title: Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.
abstract: Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts.
authors.affiliation: Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science and University of Maryland Baltimore County, Baltimore, MD, 21202, USA.|Department of Energy and Resources Engineering, College of Engineering, Peking University, Beijing, 100871, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
authors.forename: Danxiang
authors.initials: D
authors.lastname: Han
authors.affiliation: Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
authors.forename: Kangsup
authors.initials: K
authors.lastname: Yoon
authors.affiliation: Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science and University of Maryland Baltimore County, Baltimore, MD, 21202, USA.
authors.forename: Yantao
authors.identifier: http://orcid.org/0000-0001-7545-1883
authors.initials: Y
authors.lastname: Li
mesh_terms: D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005227:Fatty Acids / Q000378:metabolism; D050356:Lipid Metabolism; D013379:Substrate Specificity; D014280:Triglycerides / Q000096:biosynthesis*
chemical_list: C011439:1,2-diacylglycerol; D020418:Algal Proteins; D004075:Diglycerides; D005227:Fatty Acids; D014280:Triglycerides; D051048:Diacylglycerol O-Acyltransferase
keywords: Chlamydomonas reinhardtii; algae; biofuels; diacylglycerol acyltransferase; lipid metabolism; triacylglycerol