Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae

Background: Sesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon alpha-santalene is a precursor of sesquiterpenes with relevant commercial applications. Here, we construct an efficient Saccharomyces cerevisiae cell factory for alpha-santalene production. Results: A multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product. To do so, genetic modifications were introduced acting to optimize the farnesyl diphosphate branch point, modulate the mevalonate pathway, modify the ammonium assimilation pathway and enhance the activity of a transcriptional activator. The approach employed resulted in an overall alpha-santalene yield of a 0.0052 Cmmol (Cmmol glucose)(-1) corresponding to a 4-fold improvement over the reference strain. This strategy, combined with a specifically developed continuous fermentation process, led to a final alpha-santalene productivity of 0.036 Cmmol (g biomass)(-1) h(-1). Conclusions: The results reported in this work illustrate how the combination of a metabolic engineering strategy with fermentation technology optimization can be used to obtain significant amounts of the high-value sesquiterpene alpha-santalene. This represents a starting point toward the construction of a yeast "sesquiterpene factory" and for the development of an economically viable bio-based process that has the potential to replace the current production methods.