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Journal of Applied Phycology

, Volume 29, Issue 1, pp 323–333 | Cite as

A simple and reproducible non-radiolabeled in vitro assay for recombinant acyltransferases involved in triacylglycerol biosynthesis

  • Jin LiuEmail author
  • Yi-Ying Lee
  • Xuemei Mao
  • Yantao LiEmail author
Article

Abstract

Diacylglycerol acyltransferase (DGAT) is considered as a rate-limiting enzyme of triacylglycerol (TAG) biosynthesis in many organisms including algae. Many algae have multiple DGAT genes in their genomes. It is crucial to clarify substrate specificity and activity of different DGATs for understanding their biological roles. The current in vitro DGAT assays involve predominantly the use of radiolabeled substrates, either acyl-CoA or diacylglycerol (DAG). The availability of limited radiolabeled substrates and technical difficulties to conduct radiolabeled experiments have limited the use of these assays. Therefore, an assay without the involvement of radiolabeled substrates is needed. In the present study, we developed a novel in vitro DGAT assay using non-radiolabeled substrates and optimized its conditions including buffer pH and concentration, Mg2+ concentration, microsomal protein amount, acyl-CoA concentration, and incubation time. CrDGTT1, a type 2 DGAT from Chlamydomonas reinhardtii, was used to assess the feasibility of our non-radiolabeled in vitro assay toward different acyl-CoAs and DAGs. In addition, the substrate preference and activity of ScDGA1, a yeast-derived type 2 DGAT, were evaluated with our assay method, and the results obtained were consistent with those from a previous radiolabeled assay. We also demonstrated the suitability of this assay for the activity of phospholipid:diacylglycerol acyltransferase, an enzyme responsible for the acyl-CoA-independent TAG biosynthesis. Taken together, the in vitro acyltransferase assay developed here eliminates the use of radiolabeled substrates, is simple and reproducible, and allows the investigation of enzyme specificity and activity over a wide range of substrates.

Keywords

Diacylglycerol acyltransferase In vitro assay Non-radiolabeled Microalgae Triacylglycerol 

Notes

Acknowledgments

We thank Professor Sten Stymne at Swedish University of Agricultural Sciences for providing the H1246 yeast mutant and Professor Jian Xu at Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences for providing the H1246 strain carrying ScDGA1. This research is partially supported by a grant to J.L. from the National Natural Science Foundation of China (31571807), a start-up grant from the National Youth Thousand Talents Program of China, awards to Y.L. from the US National Science Foundation (CBET 1511939), Climate Change and Emissions Management Corporation (CCEMC), Office of Naval Research (N00014-15-1-2219), and a seed grant from Institute of Marine and Environmental Technology, University System of Maryland.

Author contributions

Jin Liu and Yantao Li conceived the study and designed the experiments. Jin Liu, Yi-Ying Lee, and Xuemei Mao conducted the experiments. Jin Liu and Yantao Li wrote the manuscript. All authors read and approved the final manuscript.

Supplementary material

10811_2016_949_MOESM1_ESM.pdf (362 kb)
ESM 1 (PDF 362 kb)

