Pathway Design And Construction For Biosynthesis Of Plant Sesquiterpenoids Nootkatone In Saccharomyces Cerevisiae

Valencene and its oxidative product nootkatone belong to the family of sesquiterpene,which are composed of three molecules of isoprene.Both valencene and nootkatone have strong citrus fruit aroma and possess typical grapefruit aroma at low concentrations.They can be used as flavor in food and cosmetics industry.In addition,nootkatone has been reported to exhibit a variety of biological activities,including anticancer,antiplatelet aggregation and metabolism activation et al Thus,nootkatone has attracted great interest as a drug precursor.However,production of valencene and nootkatone through natural extraction or chemical synthesis is very inefficient,limiting their commercial applications.Our present study aims to achieve the heterogolous biosynthesis of valencene and nootkatone in Saccharomyces cerevisiae by metabolic engineering.S.cerevisiae naturally synthesizes farnesyl diphosphate,the precursor of valencene,through the mevalonate pathway.We firstly heterogenously expressed the valencenee synthase CnVS of Alaska cedar in S.cerevisiae,yielding 11.6 mg/L of valencene as quantified by gas chromatography.In order to further improve the yield of valencene,CnVS and farnesyl diphosphate synthase FPPS were expressed as a fusion protein,and a truncated form of the enzyme 3-hydroxy-3-methyl glutaryl coenzyme A reductase(tHMG1)catalyzing a rate-limiting step in the mevalonic pathway,was simutanously overexpressed.The production of valencene was significantly increased to 156.95 mg/L in flask fermentation.Furthermore,the competition pathway for FPP was down-regulated by replacing the promoter of squalene synthase ERG9 with PHXTI,which further increased the production of valencene to 217.95 mg/L.Aiming to synthesize nootkatone,we separately overexpressed a lipoxygenase LOX1 from Pleurotus sapidus and a cytochrome P450 enzyme HPO(CYP71D55)from Hyoscyamus muticus in the strains with high production of valencene.The production of nootkatol and nootkatone was only detected in the strains expressing HPO.However,the yield of nootkatone was only 9.66 mg/L while a large amout of nootkatol was produced.Then,various dehydrogenases,including ADH2 and ADH6 from S.cerevisiae,ADH-C3 from P.pastoris,ZND and CND from plants,were selected for overexpression,of which ADH-C3,ZND and CND successfully converted nootkatol to nootkatone.The yield of nootkatone was increased by five fold to 59.78 mg/L.Our present study provided a basis for the industrial production of valencene and nootkatone.