Engineering of Pichia pastoris for the De Novo Synthesis of the Sesquiterpene Zealexin A1 from Methanol

Methanol is a promising one-carbon feedstock for the biomanufacturing of chemicals. In this study, we described the engineering of a Pichia pastoris strain capable of using methanol as a substrate to efficiently produce sesquiterpene phytoalexin zealexin A1. First, the cellular metabolism of P. pastoris was reconstructed to achieve high-level production of (S)-β-macrocarpene, the precursor of zealexin A1, by optimizing the MVA pathway, increasing the gene copy number of (S)-β-macrocarpene synthase, and downregulating the corresponding competitive pathway. The engineered strain produced 62.3 mg/L of (S)-β-macrocarpene. Second, cytochrome P450 enzyme (P450) CYP71Z18 was introduced to the strain to oxidize (S)-β-macrocarpene to zealexin A1, and 3.8 mg/L zealexin A1 was obtained from this approach. Finally, the supply of NADPH and methanol assimilation was enhanced to further improve the production of zealexin A1 by 24.8 mg/L. Subsequently, the engineered strain NT16 could produce 102.5 mg/L zealexin A1 under fed-batch fermentation carried out in a 5-L bioreactor. So far, this is the first time that zealexin A1 has been synthesized from methanol in P. pastoris.