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Overexpression of YbeD in Escherichia coli Enhances Thermotolerance

  • Kim, Sinyeon (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Kim, Youngshin (Department of Bioscience and Biotechnology, Konkuk University) ;
  • Yoon, Sung Ho (Department of Bioscience and Biotechnology, Konkuk University)
  • Received : 2019.01.16
  • Accepted : 2019.03.11
  • Published : 2019.03.28

Abstract

Heat-resistant microbial hosts are required for bioprocess development using high cell density cultivations at the industrial scale. We report that the thermotolerance of Escherichia coli can be enhanced by overexpressing ybeD, which was known to encode a hypothetical protein of unknown function. In the wild-type E. coli BL21(DE3), ybeD transcription level increased over five-fold when temperature was increased from $37^{\circ}C$ to either $42^{\circ}C$ or $46^{\circ}C$. To study the function of ybeD, a deletion strain and an overexpression strain were constructed. At $46^{\circ}C$, in comparison to the wild type, the ybeD-deletion reduced cell growth half-fold, and the ybeD-overexpression promoted cell growth over two-fold. The growth enhancement by ybeD-overexpression was much more pronounced at $46^{\circ}C$ than $37^{\circ}C$. The ybeD-overexpression was also effective in other E. coli strains of MG1655, W3110, DH10B, and BW25113. These findings reveal that ybeD gene plays an important role in enduring high-temperature stress, and that ybeD-overexpression can be a prospective strategy to develop thermotolerant microbial hosts.

