The yeast peroxisome: A dynamic storage depot and subcellular factory for squalene overproduction

Engineering microbes to produce terpenes from renewable feedstock is a promising alternative to traditional production approaches. Generally, terpenes are not readily secreted by microbial cells, and their distribution within cells is usually obscure and often a restricting factor for the overproduction of terpenes due to the storage limitation. Here, we determined that squalene overproduced in the cytoplasm of Saccharomyces cerevisiae was distributed in a form similar to oil droplets. Interestingly, these suspected oil droplets were confirmed to be inflated peroxisomes that were swollen along with the production of squalene, indicating that peroxisomes in S. cerevisiae are dynamic depots for the storage of squalene. In view of this, harnessing peroxisomes as subcellular compartments for squalene synthesis was performed, achieving a 138-fold improvement in squalene titer (1312.82 mg/L) relative to the parent strain, suggesting that the peroxisome of S. cerevisiae is an efficient subcellular factory for the synthesis of terpenes. By dual modulation of cytoplasmic and peroxisomal engineering, the squalene titer was further improved to 1698.02 mg/L. After optimizing a two-stage fed-batch fermentation method, the squalene titer reached 11.00 g/L, the highest ever reported. This provides new insight into the synthesis and storage of squalene in peroxisomes and reveals the potential of harnessing peroxisomes to overproduce terpenes in S. cerevisiae through dual cytoplasmic-peroxisomal engineering.