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

作者：Tingting Niu, Xiaoguang Yan, Jingru Wang, Hongjian Song, Yi Cui, Xiangping Cai, Alan K. Chang, Jianjun Qiao, Mingzhang Wen, Jianwei He · 发表于：ACS Sustainable Chemistry & Engineering · 年份：2024 · DOI：10.1021/acssuschemeng.4c02722 · 被引用次数：11 · 研究领域：Plant biochemistry and biosynthesis、Microbial Metabolic Engineering and Bioproduction、Microbial Natural Products and Biosynthesis

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 .