Lipid Biosynthesis In The Engineered Yarrowia Lipolytica

With the increasing scarcity of fossil resources,microbial oil as the new renewable resource,has been gained much attention.Among the oleaginous microorganisms,the oleaginous yeast has the advantages of fast growth rate,large biomass,high lipid content and so on.However,the effect of fatty acid desaturase to the lipid synthesis in oleaginous yeast is not clear.The previous study showed that expression of SCD in Yarrowia lipolytica,the cell grew rapidly and the lipid production increased significantly.The fatty acid synthesis pathway comprises a variety of desaturases with substrate specificities in different carbon position in fatty chain and lipid moiety.It is essential to consider SCD is a specificity enzyme or a class desaturase towards liquid accumulation.This study construct the high-yield lipid strain YL-AD10 through a combination of three genetic engineered;based on the engineered strain,explore the key role of fatty acid desaturase(SCD,12D,15D)on lipid synthesis;and explore the fermentation in a 5-L bioreactor.The main results were as follows:(1)Based on the homologous recombination,we delete the pex10,mfe gene in Y.lipolytica polf to block the degradation pathway of fatty acid.The results shows that the pex10-strain accumulate more intracellular lipid content than mfe-strain,which yield a 1-fold increase in lipid content over the control strain,to a lipid content of 15.5%of dry cell weight(DCW);and the inhibitory effect on strain growth is relatively small.We then used the pex10-platform for overexpression of diacylglycerol acyltransferase(DGA1),the final step of triglyceride(TAG)synthesis pathway,increased of 47%over the control strain pex10-,or 20.5%lipid content;and overexpression of DGA1 have little influence on the composition of fatty acids.Next we used the platform of pex10-,DGA1+ for overexpression of acetyl-CoA carboxylase(ACC1)yielded the strain YL-AD10,which featured a 32%increase of lipid content over control,to a lipid content of 29.87%DCW,and the fatty acid composition changed little.(2)To explore the effect of fatty acid desaturase on the lipid production and fatty acid composition,overspression of delta-9 stearoy-CoA desaturase(SCD),12D and 15D(exogenous,form flax)in YL-AD10(pex10-,DGA1+,ACC1+)showed that the three desaturases intracellular lipid content increased.The maximal lipid content of 37.56%is obtained,when overexpression of 15D.And the linoleic acid production increase significantly from 18.2%to 23.6%,while simultaneously the oleic acid(OA)reduce slightly.Furthermore,we showed that increasing of the cobies of fatty acid desaturase towards the increasing of lipid production.Simultaneous overexpression of 12D and 15D yielded the engineered strain(YL-12D15DA)exhibiting the lipid overproduction 48.7%DCW,increased of 63%over the control strain YL-AD10.(3)The lipid production characteristics of the engineered strain YL-12D15DA and the control polf were explored in a 5-L bioreactor fermentation,achieving 33.7%and 7.7%lipid content after 110 h,respectively.From the imaging analyes of the morphology of the stains,the control showed two atypial phenotypes during the log phase culture;the YL-12D15DA cells retaind sphericalphenotype and lipid body preserve approximately 3/4rd cell volume with intacellular oil.This study constructed the high-yield lipid strain YL-AD 10 through the genetic engineered;On the basis of the engineered strain overexpression of fatty acid desaturase(12D,15D,SCD)found that the lipid production significantly increased,and simultaneous overexpression of 12D and 15D yielded the engineered strain YL-12D15DA exhibitied more efficient lipid synthesis.The study laid the foundation of better understanding of lipid metabolism and the industrialization of microbial oils.