Manipulation Of Lipid Metabolism In Chlamydomonas Reinhardtii Through Genetic Engineering Of Key Enzymes In Kennedy Pathway

Triacylglycerol(TAG)is the major storage lipid of animals,plants,microorganism and microalgae.TAG can be applied to food,light industry and biofuels,it is the raw material of biodiesel,which is a new kind of renewable energy.Comparing to oil crops,microalgae has the advantage of quick growing speed,high oil production and the ability of adapting to a variety of growth environment,it is a potential raw material of biodiesel.Chlamydomonas reinhardtii is a model single cell organism alga,and perfect for studying the genes involved in lipid metabolism.Current research shows that TAG is synthesis through Kennedy Pathway,in which both glycerol-3-phosphate acyltransferase(GPAT)and phospholipid: diacylglycerol acyltransferase(PDAT)are key speed-control enzymes.In this study,we cloned the genes of these two enzymes,GPAT1,GPAT2 and PDAT,and overexpression these three genes respectively in Chlamydomonas reinhardtii,which led to the increase of TAG content.The following are the results of this study:1.We successfully cloned the complete sequence of GPAT1,GPAT2 and PDAT.Through bioinformatics analysis,we found that the CDS of GPAT1,GPAT2 and PDAT are 1233 bp,1371bp and 3123 bp long,they encode 410,456 and 1040 amino acids separately.2.The HSP70A-RBCS2 promoter and Zeomycin gene in p JD carrier were switched to Psa D promoter and Hygromycin resistance gene,and the CDS of GPAT1,GPAT2 and PDAT were ligated to carrier separately to build PPH-GPAT1,PPH-GPAT2 and PPH-PDAT vectors.These three expression vectors were transformed and expressed in CC849 which is a wild type of Chlamydomonas reinhardtii.Three transformats G1-2,G2-2 and P-9 were screened by BODIPY 505/515.Fluorescence quantitative PCR showed that the expression level of GPAT1 in G1-2 were increased by 2.4 folds,the expression level of GPAT2 in G2-2 were increased by 40%,the expression level of PDAT in P-9 were increased by 60%,all compared with the wild type strain CC849.3.The GC-MS was used to analyze the total fatty acid content and components.Total fatty acid content of G1-2 was 73.4 ?g/mg,it's 35% higher than CC849,the most improved component was C18:3,56% higher compared with CC849.Total fatty acid content of G2-2 was 76.25 ?g/mg,it's 40% higher than CC849,the most improved component in G2-2 was also the C18:3,increase by 53% compared with CC849.Total fatty acid content of the last transformat P-9 was 77.1 ?g/mg,41% higher than CC849 which was the highest among all transformats,the most improved component was C18:2,it was upregulated by 42% compared with CC849.In this study,the genes of two key enzymes GPAT and PDAT in Kennedy Pathway were transformed into Chlamydomonas reinhardtii,in a vector that has promoter of continuous expression.Through overexpressed these genes,the TAG content and total fatty acid content in cells were upregulated,suggested that both GPAT and PDAT genes can improved the TAG in Chlamydomonas reinhardtii.We hope this study can provide a new idea in building high oil content transgenic alga.