Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast
Yuanwei Gou, Dongfang Li, Minghui Zhao, Mengxin Li, Jiaojiao Zhang, Yi‐Lian Zhou, Feng Xiao, Gaofei Liu, Haote Ding, Chen‐Fan Sun, Cuifang Ye, Chang Dong, Jucan Gao, Di Gao, Zehua Bao, Lei Huang, Zhinan Xu, Jiazhang Lian
Abstract
Abstract While sanguinarine has gained recognition for antimicrobial and antineoplastic activities, its complex conjugated structure and low abundance in plants impede broad applications. Here, we demonstrate the complete biosynthesis of sanguinarine and halogenated derivatives using highly engineered yeast strains. To overcome sanguinarine cytotoxicity, we establish a splicing intein-mediated temperature-responsive gene expression system (SIMTeGES), a simple strategy that decouples cell growth from product synthesis without sacrificing protein activity. To debottleneck sanguinarine biosynthesis, we identify two reticuline oxidases and facilitated functional expression of flavoproteins and cytochrome P450 enzymes via protein molecular engineering. After comprehensive metabolic engineering, we report the production of sanguinarine at a titer of 448.64 mg L −1 . Additionally, our engineered strain enables the biosynthesis of fluorinated sanguinarine, showcasing the biotransformation of halogenated derivatives through more than 15 biocatalytic steps. This work serves as a blueprint for utilizing yeast as a scalable platform for biomanufacturing diverse benzylisoquinoline alkaloids and derivatives.