Biochemical Characterization Of Thraustochytrium Roseum WS/DGAT TrWSD4,TrWSD5 And Utilization Of Microbial Biosynthesis Of Wax Ester

Wax esters and triglyceride are natural lipids that composed of long-chain fatty alcohols and fatty acids.The wax esters are widely distributed in nature and are intracellular storage lipids that are found in animals and plants as well as microorganisms.They are considerable important commercial lipids with technical,cosmetic,food and medical applications.The resource of natural wax esters are limited and relatively expensive.In recent years,these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels.The final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A?acyl-CoA?:diacylglycerol acyltransferase?WS/DGAT?.This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms.Here,we did a research on WS/DGATs from Thraustochytrium roseum whereas little is known in eukaryotic microorganisms.In this thesis,the two ws/dgat genes TrWSD4,TrWSD5 were cloned from Thraustochytrium roseum.We did a biochemical characterization of TrWSD4,TrWSD5 and utilization of yeast biosynthesis of wax ester.The results and experiment process were list as follows:The two ws/dgat genes cloned from Thraustochytrium roseum were named as Tr WSD4and TrWSD5.The bioinformatics analysis results showed that the two protein sequences of TrWSD4 and TrWSD5 contain a WS-like acyl-Co A acyltransferase domain.Phylogenetic tree analysis showing the Tr WSD4 and TrWSD5 were clustered into a ws/dgat branch.As a result,we conclude that the TrWSD4 and TrWSD5 belong to the WS/DGAT family.The TrWSD4,TrWSD5 genes were heterogenous expressed into E.coli.The two protein were purified for characterization their activity in vitro.TrWSD4 and TrWSD5 have substantial WS and lower DGAT activity.They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs.TrWSD4 displays WS activity with the lowest K_m value of 0.14mM lauroyl-CoA(C_12:0).TrWSD5 displays WS activity with the lowest K_m value of 0.96mM decanoyl-CoA(C_10:0).The enzymatic characteristics showed the two WS/DGAT enzymes have the highest WS activity at 37? and 47?,and WS activity was greatly decreased when temperature exceeds47?.TrWSD4 and TrWSd5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mMExpression of the TrWSD4 and TrWSD5 genes in Saccharomyces cerevisiae H1246resulted in the accumulation of wax esters,but not triacylglycerol,indicating that TrWSD4and TrWSD5 were predominantly functions as wax esters synthase.GC-MS analysis of the wax esters produced by recombinant yeast H1246 expressing the TrWSD4 and TrWSD5genes revealed that the wax ester were predominantly composite of medium and short chain acyl-CoAs,hexadecyl ester.In this research,the result showed that the two enzymes TrWSD4,TrWSD5 were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs as substrate for WS activity in vitro and utilizing of yeast biosynthesis of wax ester in vivo.This research lay a solid theory foundation for application.