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Applied Microbiology and Biotechnology

, Volume 99, Issue 5, pp 2243–2253 | Cite as

Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase

  • Michael S. Greer
  • Martin Truksa
  • Wei Deng
  • Shiu-Cheung Lung
  • Guanqun Chen
  • Randall J. WeselakeEmail author
Applied genetics and molecular biotechnology

Abstract

Diacylglycerol acyltransferase (DGAT) catalyzes the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol to produce triacylglycerol (TAG). This enzyme, which is critical to numerous facets of oilseed development, has been highlighted as a genetic engineering target to increase storage lipid production in microorganisms designed for biofuel applications. Here, four transcriptionally active DGAT1 genes were identified and characterized from the oil crop Brassica napus. Overexpression of each BnaDGAT1 in Saccharomyces cerevisiae increased TAG biosynthesis. Further studies showed that adding an N-terminal tag could mask the deleterious influence of the DGATs’ native N-terminal sequences, resulting in increased in vivo accumulation of the polypeptides and an increase of up to about 150-fold in in vitro enzyme activity. The levels of TAG and total lipid fatty acids in S. cerevisiae producing the N-terminally tagged BnaDGAT1.b at 72 h were 53 and 28 % higher than those in cultures producing untagged BnaA.DGAT1.b, respectively. These modified DGATs catalyzed the synthesis of up to 453 mg fatty acid/L by this time point. The results will be of benefit in the biochemical analysis of recombinant DGAT1 produced through heterologous expression in yeast and offer a new approach to increase storage lipid content in yeast for industrial applications.

Keywords

Yeast DGAT Storage lipid synthesis Brassica napus Biofuel 

Notes

Acknowledgments

The authors thank Drs. G. Séguin-Swartz and G. Rakow for providing B. napus line DH12075. RJW acknowledges the support provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, Alberta Enterprise and Advanced Education, Alberta Innovates Bio Solutions, and the Canada Research Chairs Program. MSG is a recipient of the NSERC Graham Bell Canada Graduate Scholarship, the Alberta Innovates Graduate Student NSERC Top-up Award, and the President’s Doctoral Prize of Distinction.

Conflict of interest

The authors have no conflicts of interests to declare.

Supplementary material

253_2014_6284_MOESM1_ESM.pdf (145 kb)
ESM 1 (PDF 144 kb)

