Haiyan Ma1, Xiaoying Wu1, Ziwang Wei1,2, Liang Zhao1, Zhongze Li1,3, Qing Liang1, Jie Zheng1,3, Yu Wang1, Yanhua Li1, Linfei Huang1,3, Qiang Hu1,4,5,6,7,8, Danxiang Han1,6,7. 1. Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. 2. Department of Chemistry, The University of British Columbia, Vancouver, BC, Canada. 3. University of Chinese Academy of Sciences, Beijing, China. 4. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China. 5. Institute for Advanced Study, Shenzhen University, Shenzhen, China. 6. Key Laboratory for Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. 7. The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China. 8. Beijing Key Laboratory of Algae Biomass, SDIC Biotech Investment Corporation, Beijing, China.
Abstract
Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final committed step in triacylglycerol biosynthesis in eukaryotes. In microalgae, the copy number of DGAT genes is extraordinarily expanded, yet the functions of many DGATs remain largely unknown. This study revealed that microalgal DGAT can function as a lysophosphatidic acyltransferase (LPAAT) both in vitro and in vivo while losing its original function as DGAT. Among the five DGAT-encoding genes identified and cloned from the green microalga Haematococcus pluvialis, four encoded HpDGATs that showed triacylglycerol synthase activities in yeast functional complementation analyses; the exception was one of the type II DGAT encoding genes, HpDGTT2. The hydrophobic recombinant HpDGTT2 protein was purified in soluble form and was found to function as a LPAAT via enzymatic assay. Introducing this gene into the green microalga Chlamydomonas reinhardtii led to retarded cellular growth, enlarged cell size, and enhanced triacylglycerol accumulation, identical to the phenotypes of transgenic strains overexpressing CrLPAAT. This study provides a framework for dissecting uncharacterized DGATs, and could pave the way to decrypting the structure-function relationship of this large group of enzymes that are critical to lipid biosynthesis.
n class="Chemical">Acyl-CoA:n>n class="Gene">diacylglycerol acyltransferase (DGAT) catalyzes the final committed step in triacylglycerol biosynthesis in eukaryotes. In microalgae, the copy number of DGAT genes is extraordinarily expanded, yet the functions of many DGATs remain largely unknown. This study revealed that microalgal DGAT can function as a lysophosphatidic acyltransferase (LPAAT) both in vitro and in vivo while losing its original function as DGAT. Among the five DGAT-encoding genes identified and cloned from the green microalga Haematococcus pluvialis, four encoded HpDGATs that showed triacylglycerol synthase activities in yeast functional complementation analyses; the exception was one of the type II DGAT encoding genes, HpDGTT2. The hydrophobic recombinant HpDGTT2 protein was purified in soluble form and was found to function as a LPAAT via enzymatic assay. Introducing this gene into the green microalga Chlamydomonas reinhardtii led to retarded cellular growth, enlarged cell size, and enhanced triacylglycerol accumulation, identical to the phenotypes of transgenic strains overexpressing CrLPAAT. This study provides a framework for dissecting uncharacterized DGATs, and could pave the way to decrypting the structure-function relationship of this large group of enzymes that are critical to lipid biosynthesis.
Authors: E-Ming Rau; Zdenka Bartosova; Kåre Andre Kristiansen; Inga Marie Aasen; Per Bruheim; Helga Ertesvåg Journal: Front Microbiol Date: 2022-01-25 Impact factor: 5.640