Journal of Applied Phycology

, Volume 27, Issue 4, pp 1499–1507 | Cite as

Diacylglycerol acyltransferase type 2 cDNA from the oleaginous microalga Neochloris oleoabundans: cloning and functional characterization

  • Wipa Chungjatupornchai
  • Akaraphol Watcharawipas


Currently, biodiesel production from microalgae is technically, but not yet economically, feasible. Increasing microalgal triacylglycerol (TAG) content via genetic engineering could be a potential approach to improve the efficiency of biodiesel production. In all organisms studied so far, diacylglycerol acyltransferase (DGAT) is the primary enzyme of TAG biosynthesis. In this study, we cloned the cDNA encoding DGAT type 2 (NeoDGAT2) from the oleaginous microalga Neochloris oleoabundans. The open reading frame of NeoDGAT2 cDNA was 978 bp encoding a protein of 325 amino acids. Although N. oleoabundans has been classified in the class Chlorophyceae, NeoDGAT2 was closely related to Treboxiophycean DGAT2 as it shared 80 % amino acid identity with CvDGAT2-2 of Chlorella variabilis, but distantly related to Chlorophycean DGAT2 as it shared only 34 % identity with CrDGAT2A (DGTT4) of Chlamydomonas reinhardtii. As suggested by hydropathy analysis, NeoDGAT2 might contain 4 to 5 transmembrane domains. We tested whether NeoDGAT2 cDNA encoded a protein with DGAT activity by functional complementation assay in yeast Saccharomyces cerevisiae mutant H1246. NeoDGAT2 was able to compensate the endogenous DGAT2 activity of mutant H1246 and restore the lipid body formation and TAG synthesis. The successful cloning of cDNA encoding a protein with DGAT activity provides a candidate for genetic manipulation in microalgae to increase TAG content for biodiesel production.


Microalgae Neochloris oleoabundans Biodiesel Triacylglycerol biosynthesis Diacylglycerol acyltransferase (DGAT) 



We thank Prof. Sten Stymne (Scandinavian Biotechnology Research, Alnarp, Sweden) for providing the yeast strains H1246 and SCY62 and Sirirat Fa-aroonsawat for technical assistance. This work was supported by Mahidol University and The Thailand Research Fund to Wipa Chungjatupornchai. Akaraphol Watcharawipas was supported by the 60th Year Supreme Reign of His Majesty King Bhumibol Adulyadej Scholarship.


