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.

工程微生物从可再生原料生产萜烯是传统生产方法的有前途的替代方法。通常，萜烯不容易被微生物细胞分泌，并且它们在细胞内的分布通常是模糊的，并且由于储存限制而常常是萜烯过量生产的限制因素。在这里，我们确定在酿酒酵母的细胞质中过量产生的角鲨烯以类似于油滴的形式分布。有趣的是，这些疑似油滴被证实是膨胀的过氧化物酶体，随着角鲨烯的产生而膨胀，表明酿酒酵母中的过氧化物酶体。是用于储存角鲨烯的动态仓库。有鉴于此，利用过氧化物酶体作为角鲨烯合成的亚细胞区室，与亲本菌株相比，角鲨烯效价提高了138倍（1312.82 mg / L），这表明酿酒酵母的过氧化物酶体是一种有效的亚细胞工厂。用于合成萜烯。通过细胞质和过氧化物酶体工程的双重调节，角鲨烯效价进一步提高至1690.22 mg / L。优化两阶段分批补料发酵方法后，角鲨烯效价达到11.00 g / L，是有史以来最高的。这为过氧化物酶体中角鲨烯的合成和储存提供了新的见解，并揭示了利用过氧化物酶体在酿酒酵母中过量生产萜烯的潜力。 通过双重胞质过氧化物酶体工程。