Production of the anticancer drug intermediate strictosidinic acid in engineered yeast

Strictosidinic acid is a key intermediate in the biosynthetic pathway of camptothecin, a plant alkaloid that serves as a precursor for semisynthetic anticancer drugs. At the moment, camptothecin is mainly sourced from trees, causing limited supply and high costs. Improving access to strictosidinic acid would help to elucidate yet unknown biosynthetic steps downstream of this intermediate and in the long term enable sustainable production of camptothecin in heterologous hosts. While structurally similar to the common monoterpene indole alkaloid precursor strictosidine, strictosidinic acid has not been the target of metabolic engineering efforts before. Here, we present a strategy to produce strictosidinic acid from glucose and tryptophan in engineered yeast. First, we create a basic strain that generates 75 mg/L strictosidine. We further optimise this strain by introducing a membrane steroid binding protein and a second copy of the farnesyl pyrophosphate synthase mutant gene ERG20^WW, boosting strictosidine levels by 5.5-fold to 398 mg/L. At these higher titres, a previously overlooked shunt product, (2E,6E)-2,6-dimethylocta-2,6-dienedioic acid (DOA), was identified that diverts flux from the pathway. Lastly, we reprogrammed our strictosidine strain to strictosidinic acid production by four genomic modifications. Final fed-batch cultivation in shake flasks resulted in 843 mg/L strictosidine or 548 mg/L strictosidinic acid, respectively, after 168 h. Taken together, our work now grants access to strictosidinic acid by metabolic engineering, while revealing strategies to further enhance the production of strictosidine and related monoterpene indole alkaloids. These findings will help to produce plant alkaloids in microbial cell factories in the future at scale.