Modular Metabolic Engineering of Saccharomyces cerevisiae for Enhanced Production of Ursolic Acid

Ursolic acid, a plant-derived pentacyclic triterpenoid with anti-inflammatory, antioxidant, and other bioactive properties, holds significant potential for use in nutritional supplements and drug development. However, its extraction from medicinal plants is inefficient due to low yield and dependence on seasonality and geography. Herein, we use modular metabolic engineering to enhance ursolic acid production in Saccharomyces cerevisiae by dividing the biosynthetic pathway into five modules. First, the heterologous ursolic acid biosynthesis module was established using Catharanthus roseus α-amyrin synthase (CrMAS) and a fused α-amyrin oxidase (CrOAS) with cytochrome P450 reductase (CPR). Next, the full hybrid mevalonate pathway was overexpressed, and the copy number of CrMAS was optimized. The sterol pathway was further optimized by introducing N-degron tags to relieve the competition pathway and deleting the SSM4 gene to enhance the ERG1 stability. Acetyl-CoA supply was improved via phosphoketolase and acetyl-CoA synthase pathways, combined with fine-tuning of mitochondrial and cytosolic carbon flux. The final engineered strain produced 1083.62 mg/L of ursolic acid in shake-flask cultures and 8.59 g/L in a 5 L bioreactor via fed-batch fermentation, achieving the highest microbial ursolic acid titer reported to date. This study not only demonstrates the potential for efficient biosynthesis of triterpenoid compounds but also provides ideas that can be extended to other microbial hosts for the concentrated use of intracellular carbon sources in the synthesis of complex natural products.