De novo biosynthesis of diverse plant-derived styrylpyrones in Saccharomyces cerevisiae

Plant styrylpyrones exerting well-established neuroprotective properties have attracted increasing attention in recent years. The ability to synthesize each individual styrylpyrone in engineered microorganisms is important to understanding the biological activity of medicinal plants and the complex mixtures they produce. Microbial biomanufacturing of diverse plant-derived styrylpyrones also provides a sustainable and efficient approach for the production of valuable plant styrylpyrones as daily supplements or potential drugs complementary to the prevalent agriculture-based approach. In this study, we firstly demonstrated the heterogenous biosynthesis of two 7,8-saturated styrylpyrones (7,8-dihydro-5,6-dehydrokavain (DDK) and 7,8-dihydroyangonin (DHY)) and two 7,8-unsaturated styrylpyrones (desmethoxyyangonin (DMY) and yangonin (Y)), in Saccharomyces cerevisiae. Although plant styrylpyrone biosynthetic pathways have not been fully elucidated, we functionally reconstructed the recently discovered kava styrylpyrone biosynthetic pathway that has high substrate promiscuity in yeast, and combined it with upstream hydroxycinnamic acid biosynthetic pathways to produce diverse plant-derived styrylpyrones without the native plant enzymes. We optimized the de novo pathways by engineering yeast endogenous aromatic amino acid metabolism and endogenous double bond reductases and by CRISPR-mediated δ-integration to overexpress the rate-limiting pathway genes. These combinatorial engineering efforts led to the first three yeast strains that can produce diverse plant-derived styrylpyrones de novo, with the titers of DDK, DMY and Y at 4.40 μM, 1.28 μM and 0.10 μM, respectively. This work has laid the foundation for larger-scale styrylpyrone biomanufacturing and the complete biosynthesis of more complicated plant styrylpyrones.