Engineering Pichia pastoris for high-level biosynthesis of squalene

High-value-added chemicals and advanced biofuels can be feasibly produced from renewable feedstocks through microbial metabolic engineering. Methanol is produced with high quantities from CO2 using green energy sources such as solar energy, thereby making it an ideal feedstock. However, little has been documented on the microbial production of terpenoids utilising methanol. This study aimed to engineer Pichia pastoris to overproduce triterpene squalene from methanol by optimising and reconstructing the utilisation pathways of mevalonate and methanol. In addition, we explored the exocytosis of squalene and promoted squalene secretion successfully. Furthermore, we identified a key bottleneck of squalene synthesis in P. pastoris through transcriptomic analysis. The final engineered strain produced 20.80 +/- 0.02 g/L squalene during fed-batch fermentation, which is the highest squalene production using organic one-carbon as carbon source reported so far. Our findings set the stage for applying P. pastoris as an organic one-carbon platform for producing biochemicals and biofuels.