Ethanol is a more reduced substrate than sugars. Here, C-13-metabolic flux analysis (MFA) revealed that ethanol catabolism could supply sufficient acetyl-CoA and reducing equivalent for PPD biosynthesis. Then, we described modular engineering strategy to optimize a multigene pathway for protopanaxadiol (PPD) production from ethanol in Saccharomyces cerevisiae. PPD biosynthesis was divided into four modules: mevalonate (MVA) pathway module, triterpene biosynthesis module, sterol biosynthesis module, and acetyl-CoA formation module. Combinatorially overexpressing every gene in MVA pathway and optimizing metabolic balance in triterpene biosynthesis module led to significantly enhanced PPD production (42.34 mg/L/OD600). In sterol biosynthesis module, fine-tuning lanosterol synthase gene (ERG7) expression using TetR-TetO gene regulation system enabled further production improvement (51.26 mg/L/OD600). Furthermore, increasing cytoplasmic acetyl-CoA supply by overexpressing a Salmonella ACS (acetyl-CoA synthetase gene) mutant ACS(seL641P) improved PPD production to 66.55 mg/L/OD600. In 5 L bioreactor, PPD production of the best-performing strain WLT-MVA5 reached 8.09 g/L, which has been the highest titer of plant triterpene produced in yeast. (c) 2018 American Institute of Chemical Engineers AIChE J, 65: 866-874, 2019