1887

Microbiology

Volume 164, Issue 1

Molecular cloning and overexpression of , an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast DMKU-RK253 Free

Abstract

Triacylglycerol (TAG) is a major component of lipid storage in yeast. The acyl CoA: diacylgycerol acyltransferase (DGAT) that catalyzes the final and rate-limiting step in the production of TAG is rather interesting. Consequently, cloning and analysis of the gene-encoding TAG synthase, diacylglycerol acyltransferase gene (), of the oleaginous yeast DMKU-RK253 were undertaken. Analysis of the deduced amino acid sequence of DGA1 from DMKU-RK253 () showed similarity with the acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) from other organisms. The cDNA of was cloned into the yeast expression vector pYES2 and heterologously overexpressed in . One of the transformants showed a 1.6-fold increase in lipid content compared with the wild-type strain harbouring the pYES2 empty vector. Furthermore, overexpression in DMKU-RK253 resulted in a 2.5-fold increase in lipid content when compared with the wild-type strain, and no significant differences in fatty acid composition were observed between -overexpressed and wild-type strains. Taken together, our results supported our hypothesis that the is a genetic factor that can be used for the development of a strain with improved lipid accumulation capabilities.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): diacylglycerol acyltransferase gene , lipid accumulation , oleaginous yeast , Rhodosporidiobolus fluvialis and Saccharomyces cerevisiae
© 2018 The Authors
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2018-01-01
2021-05-19
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References

