Abstract

Monoterpenes and their indole alkaloid derivates are plant secondary metabolites valuable to the pharmaceutical and industrial sectors for their bioactivities in humans, cats and insects. The biosynthetic pathway to strictosidine, the central monoterpene indole alkaloid scaffold, proceeds via an industrially relevant intermediate – nepetalactol. The oxidized counterpart of this monoterpene, nepetalactone, is known for its euphoric influence on cats but is also a potent insect repellent. Unfortunately, low in planta levels of these metabolites have resulted in high production costs and environmentally unfeasible cultivation strategies for an industrial product with mass market appeal. Engineering a high-producing nepetalactol or strictosidine yeast would benefit several commercial sectors, however recent studies attempting de novo strictosidine in S. cerevisiae only achieved trace titers (0.5 mg/L). Activity of geraniol-10-hydroxylase, a cytochrome P450, was an identified bottleneck in nepetalactol/strictosidine production. This thesis presents the application of a combination of strategies aimed at overcoming the geraniol-10-hydroxylase bottleneck and improving titres of 10-hydroxygeraniol. Geraniol-10-hydroxylase and cytochrome P450 reductase variants were explored to increase enzyme efficiency, wherein several reductases were shown to beneficially influence hydroxylation activity. We further demonstrated that a cytochrome P450-reductase fusion increased carbon flux towards 10-hydroxygeraniol by 1.7-fold. Accumulation of a heterologous metabolite, putatively identified as isopulegol, was observed, implying inefficient channeling of carbon into the heterologous pathway. Deletion of two native reductases, oye2 and oye3, resulted in a 4.6-fold increase in carbon channeled to 10-hydroxygeraniol, resulting in a final accumulation of 144 mg/L of 10-hydroxygeraniol. These pathway optimization steps will aid in the development of high yielding monoterpene S. cerevisiae strains.