Key Issues In The Metabolic Engineering Of Saccharomyces Cerevisiae For Monoterpene Production

In this dissertation, a haploid Saccharomyces cerevisiae yeast strain CEN.PK2-1C wasemployed to express genes encoding monocyclic monoterpene synthase (LIS1) and acyclicmonoterpene (GES1) from plant, and a new biosynthesis mechanism of monoterpenes in S.cerevisiae was also deduced. Metabolic engineering the mevalaonate (MVA) pathway in S.cerevisiae increased monoterpene production from0.68mg/L to44.13mg/L, whereas poortolerance of S. cerevisiae restrained the increase in monoterpene production. To construct arobust yeast strain with higher monoterpene resistance, the present study tried to uncover themechanisms that protect S. cerevisiae from monoterpene stress. The main results weredescribed as follows:1) Unlike monocyclic monoterpene synthase, acyclic monoterpene geraniol synthase couldbe directly expressed in S. cerevisiae. Coexpression of D-limonene synthase and NPPsynthase succeeded in the yield of D-limonene, whereas coexpression of NPP synthaseand geraniol synthase resulted in decreased geraniol production. The results obtained inthis study revealed that GPP is the precursor of acyclic monoterpene, and NPP is theprecursor of monocyclic monoterpene. Finally, a new mechanism of monoterpenebiosynthesis was deduced.2) Metabolic engineering key regular points of the pyruvate dehydrogenase and themevalonate pathway in S. cerevisiae increased geraniol production from0.68mg/L to15.81mg/L. Overexpress a mutated ERG20K197G, which encoding FPP synthase in S.cereviaise, further increased monoterpene geraniol production to37.86mg/L. Besides, theoverexpression of MAF1, encoding a negative regulator of the biosynthesis of tRNA,further increased geraniol production to44.13mg/L.3) The limited tolerance of S. cerevisiae to monoterpenes restrained efficiency yield of thesecompounds. To construct a robust yeast strain, microarray was conducted to investigatethe mechanisms that S. cerevisiae employed in resistance to monoterpene stress. Theresults revealed genes encoding energy, NADPH regeneration, antioxidant enzymes andDNA repairment were upregulated at transcription level. Besides, genes encoding thebiosynthesis of membrane and redox regulation upregulated at transcription level undermonoterpene stress. Thus, it could be deduced that monoterpene treatment inducedoxidative stress in S. cerevisiae.4) Exogenous ergosterol enhanced survival of S.cerevisiae to monoterpene D-limonene stress by recover membrane impairment. qPCR revealed the upregulation of genes encoding thebiosynthesis of ergostreol. Intracellular ergosterol concentration and ability of S.cerevisiae resistance to monoterpene stress exhibited an concentration-depended manner.5) The physiological changes in monoterpene treated S. cerevisiae cells suggested it has beensuffered oxidative stress. Further research verified that monoterpene induced reactiveoxygen species (ROS) accumulation and lead to cell apoptosis. In response to scavengeROS, S. cerevisiae activated its antioxidant mechanisms. Besides, more NADPH wassynthesized to fulfill increased consuming of reducing agents and cofactors needed forantioxidant mechanisms. qPCR also verified the upregulation of genes involved inantioxidant machanisms in S. cerevisiae.