Skip to main content

Advertisement

Log in

Identification and characterization of a type-2 diacylglycerol acyltransferase (DGAT2) from Rhodosporidium diobovatum

  • Original Paper
  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

Triacylglycerols (TAGs), synthesized in the microsomal membranes of eukaryotes, serve as a primary storage form of carbon and energy in microorganisms. For this reason, TAGs produced by organisms have great potential to become biofuels and facilitate researchers to look for alternative renewable sources of energy. The present study describes the identification and functional characterization of a type-2 diacylglycerol acyltransferase from Rhodosporidium diobovatum, designated as RdDGAT, which catalyzed the final step of TAG synthesis. A full-length cDNA clone for RdDGAT was obtained, and its biological activity was proven by being expressed in a Saccharomyces cerevisiae quadruple mutant that was defective in TAG synthesis. Enzymatic assays were performed and finally the existence of TAGs in the transformed Saccharomyces cerevisiae quadruple mutant was determined using the method of thin-layer chromatography. Substrate preference experiments revealed that RdDGAT preferred unsaturated fatty acids over saturated ones. Through further analysis, we assume that the evolution and expression characteristics of the RdDGAT gene perhaps is the result of adaption to its oligotrophic and cold living environment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG (2011) Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 90:1219–1227

    Article  PubMed  CAS  Google Scholar 

  • Boyle NR, Page MD, Liu B, Blaby IK, Casero D, Kropat J, Cokus SJ, Hong-Hermesdorf A, Shaw J, Karpowicz SJ, Gallaher SD, Johnson S, Benning C, Pellegrini M, Grossman A, Merchant SS (2012) Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas. J Biol Chem 287:15811–15825

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Butinar L, Spencer-Martins I, Gunde-Cimerman N (2007) Yeasts in high Arctic glaciers: the discovery of a new habitat for eukaryotic microorganisms. Anton Leeuw 91:277–289

    Article  Google Scholar 

  • Cao HP (2011) Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Res 4:249

    Article  CAS  Google Scholar 

  • Cases S, Smith SJ, Zheng YW, Myers MH, Lear SR, Sande E, Novak S, Collins C, Welch CB, Lusis AJ, Erickson SK, Farese RV Jr (1998) Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Cell biol 95:13018–13023

    CAS  Google Scholar 

  • Chen HC, Rao M, Sajan MP, Standaert M, Kanoh Y, Miura A, Farese RV Jr, Farese RV (2004) Role of adipocyte-derived factors in enhancing insulin signaling in skeletal muscle and white adipose tissue of mice lacking Acyl CoA: diacylglycerol acyltransferase 1. Diabetes 53:1445–1451

    Article  PubMed  CAS  Google Scholar 

  • Granger LM, Perlot P, Goma G, Pareilleux A (1993) Effect of various nutrient limitations on fatty acid production by Rhodotorula glutinis. Appl Microbiol Biotechnol 38:784–789

    Article  CAS  Google Scholar 

  • Guihéneuf F, Leu S, Zarka A, Khozin-Goldberg I, Khalilov I, Boussiba S (2011) Cloning and molecular characterization of a novel acyl-CoA/diacylglycerol acyltransferase 1-like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum. FEBS J 278:3651–3666

    Article  PubMed  Google Scholar 

  • Hobbs D, Lu C, Hills M (1999) Cloning of a cDNA encoding diacylglycerol acyltransferase from Arabidopsis thaliana and its functional expression. FEBS Lett 452:145–149

    Article  PubMed  CAS  Google Scholar 

  • Iassonova DR, Hammond EG, Beattie SE (2008) Oxidative stability of polyunsaturated triacylglycerols encapsulated in oleaginous yeast. J Am Oil Chem Soc 85:711–716

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Kennedy EP (1961) Biosynthesis of complex lipids. Fed Proc 20:934–940

    PubMed  CAS  Google Scholar 

  • Kroon KMJ, Wei W, Simon JW, Slabas RA (2006) Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry 67:2541–2549

