Biosynthesis Of Geraniol Based On Recombinant Saccharomyces Cerevisiae

Geraniol,a monoterpene compound,is one of the most important spices.As to Saccharomyces cerevisiae,the synthesis of terpenoids is carried out by the mevalonate(MVA)pathway,which is carried out in the cytoplasm.The recombinant S.cerevisiae in this study is the key enzyme gene of MVA pathway overexpressed in the cytoplasm,and the complete MVA pathway was constructed in the mitochondria of the strain,realizing the dul-regulation of both cytoplasm and mitochondria to produce the precursor of terpenoid synthesis.In this study,recombinant S.cerevisiae was used as the host by transplanting geraniol synthase(GES)gene.Overexpression of farnesyl diphosphate synthase(ERG20)gene and isopentenyl diphosphate isomerase(IDI)gene in cytoplasm.The effect of antioxidant of bamboo leaves on biosynthesis of geraniol by S.cerevisiae was also studied.The aim of this study is to lay a foundation for the biosynthesis of geraniol and other terpenoids.The main research results are as follows:(1)Construction and expression of the co-expression vector of GES and ERG20 genesThe GES gene sequence from Catharanthus roseus(KF561459)and the ERG20 gene sequence from S.cerevisiae were synthesized and cloned into the plasmid PESC-URA.The metabolic pathway of geranol biosynthesis was constructed by co-expression of GES and ERG20 by double promoters(Gal10 and Gal1)in pESC-URA vector.The recombinant plasmid was transformed into two recombinant S.cerevisae(BY4742-C-04 and BY4742-C-05).The results showed that the content of geranitol was 4.08 mg/L(BY4742-C-05)and 3.04 mg/L(BY4742-C-04)in fermentation broth after fermentation for 60 h.Therefore,the biosynthesis of geranitol was successfully realized through the construction of the GES and ERG20 co-expression vector.(2)Cloning and expression analysis of IDI geneThe cloning of IDI gene was based on Cinnamomum camphora plant.Reverse transcriptase-polymerase chain reaction(RT-PCR)technology was used to clone the IDI gene of C.camphora,and the protein encoded by IDI gene was characterized by bioinformatics method.The results showed that the length of the IDI gene was 868 bp,the open reading frame was 708 bp,and it encoded 235 amino acids.The molecular weight of the encoding protein was 27.04 kD,and the isoelectric point was 5.13.The secondary sequence structure of the protein was mainly a-helix.It is predicted that C.camphorum IDI is a hydrophilic stable protein located in cytoplasm without signal peptide and has the characteristic domain of isopentenyl pyrophosphate isomerase.We successfully cloned IDI gene,which laid a foundation for further study on the function of IDI gene in terpenoid biosynthesis.(3)The effect of IDI overexpression and mitochondrial MVA pathway on geraniol synthesis in S.cerevisiaeTwo S.cereviscera strains BY4742-MC-01 and BY4742-MC-02(overexpressed IDI)were used as the host,and the GES and ERG20 co-expression vector with mitochondrial targeting peptide was transferred into the two strains.The results showed that the yield of geranitol produced by the overexpression of IDI gene was significantly increased after fermentation.Two copies of GES and ERG20 were transferred to the strains that the MVA pathway constructed in mitochondria to realize the co-regulation of geranitol production in mitochondria and cytoplasm.The maximum yield of geranitol detected in fermentation broth was 10.77 mg/L.(4)Effect of antioxidant of bamboo leaves on geraniol yieldThe yield of geraniol was not increased by adding antioxidant of bamboo leaves to the medium,but inhibited the biosynthesis of geraniol with the increase of the concentration of antioxidant of bamboo leaves.In addition,an automatic online solid phase extraction-high performance liquid chromatography(online-SPE-HPLC)method was established to determine the main antioxidant components in the antioxidant of bamboo leaves.In summary,both the overexpression of IDI gene and the construction of MVA pathway in mitochondria can promote the synthesis of geraniol,and the exogenous addition of the antioxidant of bamboo leaves could inhibit the synthesis of geraniol.This study lays a foundation for the study of biosynthesis of monophilic compounds such as geraniol.