Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Effect of Expression of Genes in the Sphingolipid Synthesis Pathway on the Biosynthesis of Ceramide in Saccharomyces cerevisiae

  • Kim, Se-Kyung (Department of Biological Engineering, Inha University) ;
  • Noh, Yong-Ho (Department of Biological Engineering, Inha University) ;
  • Koo, Ja-Ryong (Department of Biological Engineering, Inha University) ;
  • Yun, Hyun-Shik (Department of Biological Engineering, Inha University)
  • Published : 2010.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

Ceramide is important not only for the maintenance of the barrier function of the skin but also for the water-binding capacity of the stratum corneum. Although the exact role of ceramide in the human skin is not fully understood, ceramide has become a widely used ingredient in cosmetic and pharmaceutical industries. Compared with other microorganisms, yeast is more suitable for the production of ceramide because yeast grows fast and is non-toxic. However, production of ceramide from yeast has not been widely studied and most work in this area has been carried out using Saccharomyces cerevisiae. Regulating the genes that are involved in sphingolipid synthesis is necessary to increase ceramide production. In this study, we investigated the effect of the genes involved in the synthesis of ceramide, lcb1, lcb2, tsc10, lac1, lag1, and sur2, on ceramide production levels. The genes were cloned into pYES2 high copy number vectors. S. cerevisiae was cultivated on YPDG medium at $30^{\circ}C$. Ceramide was purified from the cell extracts by solvent extraction and the ceramide content was analyzed by HPLC using ELSD. The maximum production of ceramide (9.8 mg ceramide/g cell) was obtained when the tsc10 gene was amplified by the pYES2 vector. Real-time RT-PCR analysis showed that the increase in ceramide content was proportional to the increase in the tsc10 gene expression level, which was 4.56 times higher than that of the control strain.

