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Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis
Author(s) -
Liu Jin,
Han Danxiang,
Yoon Kangsup,
Hu Qiang,
Li Yantao
Publication year - 2016
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13143
Subject(s) - chlamydomonas reinhardtii , biosynthesis , acyltransferases , biochemistry , diacylglycerol kinase , chlamydomonas , biology , chloroplast , phosphatidic acid , enzyme , gene , mutant , phospholipid , protein kinase c , membrane
Summary Diacylglycerol acyltransferases ( DGAT s) 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 DGAT s ( DGTT s). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTT s. In vitro enzyme analysis using a radiolabel‐free assay revealed distinct substrate specificities of three DGTT s: Cr DGTT 1 preferred polyunsaturated acyl CoAs, Cr DGTT 2 preferred monounsaturated acyl CoAs, and Cr DGTT 3 preferred C16 CoAs. When diacylglycerol was used as the substrate, Cr DGTT 1 preferred C16 over C18 in the sn‐ 2 position of the glycerol backbone, but Cr DGTT 2 and Cr DGTT 3 preferred C18 over C16. In vivo knockdown of Cr DGTT 1 , Cr DGTT 2 or Cr DGTT 3 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 Cr DGTT 1, Cr DGTT 2 and Cr DGTT 3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii . Cr DGTT 1 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 Cr DGTT 1 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.