The final, formatted version of the article will be published soon.
ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Microbiotechnology
Volume 15 - 2024 |
doi: 10.3389/fmicb.2024.1501020
Expression and functional study of DNA polymerases from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963
Provisionally accepted- Research Institute of Tsinghua University in Shenzhen (RITS), Shenzhen, China
The properties of DNA polymerases isolated from thermophilic and mesophilic microorganisms, such as the thermophilic Geobacillus stearothermophilus (Bst) and mesophilic Bacillus subtilis phage (Phi29), have been widely researched. However, DNA polymerases in psychrophilic microorganisms remain poorly understood. In this study, we present for the first time the expression and functional characterization of DNA polymerases PWT-WT and FWT-WT from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963. Enzymatic activity assays revealed that FWT-WT possessed strand displacement but lacked exonuclease activity and high ionic strength tolerance, whereas PWT-WT lacked all these properties. Further protein engineering and biochemical analysis identified D423 and S490 as critical mutation sites for improving strand displacement and tolerance to high ionic strength, specifically in the presence of 0 – 0.3 M potassium chloride (KCl), sodium chloride (NaCl), and potassium acetate (KAc). Three-dimensional structural analysis demonstrated that the size and the electric charge of the single-stranded DNA (ssDNA) encapsulation entrance were pivotal factors in the binding of the ssDNA template.
Keywords: Psychrophilic polymerases, protein expression, high ionic strength tolerance, Strand displacement, Exonuclease activity
Received: 24 Sep 2024; Accepted: 06 Nov 2024.
Copyright: © 2024 Sun, Ko, Gao, Fu, Mao, He and Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Yaping Sun, Research Institute of Tsinghua University in Shenzhen (RITS), Shenzhen, China
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.