De novo biosynthesis of alpinetin enhanced by directed evolution of 5-O-methyltransferase

Alpinetin ((2 S)-7-Hydroxy-5-methoxyflavan-4-one) is a natural flavonoid found in various medicinal herbs and is frequently used in Chinese patent medicines. It exhibits a wide range of bioactivities, including anti-inflammatory, cardiovascular protective, lung protective, antiviral, hepatoprotective, and antitumor effects. Alpinetin features a 5-methyl group on the A ring, a rare characteristic among methylated flavonoids. The limited abundance of alpinetin in plant biomass, its laborious extraction and purification from this biomass, and the toxicity and lack of regio- and chemoselectivity in organic synthesis render its biosynthesis in engineered microbes an attractive alternative. In this study, we aimed to achieve the de novo biosynthesis of alpinetin in Escherichia coli. In a series of optimization steps, we varied the selection of pathway enzymes, plasmid configurations, medium composition, and fermentation strategies. Lastly, we applied a directed evolution campaign to the 5-O-methyltransferase to successfully enable the de novo biosynthesis of alpinetin in E. coli for the first time. Moreover, we demonstrated the ability of O-methyltransferase to methylate a broad range of flavonoid substrates, leading to the production of valuable O-methylated flavonoids. Our study represents the first example of alpinetin biosynthesis in a heterologous host and paves the way to produce other valuable O-methylated flavonoids enzymatically.