Abstract

The production of biologically active natural products through microbial metabolic factories, such as Saccharomyces cerevisiae, represents a promising approach to achieving green and sustainable production. However, establishing a stable metabolic flux for natural products by disrupting the natural evolutionary pathways of S. cerevisiae remains a significant challenge. This phenomenon is often associated with strain growth stagnation, presenting a key bottleneck in metabolic engineering. Here, we successfully devised a recombinant S. cerevisiae chassis strain capable of producing levopimaradiene (LP), a critical precursor for ginkgolide biosynthesis. By systematically modifying levopimaradiene synthase (LPS), modulating the mevalonate pathway, and enhancing the supply of acetyl-coenzyme A, ATP, and NADPH, we significantly improved the titer of LP. Shake flask fermentation achieved a LP titer of 792.72 ± 69.59 mg/L, while fed-batch fermentation further elevated the titer to 1809.32 ± 72.32 mg/L. This represents a 8.3-fold increase over the previously reported S. cerevisiae chassis strain, corresponding to the highest titer reported to date. This study provides a strategy for the construction of ginkgolide chassis factories. Moreover, it offers a conceptual framework for the construction of factories for the production of diterpene, with a particular focus on the field of S. cerevisiae production.