Skip to content
1887

Microbiology Society logo Microbiology

Volume 159, Issue Pt_1

A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Free

Abstract

The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in . Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from . The DGAT activity of the protein encoded by was confirmed by the heterologous expression of cDNA in a quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an fatty acid supplementation experiment, expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.063156-0
2013-01-01
2025-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/1/155.html?itemId=/content/journal/micro/10.1099/mic.0.063156-0&mimeType=html&fmt=ahah

References

  1. Ageitos J. M., Vallejo J. A., Veiga-Crespo P., Villa T. G. ( 2011). Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 90:1219–1227 [View Article][PubMed]
     [Google Scholar]
  2. Bagnato C., Igal R. A. ( 2003). Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts. J Biol Chem 278:52203–52211 [View Article][PubMed]
     [Google Scholar]
  3. Bishop W. R., Bell R. M. ( 1988). Assembly of phospholipids into cellular membranes: biosynthesis, transmembrane movement and intracellular translocation. Annu Rev Cell Biol 4:579–610 [View Article][PubMed]
     [Google Scholar]
  4. Boyle N. R., Page M. D., Liu B., Blaby I. K., Casero D., Kropat J., Cokus S. J., Hong-Hermesdorf A., Shaw J. & other authors ( 2012). Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas . J Biol Chem 287:15811–15825 [View Article][PubMed]
     [Google Scholar]
  5. Carman G. M. ( 1997). Phosphatidate phosphatases and diacylglycerol pyrophosphate phosphatases in Saccharomyces cerevisiae and Escherichia coli . Biochim Biophys Acta 1348:45–55 [View Article][PubMed]
     [Google Scholar]
  6. Cases S., Smith S. J., Zheng Y. W., Myers H. M., Lear S. R., Sande E., Novak S., Collins C., Welch C. B. & other authors ( 1998). Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci U S A 95:13018–13023 [View Article][PubMed]
     [Google Scholar]
  7. Cases S., Stone S. J., Zhou P., Yen E., Tow B., Lardizabal K. D., Voelker T., Farese R. V. Jr ( 2001). Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J Biol Chem 276:38870–38876 [View Article][PubMed]
     [Google Scholar]
  8. Connerth M., Czabany T., Wagner A., Zellnig G., Leitner E., Steyrer E., Daum G. ( 2010). Oleate inhibits steryl ester synthesis and causes liposensitivity in yeast. J Biol Chem 285:26832–26841 [View Article][PubMed]
     [Google Scholar]
  9. Czabany T., Athenstaedt K., Daum G. ( 2007). Synthesis, storage and degradation of neutral lipids in yeast. Biochim Biophys Acta 1771:299–309 [View Article][PubMed]
     [Google Scholar]
  10. Dahlqvist A., Stahl U., Lenman M., Banas A., Lee M., Sandager L., Ronne H., Stymne S. ( 2000). Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Natl Acad Sci U S A 97:6487–6492 [View Article][PubMed]
     [Google Scholar]
  11. Daniel J., Deb C., Dubey V. S., Sirakova T. D., Abomoelak B., Morbidoni H. R., Kolattukudy P. E. ( 2004). Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture. J Bacteriol 186:5017–5030 [View Article][PubMed]
     [Google Scholar]
  12. Engvall E. ( 1980). Enzyme immunoassay ELISA and EMIT. Methods Enzymol 70:A419–439 [View Article][PubMed]
     [Google Scholar]
  13. Francki M. G., Whitaker P., Smith P. M., Atkins C. A. ( 2002). Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species (Lupinus angustifolius L. and L. mutabilis L.). Funct Integr Genomics 2:292–300 [View Article][PubMed]
     [Google Scholar]
  14. Gangar A., Karande A. A., Rajasekharan R. ( 2001). Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast. J Biol Chem 276:10290–10298 [View Article][PubMed]
     [Google Scholar]
  15. Gangar A., Raychaudhuri S., Rajasekharan R. ( 2002). Alteration in the cytosolic triacylglycerol biosynthetic machinery leads to decreased cell growth and triacylglycerol synthesis in oleaginous yeast. Biochem J 365:577–589[PubMed]
