S. cerevisiae has been used for millenniums to make wine, beers and bread. The rise and development of Synthetic Biology has turned many micro-organisms, especially S. cerevisiae, into microbiofactories allowing the production of a wide range of molecules from other organisms in a sustainable way using fermentation.
Evolva produces many important high value molecules from the terpene family (valencene, nootkatone, and rebD and rebM [components of the artificial sweetener Stevia]) which all use the same metabolic pathway to produce the isoprene precursor farnesyl pyrophosphate (FPP) used by terpene synthases.
We present an approach of combinatorial genome integrations of the mevalonate pathway genes into S. cerevisiae to maximize carbon flux towards terpene production. The levels of key metabolites are monitored during fermentation as an indicator of desired changes in the metabolic flux towards FPP. Flux imbalance can negatively affect terpene production, particularly when scaled-up. Hence, we applied a stepwise engineering method and took advantage of the push and pull strategy to minimize the accumulation of inhibitory concentrations of metabolites and maximize carbon flux to product formation.
We will also discuss strategies for biological formation of nootkatone from valencene. Nootkatone is not only important as a flavor and fragrance molecule but it also has potential applications in insect control. The CDC has shown that nootkatone both repels and kills ticks and mosquitos, making it a promising agent to control the spread of Lyme disease carried by the black-legged tick, Ixodes scapularis, and of the Zika virus carried mainly by the yellow fever mosquito, Aedes aegypti.