  1. Ratledge C. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie 2004;86:807–815 [CrossRef][PubMed]
     [Google Scholar]
  2. Liu Q, Siloto RM, Snyder CL, Weselake RJ. Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem 2011;286:13115–13126 [CrossRef][PubMed]
     [Google Scholar]
  3. Polburee P, Yongmanitchai W, Lertwattanasakul N, Ohashi T, Fujiyama K et al. Characterization of oleaginous yeasts accumulating high levels of lipid when cultivated in glycerol and their potential for lipid production from biodiesel-derived crude glycerol. Fungal Biol 2015;119:1194–1204 [CrossRef][PubMed]
     [Google Scholar]
  4. Wang QM, Yurkov AM, Göker M, Lumbsch HT, Leavitt SD et al. Phylogenetic classification of yeasts and related taxa within Pucciniomycotina. Stud Mycol 2015;81:149–189 [CrossRef][PubMed]
     [Google Scholar]
  5. Czabany T, Athenstaedt K, Daum G. Synthesis, storage and degradation of neutral lipids in yeast. Biochim Biophys Acta 2007;1771:299–309 [CrossRef][PubMed]
     [Google Scholar]
  6. Grum-Grzhimaylo OA, Debets AJ, Bilanenko EN. The diversity of microfungi in peatlands originated from the White Sea. Mycologia 2016;108:233–254 [CrossRef][PubMed]
     [Google Scholar]
  7. Wältermann M, Steinbüchel A. Neutral lipid bodies in prokaryotes: recent insights into structure, formation, and relationship to eukaryotic lipid depots. J Bacteriol 2005;187:3607–3619 [CrossRef][PubMed]
     [Google Scholar]
  8. Turchetto-Zolet AC, Maraschin FS, de Morais GL, Cagliari A, Andrade CM et al. Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis. BMC Evol Biol 2011;11:263 [CrossRef][PubMed]
     [Google Scholar]
  9. Gong Y, Zhang J, Guo X, Wan X, Liang Z et al. Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl-coenzyme A: diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum. FEBS Lett 2013;587:481–487 [CrossRef][PubMed]
     [Google Scholar]
  10. Oelkers P, Cromley D, Padamsee M, Billheimer JT, Sturley SL. The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 2002;277:8877–8881 [CrossRef][PubMed]
     [Google Scholar]
  11. Sorger D, Daum G. Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae. J Bacteriol 2002;184:519–524 [CrossRef][PubMed]
     [Google Scholar]
  12. Rani SH, Krishna TH, Saha S, Negi AS, Rajasekharan R. Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase. J Biol Chem 2010;285:38337–38347 [CrossRef][PubMed]
     [Google Scholar]
  13. Rani SH, Saha S, Rajasekharan R. A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis. Microbiology 2013;159:155–166 [CrossRef][PubMed]
     [Google Scholar]
  14. Sandager L, Gustavsson MH, Ståhl U, Dahlqvist A, Wiberg E et al. Storage lipid synthesis is non-essential in yeast. J Biol Chem 2002;277:6478–6482 [CrossRef][PubMed]
     [Google Scholar]
  15. Athenstaedt K. YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica. Biochim Biophys Acta 2011;1811:587–596 [CrossRef][PubMed]
     [Google Scholar]
  16. Chen Z, Liu P, Liu Y, Tang H, Chen Y et al. Identification and characterization of a type-2 diacylglycerol acyltransferase (DGAT2) from Rhodosporidium diobovatum. Antonie van Leeuwenhoek 2014;106:1127–1137 [CrossRef][PubMed]
     [Google Scholar]
  17. Polburee P, Yongmanitchai W, Honda K, Ohashi T, Yoshida T et al. Lipid production from biodiesel-derived crude glycerol by Rhodosporidium fluviale DMKU-RK253 using temperature shift with high cell density. Biochem Eng J 2016;112:208–218 [CrossRef]
     [Google Scholar]
  18. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual, 4th ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2012
     [Google Scholar]
  19. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–410 [CrossRef][PubMed]
     [Google Scholar]
  20. Kamisaka Y, Tomita N, Kimura K, Kainou K, Uemura H. DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Deltasnf2 disruptant of Saccharomyces cerevisiae. Biochem J 2007;408:61–68 [CrossRef][PubMed]
     [Google Scholar]
  21. Kawai S, Hashimoto W, Murata K. Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism. Bioeng Bugs 2010;1:395–403 [CrossRef][PubMed]
     [Google Scholar]
  22. Tsai YY, Ohashi T, Kanazawa T, Polburee P, Misaki R et al. Development of a sufficient and effective procedure for transformation of an oleaginous yeast, Rhodosporidium toruloides DMKU3-TK16. Curr Genet 2017;63:359–371 [CrossRef][PubMed]
     [Google Scholar]
  23. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–917 [CrossRef][PubMed]
     [Google Scholar]
  24. Holub BJ, Skeaff CM. Nutritional regulation of cellular phosphatidylinositol. Nutritional regulation of cellular phosphatidylinositol. 1987;141234–244
  25. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK et al. CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 2011;39:D225–D229 [CrossRef][PubMed]
     [Google Scholar]
  26. Cao H. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Res Notes 2011;4:249 [CrossRef][PubMed]
     [Google Scholar]
  27. Guo Z, Cromley D, Billheimer JT, Sturley SL. Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences. J Lipid Res 2001;42:1283–1291[PubMed]
     [Google Scholar]
  28. Wagner M, Hoppe K, Czabany T, Heilmann M, Daum G et al. Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri. Plant Physiol Biochem 2010;48:407–416 [CrossRef][PubMed]
     [Google Scholar]
  29. Athenstaedt K, Jolivet P, Boulard C, Zivy M, Negroni L et al. Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source. Proteomics 2006;6:1450–1459 [CrossRef][PubMed]
     [Google Scholar]
  30. Kamisaka Y, Kimura K, Uemura H, Shibakami M. Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the Deltasnf2 disruptant produces a significant increase in its enzyme activity. Appl Microbiol Biotechnol 2010;88:105–115 [CrossRef][PubMed]
     [Google Scholar]
  31. Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio AL et al. Engineering of a high lipid producing Yarrowia lipolytica strain. Biotechnol Biofuels 2016;9:77 [CrossRef][PubMed]
     [Google Scholar]
  32. Guihéneuf F, Leu S, Zarka A, Khozin-Goldberg I, Khalilov I et al. Cloning and molecular characterization of a novel acyl-CoA:diacylglycerol acyltransferase 1-like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum. Febs J 2011;278:3651–3666 [CrossRef][PubMed]
     [Google Scholar]
  33. Boyle NR, Page MD, Liu B, Blaby IK, Casero D et al. Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas. J Biol Chem 2012;287:15811–15825 [CrossRef][PubMed]
     [Google Scholar]
  34. Zhou YJ, Gao W, Rong Q, Jin G, Chu H et al. Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J Am Chem Soc 2012;134:3234–3241 [CrossRef][PubMed]
     [Google Scholar]
  35. Silverman AM, Qiao K, Xu P, Stephanopoulos G. Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 2016;100:3781–3798 [CrossRef][PubMed]
     [Google Scholar]
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Molecular cloning and overexpression of DGA1, an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast Rhodosporidiobolus fluvialis DMKU-RK253
Microbiology 164, 1 (2018); https://doi.org/10.1099/mic.0.000584
/content/journal/micro/10.1099/mic.0.000584
/content/journal/micro/10.1099/mic.0.000584
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