Lipase Subcellular Localization For Biodiesel Production Through Biocatalysis And Biosynthesis

Biodiesel?the main component is fatty acid methyl ester?is a representative of biofuels,and its biocatalysis and biosynthesis are one of the focuses of academic and industrial.At present,the biocatalysis of biodiesel is mostly adopteds the two-step strategy including separating lipase catalyst preparation and transesterification reaction,but the reaction process is susceptible to inhibition of by-product glycerol.And biosynthesis of biodiesel is mainly synthesized de novo in the cytoplasm of Escherichia coli or Saccharomyces cerevisiae?with limited oil production?by modifying fatty acid metabolism pathways.In this study,in order to solve the current constraints in the biocatalysis and biosynthesis of biodiesel,oleaginous Yarrowia lipolytica was used as host for lipase biocatalysis in vitro and biosynthesis in vivo to construct a new in situ carbon source system for the integration of lipase surface display and lipase-catalyzed biodiesel production,and a novel system for biosynthesis of biodiesel by subcellular localization of lipase.These provide new ideas for the biocatalysis and biosynthesis of biodiesel.In the present study,Thermomyces lanuginosus lipase?TLL?and Y.lipolytica expression vector were combined and respectively located on the surface of yeast cell wall,cytoplasm and organelles related to oll metabolism?endoplasmic reticulum,peroxisome and lipid droplet?,adding methanol in the shake flask fermentation process?the surface display system also requires the addition of oil?to evaluate the titer of FAMEs from biocatalysis and biosynthesis.The main research results are as follows:?1?TLL was localized on the surface of the yeast cell wall by anchoring protein CWP110,and the lipase expression was integrated with the catalytic biodiesel in the shake flask fermentation process by adding additional oil and methanol.By-product glycerol could further improve cell growth and lipase expression as an in situ carbon source.The consumption of glycerol would reduce the feedback inhibition of the product and improves the reaction to generate FAMEs,thereby an integrated biocatalytic system for the continuous synthesis of FAMEs with glycerol as the in situ carbon source is formed.Compared with surface-displayed resting cells,the conversion rate of FAMEs in the in situ carbon source integration system increased from 81%to 91%,the content of by-product glycerol decreased from 14%to 3%,and the cell density(OD₆₀₀)increased from 62 to 71,the enzyme activity increased from 1.21 U/mg to 1.58 U/mg,indicating that the in situ carbon source integration system can effectively catalyze the synthesis of biodiesel.?2?TLL was localized to the cytoplasm,peroxisome,endoplasmic reticulum,and lipid droplet by specific tags to construct four engineering strains.FAMEs were catalyzed by lipase in yeast using externally added methanol and endogenous oils.The results showed that when the lipase was localized to the organelles,the conversion rate of FAMEs was higher than that of lipase in the cytoplasm.Moreover,the conversion rate and titer of FAMEs were the highest when the lipase was localized to the lipid droplet,9.72% and 23.81mg/L respectively.It is shown that lipase subcellular localization is morebeneficial to the catalytic synthesis of biodiesel compared to lipase cytoplasm localization.?3?The titer of FAMEs can be improved by increasing the oil content of Y.lipolytica.There are three key enzymes in the metabolic pathway of oil accumulation:acetyl-CoA carboxylase-the first step in catalyzing the synthesis of fatty acids;diacylglycerol acyltransferase-the last step in catalyzing the synthesis of Triacylglycerol?TAG?;glyceraldehyde 3-phosphate dehydrogenase,which plays a key role in the synthesis of TAG;The co-expression of three key enzymes increased the accumulation of oil from 14.18mg/g to 25.33mg/g.After the oil content was increased,TLL was localized to the cytoplasm and organelles to compare the titer of FAMEs.The results showed that the highest titer of FAMEs was reached 41.72 mg/L when the TLL was localized to the lipid droplet.