET-28a (+), and the host cell is escherichia coli BL21 (DE 3). The polyphosphate kinases PePPK, psPPK, rbPPK and RrPPK were used to construct ATP regeneration systems. The polyphosphate kinase pure enzyme solution is used for synthesizing glutathione or 3 '-adenosine phosphate-5' -phosphosulfate. Compared with the prior art, the invention discloses the polyphosphate kinase and the application thereof in improving the ATP production, and the polyphosphate kinase is obtained through screening by an enzyme catalysis kinetic parameter prediction model EITLEM algorithm, so as to provide a plurality of high-activity wild type polyphosphate kinases. Wherein PePPK predicts a k cat/Km of 6.92s -1·mM-1, psPPK predicts a k cat/Km of 8.05s -1·mM-1, rbPPK predicts a k cat/Km of 7.24s -1·mM-1, rrPPK predicts a k cat/Km of 10.18s -1·mM-1. In practical application, the ATP generation efficiency of the wild type polyphosphate kinase is higher than that of other wi