     [Google Scholar]
  16. Ghosh A. K., Ramakrishnan G., Chandramohan C., Rajasekharan R. ( 2008a). CGI-58, the causative gene for Chanarin–Dorfman syndrome, mediates acylation of lysophosphatidic acid. J Biol Chem 283:24525–24533 [View Article][PubMed]
     [Google Scholar]
  17. Ghosh A. K., Ramakrishnan G., Rajasekharan R. ( 2008b). YLR099C (ICT1) encodes a soluble Acyl-CoA-dependent lysophosphatidic acid acyltransferase responsible for enhanced phospholipid synthesis on organic solvent stress in Saccharomyces cerevisiae . J Biol Chem 283:9768–9775 [View Article][PubMed]
     [Google Scholar]
  18. Ghosh A. K., Chauhan N., Rajakumari S., Daum G., Rajasekharan R. ( 2009). At4g24160, a soluble acyl-coenzyme A-dependent lysophosphatidic acid acyltransferase. Plant Physiol 151:869–881 [View Article][PubMed]
     [Google Scholar]
  19. Gocze P. M., Freeman D. A. ( 1994). Factors underlying the variability of lipid droplet fluorescence in MA‐10 Leyclig tumour cells. Cytometry 17:151–158[PubMed] [CrossRef]
     [Google Scholar]
  20. Heath R. J., Rock C. O. ( 1998). A conserved histidine is essential for glycerolipid acyltransferase catalysis. J Bacteriol 180:1425–1430[PubMed]
     [Google Scholar]
  21. Heath R. J., Rock C. O. ( 1999). A missense mutation accounts for the defect in the glycerol-3-phosphate acyltransferase expressed in the plsB26 mutant. J Bacteriol 181:1944–1946[PubMed]
     [Google Scholar]
  22. Hernández M. L., Whitehead L., He Z., Gazda V., Gilday A., Kozhevnikova E., Vaistij F. E., Larson T. R., Graham I. A. ( 2012). A cytosolic acyltransferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants. Plant Physiol 160:215–225 [View Article][PubMed]
     [Google Scholar]
  23. Kalscheuer R., Steinbüchel A. ( 2003). A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 278:8075–8082 [View Article][PubMed]
     [Google Scholar]
  24. Kent C., Carman G. M., Spence M. W., Dowhan W. ( 1991). Regulation of eukaryotic phospholipid metabolism. FASEB J 5:2258–2266[PubMed]
     [Google Scholar]
  25. Kohei H., Satoshi Y. ( 1984). Partial purification and properties of phosphatidate phosphatase in Saccharomyces cerevisiae . Biochim Biophys Acta 796:102–109 [View Article][PubMed]
     [Google Scholar]
  26. Lardizabal K. D., Mai J. T., Wagner N. W., Wyrick A., Voelker T., Hawkins D. J. ( 2001). DGAT2 is a new diacylglycerol acyltransferase gene family: purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J Biol Chem 276:38862–38869 [View Article][PubMed]
     [Google Scholar]
  27. Misra S., Ghosh A., Dutta J. ( 1984). Production and composition of microbial fat from Rhodotorula glutinis . J Sci Food Agric 35:59–65 [View Article]
     [Google Scholar]
  28. Müllner H., Daum G. ( 2004). Dynamics of neutral lipid storage in yeast. Acta Biochim Pol 51:323–347[PubMed]
     [Google Scholar]
  29. Murphy D. J. ( 1993). Structure, function and biogenesis of storage lipid bodies and oleosins in plants. Prog Lipid Res 32:247–280 [View Article][PubMed]
     [Google Scholar]
  30. Oelkers P., Tinkelenberg A., Erdeniz N., Cromley D., Billheimer J. T., Sturley S. L. ( 2000). A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem 275:15609–15612 [View Article][PubMed]
     [Google Scholar]
  31. Parthibane V., Rajakumari S., Venkateshwari V., Iyappan R., Rajasekharan R. ( 2012). Oleosin is bifunctional enzyme that has both monoacylglycerol acyltransferase and phospholipase activities. J Biol Chem 287:1946–1954 [View Article][PubMed]
     [Google Scholar]
  32. Raetz C. R., Dowhan W. ( 1990). Biosynthesis and function of phospholipids in Escherichia coli . J Biol Chem 265:1235–1238[PubMed]
     [Google Scholar]
  33. Rani S. H., Krishna T. H., Saha S., Negi A. S., Rajasekharan R. ( 2010). Defective in cuticular ridges (DCR) of Arabidopsis thaliana, a gene associated with surface cutin formation, encodes a soluble diacylglycerol acyltransferase. J Biol Chem 285:38337–38347 [View Article][PubMed]
