The Construction And Metabolic Engineering Of Valencene Cell Factory

Valencene,a terpenoid existed in various citrus fruits,was widely used in perfume,soap,food and beverage industry for its great biological activity and aroma.However,the nature concentration of valencene is extremely low,and current strategy to obtain valencene is complicated and expensive,which limited the extensive application of valencene.Construction of cell factory for valencene biosynthesis will be a more efficient and environmentally friendly strategy.Saccharomyces cerevisiae,a generally recognized as safe(GRAS)eukaryotic microorganisms,has been widely used in the biosynthesis of various natural compounds.The endogenous terpenoid synthetic pathway,Mevalonate(MVA)pathway,in S.cerevisiae makes it an ideal platform for construction of valencene cell factory.The valencene synthase from Callitropsis nootkatensis(CnVS)was introduced into S.cerevisiae strain firstly,resulting that the strain could synthesize valencene by using simple carbon source,and the initial yield of this strain was 1.8 mg/g DCW(dry cell weight).To increase the valencene yield,our study remoulded MVA pathway using CRISPR/Cas9 system.But this system will be limited by selection marker in gRNA expression plsmid when it comes to multiple genome editing.To solve this problem,a recyclable plasmid based on Cre/loxP system was constructed in this study.The lost of recyclable plasmid can be achieved through galactose induction,resulting in the recycle of selection marker,which was good for the next round genome editing.Based on the recyclable plasmid,gRNA expression plasmids targeting different genes were constructed to proceed modification of MVA pathway,including down-regulation of erg9,knock-out of rox1 and bts1,was proceeded using recyclable CRISPR/Cas9 system in this study.31 mutant strains involving 5 different gene loca were obtained,and the mutant combination of erg9 and rox1 showed the highest yield in valencene,achieving 5.65 mg/g DCW.The key genes in farnesyl pyrophosphate(FPP)synthetic pathway were overexpressed in yeast genome to increase the supply of valencene precursor FPP,and the yield came to 9.35 mg/g DCW.Except increasing the metabolic flux to valencene,the capacity of valencene synthase is also the limiting factor of valencene yield.A series of valencene expression cassette containing different promoters and terminators were constructed in this study.The expression cassette containing promoter HXT7 showed the best performance in valencene yield in shake flask fermentation.A yield of 22.67 mg/g DCW was achieved through introducing the best expression cassette into overexpression strain.To increase the valencene yield furthermore,fed-batch fermentation of the strain containing best yield was performed in bioreactor and the highest yield reached 539.32 mg/L(99.06 mg/g DCW).Compared to initial strain,a 55-fold increase in valencene yield was obtained.Overproduction of valencene in S.cerevisiae was realized by combination of different metabolic strategies in this study,providing good example for production of other high value compounds in microorganism.