Exploration of In Situ Extraction for Enhanced Triterpenoid Production by <scp> <i>Saccharomyces cerevisiae</i> </scp>

Plant-derived triterpenoids are in high demand due to their valuable applications in cosmetic, nutraceutical, and pharmaceutical industries. To meet this demand, microbial production of triterpenoids is being developed for large-scale production. However, a prominent limitation of microbial synthesis is the intracellular accumulation, requiring cell disruption during downstream processing. Destroying the whole-cell catalyst drives up production costs and limits productivity and product yield per cell. Here, in situ product extraction of triterpenoids into a second organic phase was researched to address this limitation. An organic solvent screening identified water-immiscible isopropyl myristate as a suitable in situ extractant, enabling extraction of up to 90% of total triterpenoids from engineered Saccharomyces cerevisiae. Combining isopropyl myristate and β-cyclodextrins improved extraction efficiency. In a first configuration, repeated batch fermentation with sequential product extraction and cell recycling resulted in 1.8 times higher production than a reference fermentation without in situ product extraction. In the second configuration, yeast cells were in contact with the second organic phase throughout a fed-batch fermentation to continuously extract triterpenoids. This resulted in 90% product extraction and an extended production phase. Further improvement of triterpenoid production was not achieved due to microbial host limitations uncovered through omics analyses.