Multi-level metabolic engineering for squalene production: from pathway optimization to systematic regulation

Squalene is a high-value unsaturated triterpene extensively used in pharmaceuticals, cosmetics, and health products. Traditional extraction methods of squalene from shark oil are environmentally unfriendly, making it imperative to explore better production approaches. Microbial engineering for squalene production has drawn much attention due to its environmentally friendly nature, sustainability, and high efficiency. However, the capacity of squalene biosynthesis is still limited by many challenges, including insufficient precursor supply, metabolic flux competition, product toxicity, and extracellular transport. To address these issues, this review systematically summarizes various metabolic engineering strategies to enhance squalene production at three hierarchies, including pathway, organelle, and cell. At the pathway level, we discuss several common metabolic engineering strategies to improve the biosynthetic pathways of squalene, including enhancing precursor supply, regulating cofactor levels, and inhibiting competing metabolic pathways. At the organelle level, we underscore the application of organelle engineering in peroxisomes, mitochondria, and endoplasmic reticulum to achieve compartmentalized storage of squalene, alleviate product cytotoxicity, reduce distribution of precursor metabolic flux, and improve catalytic efficiency. At the cell level, cellular production capacity can be comprehensively improved to achieve high-level production of squalene by regulating lipid metabolism, promoting squalene secretion, modulating transcription factors, and combining mutagenesis and evolution. These metabolic engineering strategies not only offer comprehensive guidance for the efficient squalene biosynthesis but also establish a theoretical and technical foundation for the microbial production of other high-value terpenoids.