    Article  PubMed  CAS  Google Scholar 

  • Kvasnikov EI, Nagornaia SS, Shchelokova IF (1975) Distribution of Rhodosporidium diobovatum yeasts in soil and on plants. Mikrobiologiia 44:753–756

    PubMed  CAS  Google Scholar 

  • Lardizabal KD, Mai JT, Wagner NW, Wyrick A, Voelker T, Hawkins DJ (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

    Article  PubMed  CAS  Google Scholar 

  • 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 286:13115–13126

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Mañas-Fernández A, Vilches-Ferrón M, Garrido-Cárdenas JA, Belarbi EH, Alonso DL, García-Maroto F (2009) Cloning and molecular characterization of the acyl-CoA/diacylglycerol acyltransferase 1 (DGAT1) gene from Echium. Lipids 44:555–568

    Article  PubMed  Google Scholar 

  • Newell SY, Hunter IL (1970) Rhodosporidium diobovatum sp. n., the perfect form of an asporogenous yeast (Rhodotorula sp.). J Bacteriol 104:503–508

    PubMed  CAS  PubMed Central  Google Scholar 

  • Rani SH, Krishna TH, Saha S, Negi AS, 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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Rani SH, Saha S, Rajasekharan R (2013) A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis. Microbiology 159:155–166

    Article  PubMed  CAS  Google Scholar 

  • Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51

    Article  PubMed  CAS  Google Scholar 

  • Rau U, Nguyen LA, Roeper H, Koch H, Lang S (2005) Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis. Appl Microbiol Biotechnol 80:607–613

    Article  Google Scholar 

  • Routaboul JM, Benning C, Bechtold N, Caboche M, Lepiniec L (1999) The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase. Plant Physiol Biochem 37:831–840

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Sandager L, Gustavsson MH, Stahl 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

    Article  PubMed  CAS  Google Scholar 

  • Sitepu IR, Ignatia L, Franz AK, Wong DM, Faulina SA, Tsui M, Kanti A, Boundy-Mills K (2012) An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J Microbiol Methods 91:321–328

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Smith SJ, Cases S, Jensen DR, Chen HC, Sande E, Tow B, Sanan DA, Raber J, Eckel RH, Farese RV Jr (2000) Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat. Nat Genet 25:87–90

    Article  PubMed  CAS  Google Scholar 

  • Subramaniam R, Dufreche S, Zappi M, Bajpai R (2010) Microbial lipids from renewable resources: production and characterization. J Microbiol Biotechnol 37:1271–1287

    Article  CAS  Google Scholar 

  • Taylor DC, Weber N, Barton DL, Underhill EW, Hogge LR, Weselake RJ, Pomeroy MK (1991) Triacylglycerol bioassembly in microspore-derived embryos of Brassica napus L. cv Reston. Plant Physiol 97:65–79

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Wagner M, Hoppe K, Czabany T, Heilmann M, Daum G, Feussner I, Fulda M (2010) Identification and characterization of an acyl-CoA/diacylglycerol acyltransferase 2 (DGAT2) gene from the microalga O. tauri. Plant Physiol Biochem 48:407–416

    Article  PubMed  CAS  Google Scholar 

  • Xu J, Zheng Z, Zou J (2009) A membrane-bound glycerol-3-phosphate acyltransferase from Thalassiosira pseudonana regulates acyl composition of glycerolipids. Botany 87:544–551

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Science Research Plan of Hebei Higher Schools (No. Z2010225), open fund of Key Laboratory (No. 3333112) and Bioengineering key discipline of Hebei Province.

Conflict of interest

We declare that there is no financial/commercial conflicts of interest about all authors.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Liping Zhang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 11 kb)

Supplementary material 2 (PDF 50 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, Z., Liu, P., Liu, Y. et al. Identification and characterization of a type-2 diacylglycerol acyltransferase (DGAT2) from Rhodosporidium diobovatum . Antonie van Leeuwenhoek 106, 1127–1137 (2014). https://doi.org/10.1007/s10482-014-0282-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-014-0282-5

Keywords

Navigation