An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids

- A powerful strategy for engineering yeast-based metabolite production is proposed.
- The principle relies on the transformation of subcellular morphology.
- Expansion of the endoplasmic reticulum (ER) increases yeast metabolic capacity.
- Expanded ER structures can harbour higher plant ER-associated enzyme levels.
- The principle is applicable to plant-derived triterpenoids and sesquiterpenoids.

Saponins are a structurally diverse family of triterpenes that are widely found as main constituents in many traditional plant-based medicines and often have bioactivities of industrial interest. The heterologous production of triterpene saponins in microbes remains challenging and only limited successful pathway engineering endeavors have been reported. To improve the production capacities of a Saccharomyces cerevisiae saponin production platform, we assessed the effects of several hitherto unexplored gene knockout targets on the heterologous production of triterpenoids. Here, we show that the disruption of the phosphatidic acid phosphatase-encoding PAH1 through CRISPR/Cas9 results in a dramatic expansion of the endoplasmic reticulum (ER), which stimulated the production of recombinant triterpene biosynthesis enzymes and ultimately boosted triterpenoid and triterpene saponin accumulation. Compared to the wild-type starter strain, accumulation of the oleanane-type sapogenin β-amyrin, of its oxidized derivative medicagenic acid, and its glucosylated version medicagenic-28-O-glucoside was respectively increased by eight-, six- and 16-fold in the pah1 strain. A positive effect of pah1 could also be observed for the production of other terpenoids depending on ER-associated enzymes for their biosynthesis, such as the sesquiterpenoid artemisinic acid, which increased by twofold relative to the wild-type strain. Hence, this report demonstrates that pathway engineering in yeast through transforming the subcellular morphology rather than altering metabolic fluxes is a powerful strategy to increase yields of bioactive plant-derived products in heterologous hosts.