Keywords

References

  1. Ezemaduka AN, Yu J, Shi X, Zhang K, Yin CC, Fu X, et al. 2014. A small heat shock protein enables Escherichia coli to grow at a lethal temperature of $50^{\circ}C$ conceivably by maintaining cell envelope integrity. J. Bacteriol. 196: 2004-2011. https://doi.org/10.1128/JB.01473-14
  2. Liu D, Lu Z, Mao Z, Liu S. 2009. Enhanced thermotolerance of E. coli by expressed OsHsp90 from rice (Oryza sativa L.). Curr. Microbiol. 58: 129-133. https://doi.org/10.1007/s00284-008-9288-4
  3. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodriguez-Carmona E, Baumann K, et al. 2008. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb. Cell Fact. 7: 11. https://doi.org/10.1186/1475-2859-7-11
  4. Zhang X, Liu Y, Genereux JC, Nolan C, Singh M, Kelly JW. 2014. Heat-shock response transcriptional program enables high-yield and high-quality recombinant protein production in Escherichia coli. ACS Chem. Biol. 9: 1945-1949. https://doi.org/10.1021/cb5004477
  5. Arsene F, Tomoyasu T, Bukau B. 2000. The heat shock response of Escherichia coli. Int. J. Food Microbiol. 55: 3-9. https://doi.org/10.1016/S0168-1605(00)00206-3
  6. Gross CA. 1996. Function and regulation of the heat shock proteins, pp. 1382-1399. In Neidhardt FC (ed.), Escherichia coli and Salmonella, Ed. ASM Press, Washington.
  7. Martinez-Alonso M, Garcia-Fruitos E, Ferrer-Miralles N, Rinas U, Villaverde A. 2010. Side effects of chaperone gene co-expression in recombinant protein production. Microb. Cell Fact. 9: 64. https://doi.org/10.1186/1475-2859-9-64
  8. Kolaj O, Spada S, Robin S, Wall JG. 2009. Use of folding modulators to improve heterologous protein production in Escherichia coli. Microb. Cell Fact. 8: 9. https://doi.org/10.1186/1475-2859-8-9
  9. Nonaka G, Blankschien M, Herman C, Gross CA, Rhodius VA. 2006. Regulon and promoter analysis of the E. coli heatshock factor, ${\sigma}^{32}$, reveals a multifaceted cellular response to heat stress. Genes Dev. 20: 1776-1789. https://doi.org/10.1101/gad.1428206
  10. Shimada T, Tanaka K, Ishihama A. 2017. The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase. PLoS One 12: e0179181. https://doi.org/10.1371/journal.pone.0179181
  11. Peabody MA, Laird MR, Vlasschaert C, Lo R, Brinkman F S. 2016. PSORTdb: expanding the bacteria and archaea protein subcellular localization database to better reflect diversity in cell envelope structures. Nucleic Acids Res. 44: D663-668. https://doi.org/10.1093/nar/gkv1271
  12. Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, et al. 2008. Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 9: 102. https://doi.org/10.1186/1471-2164-9-102
  13. Kozlov G, Elias D, Semesi A, Yee A, Cygler M, Gehring K. 2004. Structural similarity of YbeD protein from Escherichia coli to allosteric regulatory domains. J. Bacteriol. 186: 8083-8088. https://doi.org/10.1128/JB.186.23.8083-8088.2004
  14. Yoon SH, Jeong H, Kwon S-K, Kim JF. 2009. Genomics, biological features, and biotechnological applications of Escherichia coli B: "Is B for better?!", pp. 1-17. Systems Biology and Biotechnology of Escherichia coli, Ed. Springer, Berlin, Germany
  15. Choi JH, Keum KC, Lee SY. 2006. Production of recombinant proteins by high cell density culture of Escherichia coli Chem. Eng. Sci. 61: 876-885. https://doi.org/10.1016/j.ces.2005.03.031
  16. Studier FW, Moffatt BA. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130. https://doi.org/10.1016/0022-2836(86)90385-2
  17. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97: 6640-6645. https://doi.org/10.1073/pnas.120163297
  18. Kim SK, Lee DH, Kim OC, Kim J F, Yoon SH. 2017. Tunable control of an Escherichia coli expression system for the overproduction of membrane proteins by titrated expression of a mutant lac repressor. ACS Synth. Biol. 6: 1766-1773. https://doi.org/10.1021/acssynbio.7b00102
  19. Kim S, Jeong H, Kim EY, Kim JF, Lee SY, Yoon SH. 2017. Genomic and transcriptomic landscape of Escherichia coli BL21(DE3). Nucleic Acids Res. 45: 5285-5293. https://doi.org/10.1093/nar/gkx228
  20. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-{\Delta}{\Delta}C_T}$ Method. Methods 25: 402-408. https://doi.org/10.1006/meth.2001.1262
  21. Schagger H. 2006. Tricine-SDS-PAGE. Nat. Protoc. 1: 16-22. https://doi.org/10.1038/nprot.2006.4
  22. Chen IA, Markowitz VM, Chu K, Palaniappan K, Szeto E, Pillay M, et al. 2017. IMG/M: integrated genome and metagenome comparative data analysis system. Nucleic Acids Res. 45: D507-d516. https://doi.org/10.1093/nar/gkw929
  23. Durfee T, Nelson R, Baldwin S, Plunkett G, 3rd, Burland V, Mau B, et al. 2008. The complete genome sequence of Escherichia coli DH10B: insights into the biology of a laboratory workhorse. J. Bacteriol. 190: 2597-2606. https://doi.org/10.1128/JB.01695-07
  24. Bukau B, Walker GC. 1989. Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J. Bacteriol. 171: 2337-2346. https://doi.org/10.1128/jb.171.5.2337-2346.1989
  25. Teleha MA, Miller AC, Larsen RA. 2013. Overexpression of the Escherichia coli TolQ protein leads to a null-FtsN-like division phenotype. Microbiologyopen 2: 618-632. https://doi.org/10.1002/mbo3.101
  26. Herendeen SL, VanBogelen RA, Neidhardt FC. 1979. Levels of major proteins of Escherichia coli during growth at different temperatures. J. Bacteriol. 139: 185-194. https://doi.org/10.1128/JB.139.1.185-194.1979
  27. Ron EZ, Davis BD. 1971. Growth rate of Escherichia coli at elevated temperatures: limitation by methionine. J. Bacteriol. 107: 391-396. https://doi.org/10.1128/JB.107.2.391-396.1971
  28. Ron EZ, Shani M. 1971. Growth rate of Escherichia coli at elevated temperatures: reversible inhibition of homoserine trans-succinylase. J. Bacteriol. 107: 397-400. https://doi.org/10.1128/JB.107.2.397-400.1971
  29. Jordan SW, Cronan JE, Jr. 2003. The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase. J. Bacteriol. 185: 1582-1589. https://doi.org/10.1128/JB.185.5.1582-1589.2003
  30. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, et al. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2: 2006 0008.
  31. Fayet O, Ziegelhoffer T, Georgopoulos C. 1989. The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171: 1379-1385. https://doi.org/10.1128/jb.171.3.1379-1385.1989
  32. Teter SA, Houry WA, Ang D, Tradler T, Rockabrand D, Fischer G, et al. 1999. Polypeptide flux through bacterial Hsp70: DnaK cooperates with trigger factor in chaperoning nascent chains. Cell 97: 755-765. https://doi.org/10.1016/S0092-8674(00)80787-4
  33. Wick LM, Egli T. 2004. Molecular components of physiological stress responses in Escherichia coli. Adv. Biochem. Eng. Biotechnol. 89: 1-45.
  34. Wagner S, Baars L, Ytterberg AJ, Klussmeier A, Wagner CS, Nord O, et al. 2007. Consequences of membrane protein overexpression in Escherichia coli. Mol. Cell. Proteomics 6: 1527-1550. https://doi.org/10.1074/mcp.M600431-MCP200