Efficient Production Of α-santalene In Metabolically Engineered Saccharomyces Cerevisiae

Z-(+)-α-santalol is the main integredient of sandalwood oil which is one of the most expensive fragrents in the world.To achieve the efficient production of a-santalene,the direct precursor of santalol,this study focused on constructing the biosynthesis pathway of a-santalene in engineered Saccharomyces cerevisiae.First,we investegated the effficiency of CRISPR/Cas9 system in S.cerevisiae.The result showed that the deletion efficiency of the target fragments ranged from 2000 bp to 7000 bp could reach 100%using homologous arms of 200-500 bp.Using 40 bp homologous arms could effectively achieve the insertion of 1000-10000 bp fragments.To enhance the MVA pathway,the expression cassettes of tHMG1 and ERG20 were inserted into DPP1 and LPP1 loci respectively in both S.cerevisiae CEN.PK2-1C and BY4741.Then we construsted biosynthesis pathway of a-santalene in S.cerevisiae by expressing heterogeneous santelene synthase and investegated the influence of promoter strength,host cell,and vector copy number on santalene production.The results showed that the highest yiled of a-santalene was 12.7 mg/L in strain SCN02 using TEF1 promoter and high copy number vector.Further,the downstream squalene biosynthesis pathway was down-regulated,and the yield of a-sandalene was improved to 24.7 mg/L.Finally,we screened 5 translocation tags which target mitochondria with high efficiencies,and constructed a-santalene synthesis pathway in mitochondria using these tags.Santalene yield reached 25 mg/L in strain CEN.PK2-1C.The result proved that subcellular structural localization of metabolic pathway could effectively improve the production of target products.In conclusion,this study successfully combined metabolic engineering technology and CRISPR/Cas9 gene editing technology to construct the biosynthesis pathway ofα-santalene in S.cerevisiae.Moreover,high α-santalene production could be further obtained with the harnessing of yeast mitochondria.