(596d) Rational Protein Engineering to Identify a High 8-Hydroxy Geraniol Producing Strain for Nepetalactol Production in Yarrowia Lipolytica

Nepetalactol is an iridoid which is widely used as an insect repellant. Moreover, it is a key intermediate to over 3,000 monoterpene indole alkaloids which are of pharmaceutical importance. An important step in the nepetalactol pathway is the conversion of geraniol to 8-hydroxy geraniol (8-OH geraniol) by the action of the enzyme geraniol 8-hydroxylase (G8H) which is a P450 enzyme and often difficult to express in heterologous hosts. In a previous report of strictosidine production (Brown et al., 2015), G8H was identified as the major bottleneck in the strictosidine pathway expressed in S. cerevisiae. Hence, we have G8H from multiple plant variants. Our preliminary results indicate no conversion of geraniol to 8-OH geraniol when geraniol was spiked in the cultures. Therefore, we engineered multiple geraniol producing strains through directed integration in Yarrowia lipolytica by overexpressing mevalonate (MVA) pathway genes such as ERG10, HMGS or MVK, PMK and increasing the geraniol synthase (GES) gene copy number. The highest geraniol producing strain will be transformed with G8H variants. G8H, being a P450 enzyme, requires an additional reductase partner and in some cases cytochrome b5 (CYB5) in order to achieve full activity. These enzyme variants are also being fused and co-expressed (with and without flexible GS linkers) with cytochrome P450 reductase and CYB5. The best variant will be chosen on the basis of higher conversion of geraniol to 8-OH geraniol and will be integrated into the geraniol producing strain to obtain an 8-OH geraniol producing strain. The best variant will also be used for docking simulations based on docking of geraniol in the G8H enzyme catalytic site. Highly conserved sites identified from simulations will be used to create a site-saturation mutagenesis library and plate-based screening will be performed to find the best mutant with highest 8-OH geraniol activity. The best mutant will be integrated as an additional copy in the previous 8-OH geraniol producing strain through directed integration. Finally, enzymes downstream of geraniol 8-hydroxylase (GOR and ISY) will be integrated into the final strain to produce nepetalactol in Yarrowia lipolytica.