A variety of different applications render terpenes and terpenoids attractive research targets. A promising but so far insufficiently explored family of terpenoids are the fusicoccanes that comprise a characteristic 5-8-5 fused tricyclic ring system. Besides herbicidal effects, these compounds also show apoptotic and anti-tumour effects on mammalian cells. The access to fusicoccanes from natural sources is scarce. Recently, we introduced a metabolically engineered Saccharomyces cerevisiae strain to enable the heterologous fermentation of the shared fusicoccane-diterpenoid precursor, fusicocca-2,10(14)-diene. Here, we show experiments towards the identification of bottlenecks in this process. The suppression of biosynthetic by-products via medium optimisation was found to be an important aspect. In addition, the fermentation process seems to be improved under oxygen limitation conditions. Under fed-batch conditions, the fermentation yield was reproducibly increased to approximately 20 mg/L. Furthermore, the impact of the properties of the terpene synthase on the fermentation yield is discussed, and the preliminary studies on the engineering of this key enzyme are presented.
Keywords: anti-tumour; directed evolution; enzyme engineering; heterologous fermentation; metabolic engineering; terpenes.