Construction And Optimization Of Sabinene Producing Yarrowia Lipolitica

Sabinene is typical monoterpenoid,which has essential applications in the field of drug,perfume and jet fuel.Yarrowia lipolytica is common model host because its lipid bodies can store lipid products and it has a broad substrate spectrum.In this way,we build the synthesis pathway of the lipid product sabinene in Y.lipolytica by strategies of key enzyme screening,prolonged mevalonate（MVA）pathway enhancement,non-pathway-related gene regulation,medium optimization,to achieve efficient synthesis of sabinene,which expands the applications of synthesizing products by Y.lipolytica.We firstly make combination screen for non-truncated and truncated sabinene synthase Sp＿（t）Sab S,ERG20^WW（mutant farnesyl diphosphate synthase of F96W/N127W）and NDPS（dimethylallyl cistransferase,neryl diphosphate synthase）,and achieve the first synthesis of sabinene in Y.lipolytica.We find that coexpressing of Sp＿t Sab S and ERG20^WW can get the highest yield of sabinene,which is 1.77 mg/L.This indicates that GPP（geranyl diphosphate）is the preferred substrate of Sp＿t Sab S comparing to NPP（neryl diphosphate）.On this basis,we separately overexpress key genes of ERG12（mevalonate kinase）and HMGR（3-hydroxy-3-methylglutaryl-Co A reductase）,and find that only overexpressing HMGR can increase the production of sabinene by 2.53 fold and reach 5.19 mg/L.The effect of non-pathway-related targets on sabinene synthesis were further investigated by overexpressing 6 non-pathway-related genes,17 genes from fatty acid pathway and 5 genes from hydrocarbon oxidation pathway.We find overexpressing targets including YALI0F30987g（CTA1,catalase A）,YALI0E00814g（AOX,alternative oxidase）and YALI0A19910g（2-phenylethanol degradation related）will significantly increase the production of sabinene,among which overexpressing YALI0F30987g can increase the production of sabinene to 15.90 mg/L.Finally,we optimize the fermentation conditions by analyzing the effect of carbon source,nitrogen source,initial inoculums,and fermentation time on the production of sabinene,and find the highest production of sabinene is 26.90 mg/L,which is the highest production of sabinene in Y.lipolytica.