Department of Life Sciences, Chalmers University of Technology, Kemivägen 10, SE-412 96, Gothenburg, Sweden.
State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, 16 Neinanxiaojie, Dongcheng district, Beijing, China.
Nat Commun. 2024 Oct 9;15(1):8759. doi: 10.1038/s41467-024-53045-3.
Protoberberine alkaloids and benzophenanthridine alkaloids (BZDAs) are subgroups of benzylisoquinoline alkaloids (BIAs), which represent a diverse class of plant-specialized natural metabolites with many pharmacological properties. Microbial biosynthesis has been allowed for accessibility and scalable production of high-value BIAs. Here, we engineer Saccharomyces cerevisiae to de novo produce a series of protoberberines and BZDAs, including palmatine, berberine, chelerythrine, sanguinarine and chelirubine. An ER compartmentalization strategy is developed to improve vacuole protein berberine bridge enzyme (BBE) activity, resulting in >200% increase on the production of the key intermediate (S)-scoulerine. Another promiscuous vacuole protein dihydrobenzophenanthridine oxidase (DBOX) has been identified to catalyze two-electron oxidation on various tetrahydroprotoberberines at N7-C8 position and dihydrobenzophenanthridine alkaloids. Furthermore, cytosolically expressed DBOX can alleviate the limitation on BBE. This study highlights the potential of microbial cell factories for the biosynthesis of a diverse group of BIAs through engineering of heterologous plant enzymes.
原小檗碱类生物碱和苯并菲啶类生物碱（BZDAs）是苄基异喹啉类生物碱（BIAs）的亚组，BIAs 是一类具有多种药理学特性的植物特异性天然代谢物的多样化类别。微生物生物合成允许获得和规模化生产高价值 BIAs。在这里，我们通过工程化酿酒酵母从头合成一系列原小檗碱类和 BZDAs，包括巴马汀、小檗碱、白屈菜红碱、血根碱和白屈菜红碱。开发了内质网区室化策略来提高液泡蛋白小檗碱桥酶（BBE）的活性，导致关键中间体（S）-阿朴啡的产量增加了>200%。另一种杂泛的液泡蛋白二氢苯并菲啶氧化酶（DBOX）已被鉴定为能够在 N7-C8 位置和二氢苯并菲啶生物碱上催化各种四氢原小檗碱的两电子氧化。此外，胞质表达的 DBOX 可以缓解 BBE 的限制。这项研究通过工程化异源植物酶，突出了微生物细胞工厂用于合成多样化 BIAs 组的潜力。
