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. (C) 2016 Elsevier Ltd. All rights reserved.