Functional Identification Of Nerolidol Synthases And Reconstruction Of Nerolidol Synthesis Pathway

Terpenoids are the most abundant type of plant volatiles and common components of plant odor and volatile compounds.Nerolidol(3,7,11-trimethyldodeca-1,6,10-trien-3-ol)is a natural sesquiterpene that exists in various plants with floral fragrance.Although nerolidol can be synthesized through chemical reactions or extracted from natural plants,these two methods have the disadvantages of high cost and low yield.With the rapid development of metabolic engineering and synthetic biology,it has become an important trend of natural product biosynthesis using heterologous biosynthesis of model strain S.cerevisiae.In the previous work of my research graoup,through the transcriptomics analysis of Celastrus angulatus Max.,two gene sequences CaNES1 and CaNES2 annotated as nerolidol synthase were screened out.In this study,the coding sequences of these two enzymes were first analyzed by bioinformatics.The results show that CaNES1 and CaNES2 are highly homologous to the sequence of Tripterygium nerolidol synthase,and contain the highly conserved region of the terpene synthase subfamily TPS-g family.CaNES1 and CaNES2 can be preliminarily determined as nerolidol synthetases which located in cytoplasm according to their subcellular localization prediction.Furthermore,we constructed CaNES1 and CaNES2 recombinant strains of S.cerevisiae and E.coli,and the functional identification of CaNES1 and CaNES2 was completed through in vivo and vitro expression experiments.The vivo results show that the recombinant S.cerevisiae strains of CaNES1 and CaNES2 can produce nerolidol at 2.08 mg/L and 6.36 mg/L,respectively;the vitro results showed that CaNES1 and CaNES2 had catalytic activities for the formation of nerolidol/linalool from the sesquiterpene precursor FPP or the monoterpene precursor GPP.Finally,we used the self-assembly system of S.cerevisiae and CRISPR-Cas9 technology to optimize the endogenous MVA pathway,and the higher enzymatic activity of nerolidol synthase CaNES2 was selected to characterize the chassis cells,the nerolidol heterogeneous synthesis in S.cerevisiae was completed.Through glucose-feeding fermentation,150 mg/L nerolidol can be synthesized in S.cerevisiae,and the preliminary construction of nerolidol engineering strain is realized.This experiment laid an important foundation for the heterologous biosynthesis of other sesquiterpene compounds in S.cerevisiae.