References

  1. Byers SD, Laroche A, Smith KC, Weselake RJ (1999) Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape. Lipids 34:1143–1149CrossRefPubMedGoogle Scholar
  2. Cases S, Stone SJ, Zhou P, Yen E, Tow B, Lardizabal KD, Voelker T, Farese RV (2001) Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J Biol Chem 276:38870–38876CrossRefPubMedGoogle Scholar
  3. Fan J, Cui Y, Wan M, Wang W, Li Y (2014) Lipid accumulation and biosynthesis genes response of the oleaginous Chlorella pyrenoidosa under three nutrition stressors. Biotechnol Biofuels 7:17CrossRefPubMedPubMedCentralGoogle Scholar
  4. Greer M, Zhou T, Weselake R (2014) A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol. Lipids 49:831–838CrossRefPubMedGoogle Scholar
  5. Greer MS, Truksa M, Deng W, Lung SC, Chen G, Weselake RJ (2015) Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type 1 plant diacylglycerol acyltransferase. Appl Microbiol Biotechnol 99:2243–2253CrossRefPubMedGoogle Scholar
  6. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins A (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54:621–639CrossRefPubMedGoogle Scholar
  7. Iwai M, Ikeda K, Shimojima M, Ohta H (2014) Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus starvation-inducible promoter. Plant Biotechnol J 12:808–819CrossRefPubMedPubMedCentralGoogle Scholar
  8. Liu Q, Siloto RM, Weselake RJ (2010) Role of cysteine residues in thiol modification of acyl-CoA:diacylglycerol acyltransferase 2 from yeast. Biochemistry 49:3237–3245CrossRefPubMedGoogle Scholar
  9. 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 286:13115–13126CrossRefPubMedPubMedCentralGoogle Scholar
  10. Liu Q, Siloto RMP, Lehner R, Stone SJ, Weselake RJ (2012) Acyl-CoA:diacylglycerol acyltransferase: molecular biology, biochemistry and biotechnology. Prog Lipid Res 51:350–377CrossRefPubMedGoogle Scholar
  11. Liu J, Han D, Yoon K, Hu Q, Li Y (2016) Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis. Plant J 86:3–19CrossRefPubMedGoogle Scholar
  12. McFie PJ, Stone SJ (2011) A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity. J Lipid Res 52:1760–1764CrossRefPubMedPubMedCentralGoogle Scholar
  13. Merchant SS, Kropat J, Liu B, Shaw J, Warakanont J (2012) TAG, You’re it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation. Curr Opin Biotechnol 23:352–363CrossRefPubMedGoogle Scholar
  14. Milcamps A, Tumaney AW, Paddock T, Pan DA, Ohlrogge J, Pollard M (2005) Isolation of a gene encoding a 1, 2-diacylglycerol-sn-acetyl-CoA acetyltransferase from developing seeds of Euonymus alatus. J Biol Chem 280:5370–5377CrossRefPubMedGoogle Scholar
  15. 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 154:1737–1752CrossRefPubMedPubMedCentralGoogle Scholar
  16. Oelkers P, Cromley D, Padamsee M, Billheimer JT, Sturley SL (2002) The DGA1 gene determines a second triglyceride synthetic pathway in yeast. J Biol Chem 277:8877–8881CrossRefPubMedGoogle Scholar
  17. 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 141:1533–1543CrossRefPubMedPubMedCentralGoogle Scholar
  18. 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 277:6478–6482CrossRefPubMedGoogle Scholar
  19. Sanderson MC, Venable ME (2012) A novel assay of acyl-coa: diacylglycerol acyltransferase activity utilizing fluorescent substrate. J Phycol 48:580–584CrossRefPubMedGoogle Scholar
  20. Sanjaya MR, Durrett TP, Kosma DK, Lydic TA, Muthan B, Koo AJK, Bukhman YV, Reid GE, Howe GA, Ohlrogge J, Benning C (2013) Altered lipid composition and enhanced nutritional value of Arabidopsis leaves following introduction of an algal diacylglycerol acyltransferase 2. Plant Cell 25:677–693CrossRefPubMedPubMedCentralGoogle Scholar
  21. Shockey JM, Gidda SK, Chapital DC, Kuan JC, 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 18:2294–2313CrossRefPubMedPubMedCentralGoogle Scholar
  22. Siloto RP, Truksa M, He X, McKeon T, Weselake R (2009) Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system. Lipids 44:963–973CrossRefPubMedGoogle Scholar
  23. Vieler A, Wu G, Tsai C-H, Bullard B, Cornish AJ, Harvey C, Reca I-B, Thornburg C, Achawanantakun R, Buehl CJ, Campbell MS, Cavalier D, Childs KL, Clark TJ, Deshpande R, Erickson E, Armenia Ferguson A, Handee W, Kong Q, Li X, Liu B, Lundback S, Peng C, Roston RL, Sanjaya SJP, TerBush A, Warakanont J, Zäuner S, Farre EM, Hegg EL, Jiang N, Kuo M-H, Lu Y, Niyogi KK, Ohlrogge J, Osteryoung KW, Shachar-Hill Y, Sears BB, Sun Y, Takahashi H, Yandell M, Shiu S-H, Benning C (2012) Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet 8:e1003064CrossRefPubMedPubMedCentralGoogle Scholar
  24. 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 48:407–416CrossRefPubMedGoogle Scholar
  25. Wang D, Ning K, Li J, Hu J, Han D, Wang H, Zeng X, Jing X, Zhou Q, Su X, Chang X, Wang A, Wang W, Jia J, Wei L, Xin Y, Qiao Y, Huang R, Chen J, Han B, Yoon K, Hill RT, Zohar Y, Chen F, Hu Q, Xu J (2014) Nannochloropsis genomes reveal evolution of microalgal oleaginous traits. PLoS Genet 10:e1004094CrossRefPubMedPubMedCentralGoogle Scholar
  26. Xu J, Francis T, Mietkiewska E, Giblin EM, Barton DL, Zhang Y, Zhang M, Taylor DC (2008) Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. Plant Biotechnol J 6:799–818CrossRefPubMedGoogle Scholar
  27. 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 64:3189–3200CrossRefPubMedPubMedCentralGoogle Scholar
  28. Zhou X-R, Shrestha P, Yin F, Petrie JR, Singh SP (2013) AtDGAT2 is a functional acyl-CoA:diacylglycerol acyltransferase and displays different acyl-CoA substrate preferences than AtDGAT1. FEBS Lett 587:2371–2376CrossRefPubMedGoogle Scholar
  29. Zou J, Wei Y, Jako C, Kumar A, Selvaraj G, Taylor DC (1999) The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene. Plant J 19:645–653CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Institute for Food and Bioresource Engineering and Department of Energy and Resources Engineering, College of EngineeringPeking UniversityBeijingChina
  2. 2.Institute of Marine and Environmental TechnologyUniversity of Maryland Center for Environmental Science and University of Maryland Baltimore CountyBaltimoreUSA

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