Orchestrating multiple subcellular organelles of Saccharomyces cerevisiae for efficient production of squalene.

Squalene widely used in medicines, food, and cosmetics. Subcellular organelle engineering is an effective way to develop squalene-hyperproducing yeasts. Here, we demonstrated that synergistically modifying multiple organelles in Saccharomyces cerevisiae, including mitochondria, endoplasmic reticulum (ER), lipid droplets (LDs), and cell wall (CW), effectively increased squalene production. Based on the previously developed dual cytoplasmic-mitochondrial engineering strain SquMC13, squalene production capacity was further enhanced by improving the ER function for protein expression, expanding the LDs size for squalene storage, and increasing CW integrity to maintain high cell viability. Combinatorial modification of these organelles enhanced squalene productivity to 3.4-times that of SquMC13. NADPH generation was optimized, resulting in a further 3.9 % increase in squalene production. An efficient strain for squalene production was developed, the squalene production titer of which reached 55.8 g/L with 0.5 g/L/h productivity and specific cell production of 0.5 g/g dry cell weight, paving the way for industrial squalene production.