A simple and reproducible non-radiolabeled in vitro assay for recombinant acyltransferases involved in triacylglycerol biosynthesis
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Diacylglycerol acyltransferase (DGAT) is considered as a rate-limiting enzyme of triacylglycerol (TAG) biosynthesis in many organisms including algae. Many algae have multiple DGAT genes in their genomes. It is crucial to clarify substrate specificity and activity of different DGATs for understanding their biological roles. The current in vitro DGAT assays involve predominantly the use of radiolabeled substrates, either acyl-CoA or diacylglycerol (DAG). The availability of limited radiolabeled substrates and technical difficulties to conduct radiolabeled experiments have limited the use of these assays. Therefore, an assay without the involvement of radiolabeled substrates is needed. In the present study, we developed a novel in vitro DGAT assay using non-radiolabeled substrates and optimized its conditions including buffer pH and concentration, Mg2+ concentration, microsomal protein amount, acyl-CoA concentration, and incubation time. CrDGTT1, a type 2 DGAT from Chlamydomonas reinhardtii, was used to assess the feasibility of our non-radiolabeled in vitro assay toward different acyl-CoAs and DAGs. In addition, the substrate preference and activity of ScDGA1, a yeast-derived type 2 DGAT, were evaluated with our assay method, and the results obtained were consistent with those from a previous radiolabeled assay. We also demonstrated the suitability of this assay for the activity of phospholipid:diacylglycerol acyltransferase, an enzyme responsible for the acyl-CoA-independent TAG biosynthesis. Taken together, the in vitro acyltransferase assay developed here eliminates the use of radiolabeled substrates, is simple and reproducible, and allows the investigation of enzyme specificity and activity over a wide range of substrates.
KeywordsDiacylglycerol acyltransferase In vitro assay Non-radiolabeled Microalgae Triacylglycerol
We thank Professor Sten Stymne at Swedish University of Agricultural Sciences for providing the H1246 yeast mutant and Professor Jian Xu at Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences for providing the H1246 strain carrying ScDGA1. This research is partially supported by a grant to J.L. from the National Natural Science Foundation of China (31571807), a start-up grant from the National Youth Thousand Talents Program of China, awards to Y.L. from the US National Science Foundation (CBET 1511939), Climate Change and Emissions Management Corporation (CCEMC), Office of Naval Research (N00014-15-1-2219), and a seed grant from Institute of Marine and Environmental Technology, University System of Maryland.
Jin Liu and Yantao Li conceived the study and designed the experiments. Jin Liu, Yi-Ying Lee, and Xuemei Mao conducted the experiments. Jin Liu and Yantao Li wrote the manuscript. All authors read and approved the final manuscript.
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