Background: Propionate is widely used as a preservative in the food and animal feed industries. Propionate is currently produced by petrochemical processes, and fermentative production of propionate remains challenging. Methods and Results:In this study, a synthetic propionate pathway was constructed in the budding yeast Saccharomyces cerevisiae, for propionate production under aerobic conditions. Through expression of tdcB and aldH from Escherichia coli and kivD from Lactococcus lactis, L-threonine was converted to propionate via 2-ketobutyrate and propionaldehyde. The resulting yeast aerobically produced 0.21 g L -1 propionate from glucose in a shake flask. Subsequent overexpression of pathway genes and elimination of competing pathways increased propionate production to 0.37 g L -1 . To further increase propionate production, carbon flux was pulled into the propionate pathway by weakened expression of pyruvate kinase (PYK1), together with overexpression of phosphoenolpyruvate carboxylase (ppc). The final propionate production reached 1.05 g L -1 during fed-batch fermentation in a fermenter. Conclusions and Implications:In this work, a yeast cell factory was constructed using synthetic biology and metabolic engineering strategies to enable propionate production under aerobic conditions. Our study demonstrates engineered S. cerevisiae as a promising alternative for the production of propionate and its derivatives.

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Odd‐chain fatty acids (OCFAs) and their derivatives have attracted increasing attention due to their wide applications in the chemical, fuel, and pharmaceutical industry. However, most natural fatty acids are even‐chained, and OCFAs are rare. In this work, a novel pathway was designed and established for de novo synthesis of OCFAs via 3‐hydroxypropionic acid (3‐HP) as the intermediate in Saccharomyces cerevisiae. First, the OCFAs biosynthesis pathway from 3‐HP was confirmed, followed by an optimization of the precursor 3‐HP. After combining these strategies, a de novo production of OCFAs at 74.8 mg/L was achieved, and the percentage of OCFAs in total lipids reached 20.3%, reaching the highest ratio of de novo‐produced OCFAs. Of the OCFAs produced by the engineered strain, heptadecenoic acid (C17:1) and heptadecanoic acid (C17:0) accounted for 12.1% and 7.6% in total lipid content, respectively. This work provides a new and promising pathway for the de novo bio‐production of OCFAs.