  1. Bischoff HW, Bold HC (1963) Phycological studies. IV. Some algae from enchanted rock and related algae species. Univ Texas Pub 4:1–95Google Scholar
  2. Bligh E, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917PubMedCrossRefGoogle Scholar
  3. 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 287:15811–15825PubMedCentralPubMedCrossRefGoogle Scholar
  4. Cao H (2011) Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms. BMC Res Notes 4:249PubMedCentralPubMedCrossRefGoogle Scholar
  5. Cases S, Smith SJ, Zheng Y-W, Myers HM, Lear SR, Sande E, Novak S, Collins C, Welch CB, Lusis AJ (1998) Identification of a gene encoding an acyl CoA: diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci U S A 95:13018–13023PubMedCentralPubMedCrossRefGoogle Scholar
  6. Chen JE, Smith AG (2012) A look at diacylglycerol acyltransferases (DGATs) in algae. J Biotechnol 162:28–39PubMedCrossRefGoogle Scholar
  7. Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294–306PubMedCrossRefGoogle Scholar
  8. Deason T, Silva P, Watanabe S, Floyd G (1991) Taxonomic status of the species of the green algal genus Neochloris. Plant Syst Evol 177:213–219CrossRefGoogle Scholar
  9. Gong Y, Zhang J, Guo X, Wan X, Liang Z, Hu CJ, Jiang M (2013) Identification and characterization of PtDGAT2B, an acyltransferase of the DGAT2 acyl-coenzyme A: diacylglycerol acyltransferase family in the diatom Phaeodactylum tricornutum. FEBS Lett 587:481–487PubMedCrossRefGoogle Scholar
  10. Guiheneuf 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 278:3651–3666PubMedCrossRefGoogle Scholar
  11. Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14:378–379PubMedCrossRefGoogle Scholar
  12. Hofmann K, Stoffel W (1993) TMbase—a database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler 374:166Google Scholar
  13. Hung C-H, Ho M-Y, Kanehara K, Nakamura Y (2013) Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii. FEBS Lett 587:2364–2370PubMedCrossRefGoogle Scholar
  14. Jako C, Kumar A, Wei Y, Zou J, Barton DL, Giblin EM, Covello PS, Taylor DC (2001) Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiol 126:861–874PubMedCentralPubMedCrossRefGoogle Scholar
  15. Kennedy EP, Weiss SB (1956) The function of cytidine coenzymes in the biosynthesis of phospholipides. J Biol Chem 222:193–214PubMedGoogle Scholar
  16. Komárek R (1989) Polynuclearity of vegetative cells in coccal green algae from the family Neochloridaceae. Arch Protistenk 137:255–273CrossRefGoogle Scholar
  17. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580PubMedCrossRefGoogle Scholar
  18. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132PubMedCrossRefGoogle Scholar
  19. 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 276:38862–38869PubMedCrossRefGoogle Scholar
  20. Liu Q, Siloto RM, 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 286:13115–13126PubMedCentralPubMedCrossRefGoogle Scholar
  21. Lung SC, Weselake RJ (2006) Diacylglycerol acyltransferase: a key mediator of plant triacylglycerol synthesis. Lipids 41:1073–1088PubMedCrossRefGoogle Scholar
  22. Miller R, Wu G, Deshpande RR, Vieler A, Gartner K, Li X, Moellering ER, Zauner S, Cornish AJ, Liu B, Bullard B, Sears BB, Kuo MH, Hegg EL, Shachar-Hill Y, Shiu SH, Benning C (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 154:1737–1752PubMedCentralPubMedCrossRefGoogle Scholar
  23. Msanne J, Xu D, Konda AR, Casas-Mollano JA, Awada T, Cahoon EB, Cerutti H (2012) Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry 75:50–59PubMedCrossRefGoogle Scholar
  24. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods 8:785–786PubMedCrossRefGoogle Scholar
  25. Rismani-Yazdi H, Haznedaroglu BZ, Bibby K, Peccia J (2011) Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: pathway description and gene discovery for production of next-generation biofuels. BMC Genomics 12:148PubMedCentralPubMedCrossRefGoogle Scholar
  26. Rismani-Yazdi H, Haznedaroglu B, Hsin C, Peccia J (2012) Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation. Biotechnol Biofuels 5:74PubMedCentralPubMedCrossRefGoogle Scholar
  27. Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26:1628–1635PubMedCentralPubMedCrossRefGoogle Scholar
  28. 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 141:1533–1543PubMedCentralPubMedCrossRefGoogle Scholar
  29. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  30. 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 277:6478–6482PubMedCrossRefGoogle Scholar
  31. Shockey JM, Gidda SK, Chapital DC, Kuan J-C, Dhanoa PK, Bland JM, Rothstein 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 Online 18:2294–2313CrossRefGoogle Scholar
  32. Siloto RM, Truksa M, He X, McKeon T, Weselake RJ (2009) Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system. Lipids 44:963–973PubMedCrossRefGoogle Scholar
  33. Stobart K, Mancha M, Lenman M, Dahlqvist A, Stymne S (1997) Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds. Planta 203:58–66Google Scholar
  34. Stone SJ, Levin MC, Farese RV (2006) Membrane topology and identification of key functional amino acid residues of murine acyl-CoA: diacylglycerol acyltransferase-2. J Biol Chem 281:40273–40282PubMedCrossRefGoogle Scholar
  35. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  36. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedCentralPubMedCrossRefGoogle Scholar
  37. Tornabene T, Holzer G, Lien S, Burris N (1983) Lipid composition of the nitrogen starved green alga Neochloris oleoabundans. Enzyme Microb Technol 5:435–440CrossRefGoogle Scholar
  38. Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850PubMedCrossRefGoogle Scholar
  39. von Heijne G (1992) Membrane protein structure prediction, hydrophobicity analysis and the positive-inside rule. J Mol Biol 225:487–494CrossRefGoogle Scholar
  40. 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 48:407–416PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Wipa Chungjatupornchai
    • 1
  • Akaraphol Watcharawipas
    • 1
  1. 1.Institute of Molecular BiosciencesMahidol UniversityNakhon PathomThailand

Personalised recommendations