References

  1. Andrianov V, Borisjuk N, Pogrebnyak N, Brinker A, Dixon J, Spitsin S, Flynn J, Matyszczuk P, Andryszak K, Laurelli M, Golovkin M, Koprowski H (2010) Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. Plant Biotechnol J 8(3):277–87. doi: 10.1111/j.1467-7652.2009.00458.x CrossRefPubMedGoogle Scholar
  2. Bouvier-Nave P, Benveniste P, Oelkers P, Sturley SL, Schaller H (2000) Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase. Eur J Biochem 267:85–96. doi: 10.1046/j.1432-1327.2000.00961.x CrossRefPubMedGoogle Scholar
  3. BRAD (2011) Brassica Database. http://brassicadb.org/brad/
  4. Brown AP, Johnson P, Rawsthorne S, Hills MJ (1998) Expression and properties of acyl-CoA binding protein from Brassica napus. Plant Physiol Biochem 36:629–635. doi: 10.1016/S0981-9428(98)80011-9 CrossRefGoogle Scholar
  5. Byers SD, Laroche A, Smith KC, Weselake RJ (1999) Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape. Lipids 34:1143–1149. doi: 10.1007/s11745-999-0465-6 CrossRefPubMedGoogle Scholar
  6. Christie W, Han X (2010) Lipid analysis—isolation, separation, identification and lipidomic analysis, 4th edn. Oily Press, BridgwaterGoogle Scholar
  7. Durrett TP, Benning C, Ohlrogge J (2008) Plant triacylglycerols as feedstocks for the production of biofuels. Plant J 54(4):593–607. doi: 10.1111/j.1365-313X.2008.03442.x CrossRefPubMedGoogle Scholar
  8. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protocols 2(1):31–34. doi: 10.1038/nprot.2007.13 CrossRefGoogle Scholar
  9. Hamilton R, Watanabe CK, de Boer HA (1987) Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs. Nucleic Acids Res 15(8):3581–3593. doi: 10.1093/nar/15.8.3581 CrossRefPubMedCentralPubMedGoogle Scholar
  10. Kamisaka Y, Tomita N, Kimura K, Kainou K, Uemura H (2007) DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the ∆snf2 disruptant of Saccharomyces cerevisiae. Biochem J 408(1):61–8. doi: 10.1042/bj20070449 CrossRefPubMedCentralPubMedGoogle Scholar
  11. Kamisaka Y, Kimura K, Uemura H, Shibakami M (2010) Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the ∆snf2 disruptant produces a significant increase in its enzyme activity. Appl Microbiol Biotechnol 88(1):105–15. doi: 10.1007/s00253-010-2725-x
  12. Kamisaka Y, Kimura K, Uemura H, Yamaoka M (2013) Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast. Appl Microbiol Biotechnol 97(16):7345–55. doi: 10.1007/s00253-013-4915-9 CrossRefPubMedGoogle Scholar
  13. Knothe G (2005) Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process Technol 86(10):1059–1070. doi: 10.1016/j.fuproc.2004.11.002 CrossRefGoogle Scholar
  14. Lackey LG, Paulson SE (2011) Influence of feedstock: air pollution and climate-related emissions from a diesel generator operating on soybean, canola, and yellow grease biodiesel. Energy Fuels 26(1):686–700. doi: 10.1021/ef2011904 CrossRefGoogle Scholar
  15. Li Q, Du W, Liu D (2008) Perspectives of microbial oils for biodiesel production. Appl Microbiol Biotechnol 80(5):749–56. doi: 10.1007/s00253-008-1625-9 CrossRefPubMedGoogle Scholar
  16. Li R, Yu K, Hildebrand D (2010) DGAT1, DGAT2 and PDAT expression in seeds and other tissues of epoxy and hydroxy fatty acid accumulating plants. Lipids 45(2):145–157. doi: 10.1007/s11745-010-3385-4 CrossRefPubMedGoogle Scholar
  17. Liang MH, Jiang JG (2013) Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology. Prog Lipid Res 52(4):395–408. doi: 10.1016/j.plipres.2013.05.002 CrossRefPubMedGoogle Scholar
  18. Liu A-h, Wang J-b (2006) Genomic evolution of Brassica allopolyploids revealed by ISSR marker. Genet Resour Crop Evol 53(3):603–611. doi: 10.1007/s10722-004-2951-0 CrossRefGoogle Scholar
  19. Liu Q, Siloto RMP, Snyder CL, Weselake RJ (2011) Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase 2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem. doi: 10.1074/jbc.M110.204412 Google Scholar
  20. Liu Q, Siloto RM, Lehner R, Stone SJ, Weselake RJ (2012) Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog Lipid Res 51(4):350–77. doi: 10.1016/j.plipres.2012.06.001 CrossRefPubMedGoogle Scholar
  21. Lung SC, Weselake R (2006) Diacylglycerol acyltransferase: a key mediator of plant triacylglycerol synthesis. Lipids 41(12):1073–1088CrossRefPubMedGoogle Scholar
  22. Mcfie PJ, Stone SL, Banman SL, Stone SJ (2010) Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the N terminus in dimer/tetramer formation. J Biol Chem 285:37377–37387. doi: 10.1074/jbc.M110.163691 CrossRefPubMedCentralPubMedGoogle Scholar
  23. Nykiforuk CL, Laroche A, Weselake RJ (1999) Isolation and sequence analysis of a novel cDNA encoding a putative diacylglycerol acyltransferase from a microspore-derived cell suspension culture of Brassica napus L. cv Jet Neuf. Plant Physiol 120(1207):99–123Google Scholar
  24. O’Quin JB, Mullen RT, Dyer JM (2009) Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells. Appl Microbiol Biotechnol 83(1):117–25. doi: 10.1007/s00253-008-1826-2 CrossRefPubMedGoogle Scholar
