Terpenes are one of the largest groups of natural products. Sesquiterpenes (15-carbon terpenes) are secondary metabolites produced from farnesyl pyrophosphate (FPP). Although these compounds are normally found in plants, production levels are too low for commercial use. As a result, we have begun engineering the metabolic pathways of Saccharomyces cerevisiae to accumulate sesquiterpenes. Epi-cedrol is a sesquiterpene identified from Artemisia annua, which was used as a reporter molecule to monitor sesquiterpene production in our engineered yeast strains. Through manipulations of the ergosterol pathway, we were able to increase sesquiterpene production from 0.090 mg/L (wild type yeast) to 1.000 mg/L in yeast strains that overexpress the truncated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme (HMG1) gene in an erg9 background. This yeast strain also increased the in vivo production of farnesol and trans-nerolidol, the hydrolyzed products of FPP. Likewise, the use of a different resin to isolate the epi-cedrol allowed a further increase to 1.883 mg/L in this yeast strain.
Part II describes a valuable alternative to the specifically designed yeast strains described in Part I of this thesis by producing the yellow compound, 4,4'-diaponeurosporene. 4,4'-Diaponeurosporene is a deep yellow triterpene carotenoid identified in Staphylococcus aureus. CrtM and CrtN are two genes that are cotranscribed to convert FPP to this pigment. Because the CrtM/CrtN pair converts FPP to a colored compound, the color of yeast cells expressing these genes will provide a visual indication of the amount of FPP that can be diverted to foreign product biosynthesis.
Finally, Part III of this thesis describes the use of our engineered yeast strains to characterize Medicago truncatula sesquiterpene synthase products. Now that significant amounts of genomic and cDNA sequences have been determined for many organisms, genes of interest can be identified by comparison to other genes in that family. Seven genes were obtained and cloned into yeast expression vectors, transformed into our yeast strains and the products isolated. Several compounds were identified using GC-MS analysis.