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

BAGNATO C; PRADOS MB; FRANCHINI G; SCAGLIA N; MIRANDA S; BELLIGNI V

Ventura, CA

Congreso; Gordon Research Conference on Chloroplast Biotechnology; 2017

Gordon Research

The microalgal triglyceride (TAG) synthesis pathway has attracted considerable attention in the last decade due to the potential of these organisms for biodiesel production. Special emphasis has been put towards characterizing the algal homologs of the canonical animal, yeast and plant enzymes involved in the rate-limiting steps of TAG synthesis, such as diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltrasnferase (PDAT). These enzymes locate in the cytosol/endoplasmic reticulum in most organisms. In the green alga Chlamydomonas reinhardtii, TAGs have also been shown to accumulate in the chloroplast under nitrogen starvation1?3 and light stress4. Phospholipid remodeling, partly due to the activity of chloroplast-targeted PDAT5, likely participates in chloroplast TAG synthesis6. In addition, cytosolic TAGs might be transferred to the chloroplast through physical associations between the ER membrane and the outer envelope of the chloroplast2,7. To this date, no evidence for the existence of a chloroplast de novo TAG synthesis pathway has been provided. Through HMMER iterative data mining, we identified a novel DGAT exclusive to green algae with moderate similarity to plant soluble DGAT3. The DGAT3 clade shares a most recent common ancestor with a group of uncharacterized proteins from cyanobacteria, suggesting a cyanobacterial origin. Analysis of subcellular 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, as evidenced by thin layer chromatography. In summary, our work provides the first evidence for the presence 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 tailoring TAG synthesis using a soluble enzyme, something much more difficult to achieve with integral membrane 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.16.00718 5. 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).