Heterologous Biosynthesis of Prenylflavonoids in <i>Escherichia coli</i> Based on Fungus Screening of Prenyltransferases

Flavonoids are natural products with high biological activity and potential applications. Prenylation increases the lipophilicity of flavonoids, endowing them with specific functions, selectivity, and pharmacological properties. However, traditional methods of plant extraction and chemical synthesis are insufficient to meet the demand for prenylflavonoids. Heterologous biosynthesis of prenylflavonoids in microorganisms provides an alternative approach. Compared with plant prenyltransferases, microbial prenyltransferases showed broad substrate specificity, which is more conducive to the biosynthesis of diverse prenylflavonoids. In this study, we cloned 31 dimethylallyltryptophan synthase prenyltransferases from five fungal species and tested candidate substrates. The products of Ad03 and Ao01 were identified, resulting in two unnatural prenylflavonoids and four natural prenylflavonoids. We constructed the isopentenol utilization pathway in <i>Escherichia coli</i> to develop the efficient dimethylallyl diphosphate synthesis pathway for 6-prenylsilybin (6-PS) synthesis. By optimizing the whole cell catalysis and two-phase reaction system, the 6-PS production titer reached 176 mg/L and the yield of silybin was 88%. Our study provides an efficient method for prenylflavonoids production.