Journal of Microbiology and Biotechnology

  • 제31권6호
  • /
  • Pages.882-889
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  • 2021
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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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)
  • 투고 : 2021.03.04
  • 심사 : 2021.05.17
  • 발행 : 2021.06.28
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초록

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.

키워드

과제정보

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.

참고문헌

  1. Hitch TCA, Clavel T. 2019. A proposed update for the classification and description of bacterial lipolytic enzymes. Peer J. 8: 7.
  2. Widmann M, Juhl PB, Pleiss J. 2010. Structural classification by the lipase engineering database: a case study of Candida antarctica lipase A. BMC Genomics 11: 123. https://doi.org/10.1186/1471-2164-11-123
  3. Fischer M, Pleiss J. 2003. The lipase engineering database: a navigation and analysis tool for protein families. Nucleic Acids Res. 31: 319-321. https://doi.org/10.1093/nar/gkg015
  4. Hashim NHF, Mahadi NM, Illias RM, Feroz SR, Bakar FDA, Murad AMA. 2018. Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica. Extremophiles 22: 607-616. https://doi.org/10.1007/s00792-018-1021-z
  5. Salwoom L, Salleh AB, Convey P, Mohamad Ali MS. 2019. New recombinant cold-adapted and organic solvent tolerant lipase from psychrophilic Pseudomonas sp. LSK25, isolated from Signy Island Antarctica. Int. J. Mol. Sci. 20: 1264. https://doi.org/10.3390/ijms20061264
  6. Park SH, Kim SJ, Park S, Kim HK. 2019. Characterization of organic solvent-tolerant lipolytic enzyme from Marinobacter lipolyticus isolated from the Antarctic Ocean. Appl. Biochem. Biotechnol. 187: 1046-1060. https://doi.org/10.1007/s12010-018-2865-5
  7. Maharana AK, Singh SM. 2018. A cold and organic solvent tolerant lipase produced by Antarctic strain Rhodotorula sp. Y-23. J. Basic Microbiol. 58: 331-342. https://doi.org/10.1002/jobm.201700638
  8. Won SJ, Jeong HB, Kim HK. 2020. Characterization of novel salt-tolerant esterase isolated from the marine bacterium Alteromonas sp. 39-G1. J. Microbiol. Biotechnol. 30: 216-225. https://doi.org/10.4014/jmb.1907.07057
  9. Park CG, Kim HK. 2018. Production, immobilization, and characterization of Croceibacter atlanticus lipase isolated from the Antarctic Ross sea. Microbiol. Biotechnol. Lett. 46: 234-243. https://doi.org/10.4014/mbl.1804.04012
  10. Hough DW, Danson MJ. 1999. Extremozymes. Curr. Opin. Chem. Biol. 3: 39-46. https://doi.org/10.1016/S1367-5931(99)80008-8
  11. Adams MW, Perler FB, Kelly RM. 1995. Extremozymes: expanding the limits of biocatalysis. Biotechnology 13: 662-668.
  12. Gomes JI, Steiner W. 2004. The biocatalytic potential of extremophiles and extremozymes. Food Technol. Biotechnol. 42: 223-235.
  13. Trincone A. 2011. Marine biocatalysts: enzymatic features and applications. Mar. Drugs 9: 478-499. https://doi.org/10.3390/md9040478
  14. Demirjian DC, Mori's-Varas F, Cassidy CS. 2001. Enzymes from extremophiles. Curr. Opin. Chem. Biol. 5: 144-151. https://doi.org/10.1016/S1367-5931(00)00183-6
  15. Dror A, Shemesh E, Dayan N, Fishman A. 2014. Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus lipase T6 for enhanced stability in methanol. Appl. Environ. Microbiol. 80: 1515-1527. https://doi.org/10.1128/AEM.03371-13
  16. Bilyk B, Weber S, Myronovskyi M, Bilyk O, Petzke L, Luzhetskyy A. 2013. In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1. Appl. Microbiol. Biotechnol. 97: 351-359. https://doi.org/10.1007/s00253-012-4550-x