     [Google Scholar]
  34. Raychaudhuri S., Rajasekharan R. ( 2003). Nonorganellar acyl carrier protein from oleaginous yeast is a homologue of ribosomal protein P2. J Biol Chem 278:37648–37657 [View Article][PubMed]
     [Google Scholar]
  35. Raychaudhuri S., Reddy M. M., Rajkumar N. R., Rajasekharan R. ( 2003). Cytosolic iron superoxide dismutase is a part of the triacylglycerol biosynthetic complex in oleaginous yeast. Biochem J 372:587–594 [View Article][PubMed]
     [Google Scholar]
  36. Reddy V. S., Singh A. K., Rajasekharan R. ( 2008). The Saccharomyces cerevisiae PHM8 gene encodes a soluble magnesium-dependent lysophosphatidic acid phosphatase. J Biol Chem 283:8846–8854 [View Article][PubMed]
     [Google Scholar]
  37. Reddy V. S., Rao D. K., Rajasekharan R. ( 2010). Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana . Biochim Biophys Acta 1801:455–461 [View Article][PubMed]
     [Google Scholar]
  38. Saha S., Enugutti B., Rajakumari S., Rajasekharan R. ( 2006). Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase. Plant Physiol 141:1533–1543 [View Article][PubMed]
     [Google Scholar]
  39. Sandager L., Gustavsson M. H., Ståhl U., Dahlqvist A., Wiberg E., Banas A., Lenman M., Ronne H., Stymne S. ( 2002). Storage lipid synthesis is non-essential in yeast. J Biol Chem 277:6478–6482 [View Article][PubMed]
     [Google Scholar]
  40. Schiestl R. H., Gietz R. D. ( 1989). High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346 [View Article][PubMed]
     [Google Scholar]
  41. Siloto R. M., Truksa M., He X., McKeon T., Weselake R. J. ( 2009). Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system. Lipids 44:963–973 [View Article][PubMed]
     [Google Scholar]
  42. Sirakova T. D., Dubey V. S., Deb C., Daniel J., Korotkova T. A., Abomoelak B., Kolattukudy P. E. ( 2006). Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress. Microbiology 152:2717–2725 [View Article][PubMed]
     [Google Scholar]
  43. Sorger D., Daum G. ( 2002). Synthesis of triacylglycerols by the acyl-coenzyme A:diacyl-glycerol acyltransferase Dga1p in lipid particles of the yeast Saccharomyces cerevisiae . J Bacteriol 184:519–524 [View Article][PubMed]
     [Google Scholar]
  44. Turchetto-Zolet A. C., Maraschin F. S., de Morais G. L., Cagliari A., Andrade C. M., Margis-Pinheiro M., Margis R. ( 2011). Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis. BMC Evol Biol 11:263 [View Article][PubMed]
     [Google Scholar]
  45. Yang H., Bard M., Bruner D. A., Gleeson A., Deckelbaum R. J., Aljinovic G., Pohl T. M., Rothstein R., Sturley S. L. ( 1996). Sterol esterification in yeast: a two-gene process. Science 272:1353–1356 [View Article][PubMed]
     [Google Scholar]
  46. Yang H., Cromley D., Wang H., Billheimer J. T., Sturley S. L. ( 1997). Functional expression of a cDNA to human acyl-coenzyme A:cholesterol acyltransferase in yeast. Species-dependent substrate specificity and inhibitor sensitivity. J Biol Chem 272:3980–3985 [View Article][PubMed]
     [Google Scholar]
  47. Yen C. L., Stone S. J., Koliwad S., Harris C., Farese R. V. Jr ( 2008). Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J Lipid Res 49:2283–2301 [View Article][PubMed]
     [Google Scholar]
  48. Yu C., Kennedy N. J., Chang C. C., Rothblatt J. A. ( 1996). Molecular cloning and characterization of two isoforms of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase. J Biol Chem 271:24157–24163 [View Article][PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.063156-0
Loading
A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis
Microbiology 159, 155 (2013); https://doi.org/10.1099/mic.0.063156-0
/content/journal/micro/10.1099/mic.0.063156-0
/content/journal/micro/10.1099/mic.0.063156-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error