Identification And Characterization Of Diacylglycerol Acyltransferase In Oleaginous Yeast Rhodosporidium Toruloides

Microbial oils attract more and more attention in recent years because of their capacity of production of polyunsaturated fatty acids(PUFAs) and feedstock of biodiesel, and the mechanism of lipid accumulation is the most important field of microbial oil research. Microbial oils mainly contained nutruel neutral lipids, triglyceride(TAG) and diacylglycerol(DAG), the TAG and DAG content is more than 90% of total lipid. Intracellular lipid generally is synthesized through glycerol-3-phosphate pathway, the last step is catalyzed by diacylglycerol acyltransferase(DGAT), converting the DAG to TAG. It has been proved that DGAT play a vital role on this metabolic pathway, and may be the key factor for lipid accumulation in oleaginous microorganisms. However the role of DGAT on lipid biosynthesis in oleaginous yeast Rhodosporidium toruloides is under unclear, identification and characterization of DGATs from R. toruloides was studied in this study.Fristly, when R. toruloides CGMCC 2.1389 was cultivated in 2 L fermentor with nitrogen limited medium, at the end of fermentation biomass was up to 23.10 g/L. Lipid content in yeast cells reached the 73% of their biomass and TLC results showed that TAG content was up to 53% of their biomass. The genome of oleaginous yeast R. toruloides contains two putative DGAT genes(RtDGATa and RtDGATb). In the process of lipid accumulation, the mRNA level showed little change, but expression pattern of RtDGATb was related to the process of fatty acid biosynthesis, suggesting that RtDGATb plays an important role in lipid accumulation in R. toruloides.Secondly, according to the result of bioinformatics analysis for RtDGATa and RtDGATb with RdDGAT2 from other red yeast Rhodosporidium diobovatum as control protein showed that RtDGATb had high similarity with RdDGAT2 in physicochemical property and conserved domain, however the similarity between RtDGATa and RdDGAT2 was especially low. Phylogeny tree analysis showed that RtDGATa was clustered within DGAT1 family and had substantial similarity with Acyl-CoA: cholesterol acyltransferase from S. cervisiae(ScARE). However RtDGATb was distinctly separated into DGAT2 family and had the largest similarity with RdDGAT2 from R. diobovatum, which had been proved to belong to DGAT2 family and contained six highly conserved motifs.Finally, to affirm whether RtDGATa and RtDGATb indeed encoded proteins with DGAT activity, RtDGATa and RtDGATb were cloned from cDNA of R. toruloides and expressed individually in TAG-deficient quadruple mutant S. cervisiae H1246. And DGA1 from S. cervisiae and RdDGAT2 from R. diobovatum were also expressed in S. cervisiae H1246 as positive control. Five vectors were transformed in S. cervisiae H1246 to obtain mutant strains HY1(H1246/pYES2/NT C), HY2(H1246/pYES2-DGA1), HY3(H1246/p YES2-RdDGAT2), HY4(H1246/pYES2-RtDGATa) and HY5(H1246/pYES2-RtDGATb). The results of GC and TLC showed that TAG can not been determined in strains HY1 and HY4, but the expression of RtDGATb could completely resume TAG biosynthesis in S. cerevisiae H1246. Substrate preference experiments revealed that RtDGATb preferred unsaturated fatty acids over saturated fatty acids, but not C18:3.In conclusion, expreesion of RtDGATb completely resume TAG biosynthesis in S. cerevisiae H1246, and expression pattern of RtDGATb was related to the process of fatty acid biosynthesis in R. toruloides, suggesting that RtDGATb plays an important role in lipid accumulation in R. toruloides.