Construction And Optimization Of Saccharomyces Cerevisiae For Sabinene Overproduction

Due to its high energy density and heat of combustion,sabinene is expected to be developed into the next generation of biofuels.In this study,the biosynthesis pathway of sabinene was constructed in Saccharomyces cerevisiae.Through three strategies including N-terminal truncation,key enzymes fusion,product efflux enhancement and regulation of precursor geranyl pyrophosphate（GPP）supply,the highest sabinene productivity was achieved in yeast.In order to achieve the synthesis of sabinene,endogenous farnesyl diphosphate synthase（ERG20）mutant ERG20^wwand sabinene synthase from Salvia pomifera（Sp＿Sab S1）were expressed in S.c.By truncating the 34 amino acid residues at the N-terminus of Sp＿Sab S1（obtaining Sp＿t34Sab S1）,the yield of sabinene was 3.95times higher than the initial yield（0.44 mg/L）.Sabinene yield was further increased by 96.86%by co-expression of the forward fusion of ERG20^wwand Sp＿t34Sab S1 as well as another copy of ERG20^ww.Two efflux proteins from different sources were screened in strain harboring Sp＿t34Sab S1 to enhance efflux of sabinene,thereby reducing the effects of sabinene cytotoxicity to yeast.The efflux protein Gc＿ABCG1 from the Grosmannia clavigera worked best,resulting in a 1.04 fold increase in sabinene production.In order to reduce the intracellular consumption of GPP,the endogenous gene ERG20 expression was down-regulated by promoter replacement,resulting in a 1.06fold increase in sabinene production.In the meanwhile,Sp＿t34Sab S1 was co-located both in cytoplasm and certain sub-organelles（mitochondria or peroxisome）to fully utilize GPP.It was found that Sp＿t34Sab S1 could be synergistically expressed between cytoplasm and peroxisome,mitochondria and peroxisome,cytoplasm and mitochondria.It also demonstrated sabinene yield was increased by 36.74%,40.81%and 153%compared with the control strain,respectively.The co-localization of Sp＿t34Sab S1 between cytoplasm and mitochondria led to sabinene production at64.55 mg/L,which is the highest titer as known at shake flask level fiask in S.c.