  17. Mabizela-Mokoena NB, Limani SW, Ncube I, Piater LA, Litthauer D, Nthangeni MB. 2017. Genetic determinant of Bacillus pumilus lipase lethality and its application as positive selection cloning vector in Escherichia coli. Protein Expr. Purif. 137: 43-51. https://doi.org/10.1016/j.pep.2017.06.013
  18. Guan L, Gao Y, Li J, Wang K, Zhang Z, Yan S, et al. 2020. Directed evolution of Pseudomonas fluorescens lipase variants with improved thermostability using error-prone PCR. Front. Bioeng. Biotechnol. 8: 1034. https://doi.org/10.3389/fbioe.2020.01034
  19. Glod D. 2017. Modification of fatty acid selectivity of Candida antarctica lipase A by error-prone PCR. Biotechnol. Lett. 39: 767-773. https://doi.org/10.1007/s10529-017-2299-0
  20. Panda AK, Bisht SPS, Panigrahi AK, De Mandal S, Kumar NS. 2016. Cloning and in silico analysis of a high-temperature inducible lipase from Brevibacillus. Arab. J. Sci. Eng. 41: 2159-2170. https://doi.org/10.1007/s13369-015-1975-4
  21. Kane JF. 1995. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6: 494-500. https://doi.org/10.1016/0958-1669(95)80082-4
  22. Schwersensky M, Rooman M, Pucci F. 2020. Large-scale in silico mutagenesis experiments reveal optimization of genetic code and codon usage for protein mutational robustness. BMC Biol. 18: 146. https://doi.org/10.1186/s12915-020-00870-9
  23. Zhou WJ, Yang JK, Mao L, Miao LH. 2015. Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris. Enzyme Microb. Technol. 71: 66-72. https://doi.org/10.1016/j.enzmictec.2014.10.007
  24. Ghahremanifard P, Rezaeinezhad N, Rigi G, Ramezani F, Ahmadian G. 2018. Designing a novel signal sequence for efficient secretion of Candida antarctica lipase B in E. coli: the molecular dynamic simulation, codon optimization and statistical analysis approach. Int. J. Biol. Macromol. 119: 291-305. https://doi.org/10.1016/j.ijbiomac.2018.07.150
  25. Chakraborty S, Nag D, Mazumder TH, Uddin A. 2017. Codon usage pattern and prediction of gene expression level in Bungarus species. Gene 604: 48-60. https://doi.org/10.1016/j.gene.2016.11.023
  26. Mauger DM, Cabral BJ, Presnyak V, Su SV, Reid DW, Goodman B, et al. 2019. mRNA structure regulates protein expression through changes in functional half-life. Proc. Natl. Acad. Sci. USA 116: 24075-24083. https://doi.org/10.1073/pnas.1908052116
  27. Larsen MW, Bornscheuer UT, Hult K. 2008. Expression of Candida antarctica lipase B in Pichia pastoris and various Escherichia coli systems. Protein Expr. Purif. 62: 90-97. https://doi.org/10.1016/j.pep.2008.07.012
  28. Gutierrez-Gonzalez M, Farias C, Tello S, Perez-Etcheverry D, Romero A, Zuniga R, et al. 2019. Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli. Sci. Rep. 9: 16850. https://doi.org/10.1038/s41598-019-53200-7
  29. Thomas JG, Baneyx F. 1996. Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing heat-shock proteins. J. Biol. Chem. 271: 11141-11147. https://doi.org/10.1074/jbc.271.19.11141
  30. Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL. 2008. The Vienna RNA Websuite. Nucleic Acids Res. 36: W70-W74. https://doi.org/10.1093/nar/gkn188
  31. Lorenz R, Bernhart SH, Honer zu Siederdissen C, Tafer H, Flamm C, Stadler PF, et al. 2011. "ViennaRNA Package 2.0", Algorithms for Molecular Biology. 6: 26. https://doi.org/10.1186/1748-7188-6-26
  32. Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. 2004. Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc. Natl. Acad. Sci. USA 101: 7287-7292. https://doi.org/10.1073/pnas.0401799101
  33. Zuker M, Stiegler P. 1981. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acid Res. 9: 133-148. https://doi.org/10.1093/nar/9.1.133