Ursolic acid and oleanolic acid are pentacyclic triterpenoid compounds with a variety of biological activities. A mixture of ursolic acid and oleanolic acid has higher antitumor activity than the individual acids. We have developed a simultaneous biosynthesis pathway for different proportions of ursolic acid and oleanolic acid in Saccharomyces cerevisiae. The ScLCZ08 strain produced 175.15 mg/L of the ursolic acid precursor alpha-amyrin, the highest amount reported. Ursolic and oleanolic acid titers and proportions were optimized using Medicago truncatula amyrin C-28 oxidase and Arabidopsis thaliana cytochrome P450 reductase. Using glucose and ethanol fed-batch fermentation strategies, the final ursolic acid and oleanolic acid titers were 123.27 and 155.58 mg/L, respectively, demonstrating 4.77-fold and 4.95-fold higher production than the parent strain. The ScLCZ11 strain displayed the highest ursolic acid production obtained via microbial fermentation in fed-batch culture to date. (C) 2018 American Institute of Chemical Engineers