Enhancing Saccharomyces cerevisiae reactive oxygen species and ethanol stress tolerance for high-level production of protopanoxadiol

•A synergistic effect of P450-CPR uncoupling and ethanol stress on ROS releasing was spotted.•Cells viability was improved after enhancing ROS and ethanol stress tolerance.•PPD production of W3a-ssPy reached to 4.25 g/L in 5 L reactor, which is the highest yield reported.•This work made the production of PPD possible by fermentation instead of phytoextraction.

Protopanaxadiol (PPD) is an active compound in Panax ginseng. Recently, an optimized PPD synthesis pathway contained a ROS releasing step (a P450-type PPD synthase, PPDS) was introduced into Saccharomyces cerevisiae. Here reported a synergistic effect of PPDS-CPR (CPR, cytochrome P450 reductase) uncoupling and ethanol stress on ROS releasing, which reduced cells viability. To build a robust strain, a cell wall integrity associated gene SSD1 was high-expressed to improve ethanol tolerance, and ROS level decreased for 24.7%. Then, regulating the expression of an oxidative stress regulation gene YBP1 decreased 75.2% of ROS releasing, and improved cells viability from 71.3 ± 1.3% to 88.3 ± 1.4% at 84 h. Increased cells viability enables yeast to produce more PPD through feeding additional ethanol. In 5 L fermenter, PPD production of W3a-ssPy reached to 4.25 ± 0.18 g/L (19.48 ± 0.28 mg/L/OD600), which is the highest yield reported so far. This work makes the industrial production of PPD possible by microbial fermentation.