Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Increased mRNA Stability and Expression Level of Croceibacter atlanticus Lipase Gene Developed through Molecular Evolution Process

  • Jeong, Han Byeol (Division of Biotechnology, The Catholic University of Korea) ;
  • Kim, Hyung Kwoun (Division of Biotechnology, The Catholic University of Korea)
  • Received : 2021.03.04
  • Accepted : 2021.05.17
  • Published : 2021.06.28
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Abstract

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→ C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.

Keywords

Acknowledgement

This research was supported by the Research Fund 2020 of The Catholic University of Korea. This work was also supported by Korea Polar Research Institute (PE21150). We appreciate Ji-Woo Park and Seo-Yeon Park for their help in cloning experiment.

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