(256e) Engineering Saccharomyces Cerevisiae for Production of Iridoids

Iridoids are a precursor to the medicinally essential monoterpene indole alkaoids (MIAs), which include the anticancer compounds vinblastine and camptothecin. Due to low in planta production of many MIAs, a microbial production strategy could provide a production route towards clinically critical molecules that is both economically superior and environmentally sustainable. However, due to the complexity of the MIA pathway, microbial biosynthesis requires extensive engineering to increase the production of the MIA iridoid precursor nepetalactol. Here, we describe the strategies that we have employed to improve iridoid production in the biosynthetic workhorse Saccharomyces cerevisiae. We have developed a bioconversion-based production approach which leverages the facile organic synthesis of a biosynthetic intermediate to circumvent several metabolic bottlenecks. Using this platform, we elucidated and reconstituted two distinct shunt pathways in which endogenous yeast metabolism interferes with generation of the iridoid scaffold. Subsequent rational metabolic engineering using CRISPR/Cas9 enabled a 5.2-fold increase in the production of nepetalactol relative to a dead-end shunt product. We further describe the engineering strategies that are being investigated to establish scalable microbial production of iridoids and MIAs.