Metabolic Engineering Of Saccharomyces Cerevisiae For Geraniol Production

Terpenoids are a class of secondary metabolites that are primarily derived from plants.Generally,they are oxygenated derivatives whose molecular formulas are multiples of isoprene units.Terpenoids are widely used in the pharmaceutical,food,perfume and cosmetics industries that have a high economic value.The development of synthetic biology makes the use of microorganisms to produce terpenoids as a simpler,more efficient and cost-effective way of production.In this dissertation,the isoprenoid metabolic pathways of Saccharomyces cerevisiae were regulated by molecular biology techniques and metabolic engineering control techniques.The supply of precursors at each step was increased makes an obvious increasing of terpenoilds production.In this study,the acyclic monoterpene geraniol synthase gene was used to verify the enhancement of the synthesis ability of metabolic engineered Saccharomyces cerevisiae.The main conclusions are as follows:(1)In this dissertation,we used the shuttle vector pCTCON-dual-intra-Metab v1 with Gal1/Gal10 dual promoters as an original vector to construct a series of yeast genomic integration vectors.pINT1-TRP1-ERG8-ERG19-24 REC,pINT1-URA3-ERG13-ERG10-20 REC,pINT1-HIS3-mvaA-ERG12-21 REC,pINT1-LEU2-ERG20-IDI1-19 REC.The yeast genomic integration sites are chosen according to reported high expression yeast genomic integration site.These vectors can be integrated into the Saccharomyces cerevisiae genome for metabolic engineering after linearization and can be integrated in Saccharomyces cerevisiae after replacement of other metabolically regulated genes which providing a basis for the metabolic engineering of Saccharomyces cerevisiae.(2)In this study,we used S.cerevisiae CEN.PK2-1C as a starting strain.A series of metabolically engineered strains were obtained by integrating the yeast genomic integrated expression vectors above into yeast genome.The results of the relative expression of integrated genes in engineered yeast showed that the expression levels of the target genes ERG8,ERG19,ERG13,ERG10,mvaA,ERG12,ERG20,and IDI1 were increased by 18.51-fold,18.38-fold,8.40-fold,8.75-fold 1472667.19-fold,372.22-fold,160.90-fold and 91.77-fold respectively.(3)In order to evaluate the ability to produce terpenoilds of metabolically engineered yeasts is higher than original strain or not.We introduce a geraniol synthetase gene(GES)derived from sweet basil into the original strain and metabolically engineered yeasts.MG10 strain produced 3.08 mg/L geraniol after galactose-induced culture.The original strain and other metabolically engineered yeast can not detect any geraniol production.