Engineering a versatile yeast platform for sesquiterpene production from glucose or methanol.

Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. Pichia pastoris (syn. Komagataella phaffii), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, P. pastoris is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in P. pastoris with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules MK and PMK greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of β-farnesene from dual carbon source by replacing the α-bisabolene synthase with a β-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in P. pastoris. Moreover, it paves the way for the development of P. pastoris as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in P. pastoris.