Berberine is an extensively used pharmaceutical benzylisoquinoline alkaloid (BIA) derived from plants. Microbial manufacturing has emerged as a promising approach to source valuable BIAs. Here, we demonstrated the complete biosynthesis of berberine in Saccharomyces cerevisiae by engineering 19 genes including 12 heterologous genes from plants and bacteria. Overexpressing bottleneck enzymes, fermentation scale-up, and heating treatment after fermentation increased berberine titer by 643-fold to 1.08 mg L-1. This pathway also showed high efficiency to incorporate halogenated tyrosine for the synthesis of unnatural BIA derivatives that have higher therapeutical potentials. We firstly demonstrate the in vivo biosynthesis of 11-fluoro-tetrahydrocolumbamine via nine enzymatic reactions. The efficiency and promiscuity of our pathway also allow for the simultaneous incorporation of two fluorine-substituted tyrosine derivatives to 8, 3’-di-fluoro-coclaurine. This work highlights the potential of yeast as a versatile microbial biosynthetic platform to strengthen current pharmaceutical supply chain and to advance drug development.

小檗碱是一种广泛使用的从植物中提取的药物苄基异喹啉生物碱 (BIA)。微生物制造已成为获取有价值的 BIA 的一种有前途的方法。在这里，我们通过改造 19 个基因，包括来自植物和细菌的 12 个异源基因，展示了酿酒酵母中小檗碱的完整生物合成。过表达瓶颈酶、发酵放大和发酵后热处理使小檗碱滴度增加了 643 倍，达到 1.08 mg L-1。该途径还显示出高效率，可以掺入卤化酪氨酸来合成具有更高治疗潜力的非天然 BIA 衍生物。我们首先通过九种酶促反应证明了 11-氟-四氢鸽胺的体内生物合成。我们的途径的效率和混杂性还允许同时将两种氟取代的酪氨酸衍生物掺入8, 3'-二氟-椰壳碱中。这项工作凸显了酵母作为多功能微生物生物合成平台的潜力，可加强当前的药物供应链并推进药物开发。