  25. Ostergaard L, King G (2008) Standardized gene nomenclature for the Brassica genus. Plant Methods 4(1):10. doi: 10.1186/1746-4811-4-10 CrossRefPubMedCentralPubMedGoogle Scholar
  26. Runguphan W, Keasling JD (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng 21:103–13. doi: 10.1016/j.ymben.2013.07.003 CrossRefPubMedGoogle Scholar
  27. Sandager L, Gustavsson MH, 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(8):6478–6482. doi: 10.1074/jbc.M109109200 CrossRefPubMedGoogle Scholar
  28. Séguin-Swartz G, Gugel, R., Rakow, G., and Raney, J.P. Agronomic performance, blackleg resistance and seed quality of doubled haploid lines of Brassica napus summer rape in Canada. In: Proceedings of the 11th International Rapeseed Congress, Copenhagen, Denmark, 2003. p 452–454Google Scholar
  29. Shockey JM, Gidda SK, Chapital DC, Kuan J-C, Dhanoa PK, Bland JM, Rothsein SJ, Mullen RT, Dyer JM (2006) Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell 18(9):2294–2313CrossRefPubMedCentralPubMedGoogle Scholar
  30. Siloto RM, Madhavji M, Wiehler WB, Burton TL, Boora PS, Laroche A, Weselake RJ (2008) An N-terminal fragment of mouse DGAT1 binds different acyl-CoAs with varying affinity. Biochem Biophys Res Commun 373(3):350–354. doi: 10.1016/j.bbrc.2008.06.031 CrossRefPubMedGoogle Scholar
  31. Siloto RM, Truksa M, He X, McKeon T, Weselake RJ (2009a) Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system. Lipids 44(10):963–973. doi: 10.1007/s11745-009-3336-0 CrossRefPubMedGoogle Scholar
  32. Siloto RMP, Truksa M, Brownfield D, Good AG, Weselake RJ (2009b) Directed evolution of acyl-CoA:diacylglycerol acyltransferase: development and characterization of Brassica napus DGAT1 mutagenized libraries. Plant Physiol Biochem 47(6):456–461. doi: 10.1016/j.plaphy.2008.12.019 CrossRefPubMedGoogle Scholar
  33. Sriram SM, Kim BY, Kwon YT (2011) The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nat Rev Mol Cell Biol 12(11):735–747. doi: 10.1038/nrm3217 CrossRefPubMedGoogle Scholar
  34. Stukey JE, McDonough VM, Martin CE (1990) The OLE1 gene of Saccharomyces cerevisiae encodes the delta 9 fatty acid desaturase and can be functionally replaced by the rat stearoyl-CoA desaturase gene. J Biol Chem 265(33):20144–9PubMedGoogle Scholar
  35. Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9. doi: 10.1016/j.ymben.2012.08.007 CrossRefPubMedGoogle Scholar
  36. Tang X, Feng H, Chen WN (2013) Metabolic engineering for enhanced fatty acids synthesis in Saccharomyces cerevisiae. Metab Eng 16(0):95–102. doi: 10.1016/j.ymben.2013.01.003 CrossRefPubMedGoogle Scholar
  37. Tasaki T, Sriram SM, Park KS, Kwon YT (2012) The N-end rule pathway. Annu Rev Biochem 81:261–89. doi: 10.1146/annurev-biochem-051710-093308 CrossRefPubMedCentralPubMedGoogle Scholar
  38. Taylor DC, Zhang Y, Kumar A, Francis T, Giblin EM, Barton DL, Ferrie JR, Laroche A, Shah S, Zhu W, Snyder CL, Hall L, Rakow G, Harwood JL, Weselake RJ (2009) Molecular modification of triacylglycerol accumulation by over-expression of DGAT1 to produce canola with increased seed oil content under field conditions. Botany 87(6):533–543CrossRefGoogle Scholar
  39. Turchetto-Zolet AC, Maraschin FS, de Morais GL, Cagliari A, Andrade CM, 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. doi: 10.1186/1471-2148-11-263 CrossRefPubMedCentralPubMedGoogle Scholar
  40. Weselake RJ, Madhavji M, Szarka SJ, Patterson NA, Wiehler WB, Nykiforuk CL, Burton TL, Boora PS, Mosimann SC, Foroud NA, Thibault BJ, Moloney MM, Laroche A, Furukawa-Stoffer TL (2006) Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape. BMC Biochemistry 7:24. doi: 10.1186/1471-2091-7-24 CrossRefPubMedCentralPubMedGoogle Scholar
  41. Yu K, Li R, Hatanaka T, Hildebrand D (2008) Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis. Phytochemistry 69(5):1119–27. doi: 10.1016/j.phytochem.2007.11.015 CrossRefPubMedGoogle Scholar
  42. Yu KO, Jung J, Ramzi AB, Choe SH, Kim SW, Park C, Han SO (2013) Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate. Biotechnol Bioeng 110(1):343–7. doi: 10.1002/bit.24623 CrossRefPubMedGoogle Scholar
  43. Zhang C, Iskandarov U, Klotz ET, Stevens RL, Cahoon RE, Nazarenus TJ, Pereira SL, Cahoon EB (2013) A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds. J Exp Bot. doi: 10.1093/jxb/ert156 Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Michael S. Greer
    • 1
  • Martin Truksa
    • 1
    • 2
  • Wei Deng
    • 1
    • 3
  • Shiu-Cheung Lung
    • 1
  • Guanqun Chen
    • 1
  • Randall J. Weselake
    • 1
    Email author
  1. 1.Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional ScienceUniversity of AlbertaEdmontonCanada
  2. 2.Alberta Enterprise and Advanced EducationEdmontonCanada
  3. 3.School of Life ScienceChongqing UniversityChongqingChina

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