Engineering Acetyl-CoA Synthetic Pathway And Its Application In Geraniol Production In Saccharomyces Cerevisiae

Monoterpenes are isoprenoids with important industrial application,which widely exist in microorganisms,plants and insects.Geraniol,an acyclic monoterpene alcohol,is mainly used in food,cosmetics.It has been wildly applied as anti-cancer drug,biopesticide,antimicrobial reagent,biofuel and so on.In our previous work,the geraniol production was improved to 1.69 g/L through modular metabolic engineering,which represents the highest titer ever reported in engineered yeast.However,compared with sesquiterpenes,the synthesis of monoterpene is relatively lagging behind in Saccharomyces cerevisiae.There are two limited factors resulting in a lower production of monterpenes:(1)the shortage of precursors acetyl-CoA and geranyl pyrophosphate(GPP);(2)the toxicity of monoterpenes to cell.The present work is focused on improving the supply of cytosolic acetyl-CoA for increased geraniol production by introducing heterologous biosynthetic pathway of acetyl-CoA and regulating inherent metabolic pathway.The main results of this study are showed as follows:(1)In S.cerevisiae,acetyl-CoA is the starting compound of the mevalonate(MVA)pathway,which could provide the precursor GPP for production of monoterpenes.Therefore,the sufficient supply of acetyl-CoA is an important basis for improving the synthesis of terpenoids.However,the acetyl-CoA is highly compartmentalized in the cytosol,mitochondrion,peroxisome and nucleus,and they cannot be transported among different compartments.To increase accumulation of cytosolic acetyl-CoA,the heterologous citrate lyase(ACL),which catalyzes citrate into acetyl-CoA and oxaloacetate was introduced into cell.ACLs genes from three different sources(Yarrowia lipolytica,Mus muculus and Aspergillus nidulans)were compared in a previous geraniol-producing strain.However,the introduction of ACL did not significantly improve geraniol production.We speculated that insufficient substrate for ACL is a limited factor.(2)To increase accumulation of cytosolic citrate,the key genes from metabolic pathway of citrate were deleted,and we found that ICL1 deletion could improve geraniol production and yield with 34% and 83%,respectively.Furthermore,double deletion of ICL1 and IDH1 improved geraniol production and yield with 63% and 90%,respectively.(3)To further increase accumulation of cytosolic citrate and cyclic utilization of oxaloacetate generated from citrate lyase pathway.We enhanced transportation of citrate and oxaloacetate between cytoplasm and mitochondria.We found that the overexpression of YHM2 encoding a bidirectional transporter improved geraniol production with 1 fold in double deletion strain of ICL1 and IDH1.However,overexpression of CTP1 encoding a mitochondrial citrate transporter could not improve geraniol production.In addition,overexpression of PCK1(encoding phosphoenolpyruvate carboxykinase)and PYK1(encoding pyruvate kinase),which facilitated regeneration from oxaloacetate to pyruvate,resulted in an increase of geraniol yield by 84% in strain with ICL1 deletion.In conclusion,to increase cytosolic acetyl-CoA,we introduced a heterologous citrate lyase pathway and then engineered the citrate metabolism through regulating transportation of citrate and cyclic utilization of oxaloacetate,and geraniol production was improved by 1fold.In present study,the strategies and the constructed strains will provide a useful platform and technical reference for synthesis of other high-value compounds that required the precursor acetyl-CoA.