Abstract

The monoterpene indole alkaloids (MIAs) are a valuable family of chemicals that include the anti-cancer drugs vinblastine and vincristine. These compounds are of global significance – appearing on the World Health Organization’s list of model essential medicines – but remain exorbitantly priced due to low in planta levels. Chemical synthesis and genetic manipulation of MIA producing plants such as Catharanthus roseus have so far failed to find a solution to this problem. Synthetic biology holds a potential answer, by building the pathway into more tractable organisms such as Saccharomyces cerevisiae. Recent work has taken the first steps in this direction by producing small amounts of the intermediate strictosidine in yeast. In order to help improve on these titers, we aimed to optimize the early biosynthetic steps of the MIA pathway to the metabolite nepetalactol. We combined a number of strategies to create a base strain producing 11.4 mg/L of the precursor geraniol. We also show production of the critical intermediate 10-hydroxygeraniol and demonstrate nepetalactol production in vitro. We demonstrate that activity of the Iridiod synthase towards the intermediates geraniol and 10-hydroxygeraniol results in the synthesis of the non-productive intermediates citronellol and 10-hydroxycitronellol. As a means of improving metabolic flux in this system, we also experimented with the use of protein scaffolds to co-localize vulnerable pathway enzymes together. We demonstrate that protein scaffolds successfully bind their targets in vivo, and as a test case showed we could raise the production of citronellol up to 57.8%. These discoveries have serious implications for the reconstruction of the MIA in heterologous organisms.