Insights into potential enzymatic components of green algal chloroplast triglyceride synthesis: identification of a soluble diacylglycerol acyltransferase.

GISELA R. FRANCHINI; MARÍA VERONICA BELIGNI; CAROLINA BAGNATO; NATALIA SCAGLIA; MARÍA BELEN PRADOS; SILVIA V. MIRANDA

Ventura

Conferencia; Gordon Research Conferences on Chloroplast Biotechnology; 2017

Gordon Research Conferences

The microalgal triglyceride (TAG) synthesis pathway has attracted considerable attention in the lastdecade due to the potential of these organisms for biodiesel production. Special emphasis has been puttowards characterizing the algal homologs of the canonical animal, yeast and plant enzymes involvedin the rate-limiting steps of TAG synthesis, such as diacylglycerol acyltransferase (DGAT) andphospholipid:diacylglycerol acyltrasnferase (PDAT). These enzymes locate in the cytosol/endoplasmicreticulum in most organisms. In the green alga Chlamydomonas reinhardtii, TAGs have also beenshown to accumulate in the chloroplast under nitrogen starvation1?3 and light stress4. Phospholipidremodeling, partly due to the activity of chloroplast-targeted PDAT5, likely participates in chloroplastTAG synthesis6. In addition, cytosolic TAGs might be transferred to the chloroplast through physicalassociations between the ER membrane and the outer envelope of the chloroplast2,7. To this date, noevidence for the existence of a chloroplast de novo TAG synthesis pathway has been provided. ThroughHMMER iterative data mining, we identified a novel DGAT exclusive to green algae with moderatesimilarity to plant soluble DGAT3. The DGAT3 clade shares a most recent common ancestor with agroup of uncharacterized proteins from cyanobacteria, suggesting a cyanobacterial origin. Analysis ofsubcellular targeting predicts that most green algal DGAT3 proteins likely target to the chloroplast.Experiments to determine the subcellular localization of C. reinhardtii DGAT3 are ongoing.Heterologous expression of C. reinhardtii DGAT3 produces an increase in the accumulation of TAG, asevidenced by thin layer chromatography. In summary, our work provides the first evidence for thepresence of a DGAT3 in green algae, possibly involved in TAG de novo synthesis in the chloroplast.From a genetic engineering perspective, the identification of DGAT3 opens the possibility of tailoringTAG synthesis using a soluble enzyme, something much more difficult to achieve with integralmembrane proteins like DGAT1 and DGAT2.1. Fan, J., Andre, C. & Xu, C. FEBS Lett. 585, 1985?91 (2011).2. Goodson, C., Roth, R., Wang, Z. T. & Goodenough, U. Eukaryot. Cell 10, 1592?606 (2011).3. Goodenough, U. et al. Eukaryot. Cell 13, 591?613 (2014).4. Goold, H. D. et al. Plant Physiol. pp.00718.2016 (2016). doi:10.1104/pp.16.007185. Terashima, M., Specht, M. & Hippler, M. Curr. Genet. 57, 151?68 (2011).6. Yoon, K., Han, D., Li, Y., Sommerfeld, M. & Hu, Q. Plant Cell 24, 3708?24 (2012).7. Davidi, L., Shimoni, E., Khozin-Goldberg, I., Zamir, A. & Pick, U. Plant Physiol. 164, 2139?2156 (2014).