Engineering of Pichia pastoris for the <i>De Novo</i> Synthesis of the Sesquiterpene\nZealexin A1 from Methanol

Methanol\nis a promising one-carbon feedstock for the biomanufacturing\nof chemicals. In this study, we described the engineering of a Pichia pastoris strain capable of using methanol\nas a substrate to efficiently produce sesquiterpene phytoalexin zealexin\nA1. First, the cellular metabolism of P. pastoris was reconstructed to achieve high-level production of (<i>S</i>)-β-macrocarpene, the precursor of zealexin A1, by optimizing\nthe MVA pathway, increasing the gene copy number of (<i>S</i>)-β-macrocarpene synthase, and downregulating the corresponding\ncompetitive pathway. The engineered strain produced 62.3 mg/L of (<i>S</i>)-β-macrocarpene. Second, cytochrome P450 enzyme\n(P450) CYP71Z18 was introduced to the strain to oxidize (<i>S</i>)-β-macrocarpene to zealexin A1, and 3.8 mg/L zealexin A1 was\nobtained from this approach. Finally, the supply of NADPH and methanol\nassimilation was enhanced to further improve the production of zealexin\nA1 by 24.8 mg/L. Subsequently, the engineered strain NT16 could produce\n102.5 mg/L zealexin A1 under fed-batch fermentation carried out in\na 5-L bioreactor. So far, this is the first time that zealexin A1\nhas been synthesized from methanol in P. pastoris.