Keywords

References

  1. Bustin, S. A. 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25: 169-193. https://doi.org/10.1677/jme.0.0250169
  2. Dickson, R. C. and R. L. Lester. 1999. Yeast sphingolipids. Biochim. Biophys. Acta 1426: 347-357. https://doi.org/10.1016/S0304-4165(98)00135-4
  3. Dickson, R. C. and R. L. Lester. 2002. Sphingolipid functions in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1583: 13-25. https://doi.org/10.1016/S1388-1981(02)00210-X
  4. Dickson, R. C., E. E. Nagiec, M. Skrzypek, P. Tillman, G. B. Wells, and R. L. Lester. 1997. Sphingolipids are potential heat stress signals in Saccharomyces. J. Biol. Chem. 272: 30196-30200. https://doi.org/10.1074/jbc.272.48.30196
  5. Freeman, W., S. Walker, and K. Vrana. 1999. Quantitative RTPCR: Pitfalls and potential. Biotechniques 26: 112-125.
  6. Funato, K., B. Vallee, and H. Riezman. 2002. Biosynthesis and trafficking of sphingolipids in the yeast Saccharomyces cerevisiae. Biochemistry 41: 15105-15114. https://doi.org/10.1021/bi026616d
  7. Gietz, R. D. and R. A. Woods 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method, pp. 87-96. In C. Guthrie and G.R. Fink (eds.), Methods in Enzymology. Academic Press, San Diego.
  8. Guillas, I., P. A. Kirchman, R. Chuard, M. Pfefferli, J. C. Jiang, S. M. Jazwinski, and A. Conzelmann. 2001. C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p. EMBO J. 20: 2655-2665. https://doi.org/10.1093/emboj/20.11.2655
  9. Hannun, Y. A., C. Luberto, and K. M. Argraves. 2001. Enzymes of sphingolipid metabolism: From modular to integrative signaling. Biochemistry 40: 4893-4903. https://doi.org/10.1021/bi002836k
  10. Holm, C., D. W. Meeks-wagner, W. L. Fiangman, and D. Bofstein. 1986. A rapid, efficient method for isolating DNA from yeast. Gene 42: 169-173. https://doi.org/10.1016/0378-1119(86)90293-3
  11. Hong, S. P., C. H. Lee, S. K. Kim, H. S. Yun, H. H. Lee, and K. H. Row. 2004. Mobile phase compositions for ceramide III by normal phase high performance liquid chromatography. Biotechnol. Bioprocess Eng. 9: 47-51. https://doi.org/10.1007/BF02949321
  12. Huwiler, A., T. Kolter, J. Pfeilschifter, and K. Sandhoff. 2000. Physiology and pathophysiology of sphingolipid metabolism and signaling. Biochim. Biophys. Acta 1485: 63-99. https://doi.org/10.1016/S1388-1981(00)00042-1
  13. Kaneko, H., M. Hosohara, M. Tanaka, and T. Itoh. 1976. Lipid composition of 30 species of yeast. Lipids 11: 837-844. https://doi.org/10.1007/BF02532989
  14. Kang, D. H., S. P. Hong, and K. H. Row. 2003. Quantitative analysis of ceramide III of Saccharomyces cerevisiae by normal phase HPLC. J. Liq. Chromatogr. Relat. Technol. 26: 617-627. https://doi.org/10.1081/JLC-120017910
  15. Kim, S. K., Y. H. Now, and H. S. Yun. 2008. The ceramide contents of Saccharomyces cerevisiae in batch culture. Korean J. Biotechnol. Bioeng. 23: 449-451.
  16. Livak, K. and T. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $2-^{{\Delta}{\Delta}CT}$ method. Methods 25: 402-408. https://doi.org/10.1006/meth.2001.1262
  17. Nagiec, M. M., J. A. Baltisberger, G. B. Wells, and R. L. Lester. 1994. The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis. Proc. Natl. Acad. Sci. U.S.A. 91: 7899-7902. https://doi.org/10.1073/pnas.91.17.7899
  18. Ng, R. and J. Abelson. 1980. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 77: 3912-3916. https://doi.org/10.1073/pnas.77.7.3912
  19. Obeid, L. M., Y. Okamoto, and C. Mao. 2002. Yeast sphingolipids: Metabolism and biology. Biochim. Biophys. Acta 1585: 163-171. https://doi.org/10.1016/S1388-1981(02)00337-2
  20. Okazaki, T., T. Kondo, T. Kitano, and M. Tashima. 1998. Diversity and complexity of ceramide signalling in apoptosis. Cell. Signal. 10: 685-692. https://doi.org/10.1016/S0898-6568(98)00035-7
  21. Ovstebo, R., K. Haug, K. Lande, and P. Kierulf. 2003. PCRbased calibration curves for studies of quantitative gene expression in human monocytes: Development and evaluation. Clin. Chem. 49: 425-432. https://doi.org/10.1373/49.3.425
  22. Patton, J. L. and R. L. Lester. 1991. The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane. J. Bacteriol. 173: 3101-3108.
  23. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: 2002-2007.
  24. Rupeic, J. and V. Maric. 1998. Isolation and chemical composition of the ceramide of the Candida lipolytica yeast. Chem. Phys. Lipids 91: 153-161. https://doi.org/10.1016/S0009-3084(97)00106-0
  25. Saccharomyces Genome Database. Accessible at http://www.yeastgenome.org.
  26. Schorling, S., B. Vallee, W. P. Barz, H. Riezman, and D. Oesterhelt. 2001. Lag1p and Lac1p are essential for the acylcoA-dependent ceramide synthase reaction in Saccharomyces cerevisae. Mol. Biol. Cell 12: 3417-3427.
  27. Sherman, F. 2002. Getting started with yeast, pp. 3-41. In C. Guthrie and G. R. Fink (eds.). Methods in Enzymology. Academic Press, San Diego.

Cited by

  1. Fermentation Process Development of Recombinant Hansenula polymorpha for Gamma-Linolenic Acid Production vol.20, pp.11, 2010, https://doi.org/10.4014/jmb.1003.03004
  2. Effects of Expression of lcb1/lcb2 and lac1/lag1 Genes on the Biosynthesis of Ceramides vol.16, pp.1, 2010, https://doi.org/10.1007/s12257-010-0268-8
  3. High-level production of tetraacetyl phytosphingosine (TAPS) by combined genetic engineering of sphingoid base biosynthesis and L-serine availability in the non-conventional yeast Pichia ciferri vol.14, pp.2, 2010, https://doi.org/10.1016/j.ymben.2011.12.002
  4. Producing human ceramide-NS by metabolic engineering using yeast Saccharomyces cerevisiae vol.5, pp.None, 2010, https://doi.org/10.1038/srep16319
  5. An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity vol.216, pp.1, 2010, https://doi.org/10.1083/jcb.201606059
  6. Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome vol.355, pp.6329, 2010, https://doi.org/10.1126/science.aaf4791
  7. Seipin negatively regulates sphingolipid production at the ER–LD contact site vol.218, pp.11, 2019, https://doi.org/10.1083/jcb.201902072