Engineering yeast subcellular compartments for increased production of the lipophilic natural products ginsenosides

Eukaryotic yeasts have a variety of subcellular compartments and are ideal platform strains for the construction of complex heterologous natural product biosynthesis pathways. Improving the synthesis efficiency of microbial cell factories through the utilization and modification of subcellular compartments by synthetic biology has good application prospects. Here, we used the yeast PLN1 protein to target the normally endoplasmic reticulum (ER)-localized cytochrome P450 enzyme protopanaxadiol (PPD) synthase (PPDS) to lipid droplets (LDs), which are the storage organelles of the PPDS substrate dammarenediol-II (DD). The efficiency of converting DD to PPD was significantly increased by 394%, and the conversion rate of DD increased from 17.4% to 86.0%. Furthermore, increasing the volume of LDs can significantly enhance the production of DD and its derivatives, but the change in the ratio of the volume and surface area of LDs decreased the conversion efficiency of DD to PPD. Additionally, the biosynthetic pathways of the PPD-type saponin ginsenoside compound K (CK) was reconstituted in a PPD-producing chassis strain, and CK production reached 21.8 mg/L/OD, 4.4-fold higher compared to the native ER-expression strategy. Next, we enhanced the expression of the Pn3-29 gene module to further reduce the accumulation of PPD and increase the production of CK to 41.3 mg/L/OD. Finally, the CK titer of the resulting strain reached 5 g/L in 5 L fed-batch fermentations. This study provides a new strategy for engineering yeast to produce complex natural products.