Combinatorial metabolic engineering for overproduction of squalene in Saccharomyces cerevisiae

Abstract

Squalene, a long-chain unsaturated triterpene compound, finds extensive applications in the food, pharmaceutical, and cosmetic industries owing to its distinctive molecular structure. Traditional methods of squalene production involve extraction from shark liver and plant oils, practices that are neither environmentally sustainable nor economically viable. In light of these challenges, microbial biosynthesis has emerged as a promising alternative, offering significant environmental and economic benefits. In this study, a combinatorial metabolic engineering approach was utilized to modify Saccharomyces cerevisiae for the efficient biosynthesis of squalene. Firstly, by introducing an exogenous NADH-dependent HMGR and enhancing the endogenous synthesis pathways, resulting in a squalene titer of 56.5 mg/L. Subsequently, by integrating the mevalonate synthesis pathway into the mitochondria and introducing the ethanol utilization pathway, the supply of precursors for squalene synthesis was enhanced, resulting in a significant increase in the squalene titer to 312.5 mg/L. Furthermore, the native promoter of ERG1 gene was engineered to reduce the flux through the ergosterol biosynthesis pathway. Following optimization of the culture medium, the squalene titer of the engineered strain was improved to 595 mg/L in shake flasks and 1929.7 mg/L in 5 L fermenters, respectively. This research provides valuable insights into the metabolic engineering of microorganisms for enhanced squalene production.

Keywords

Squalene / Saccharomyces cerevisiae / Metabolic engineering / Medium optimization / Biological Sciences / Microbiology

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Funding

National Key R & D Program of China(No. 2024YFA0917900)

RIGHTS & PERMISSIONS

Jiangnan University