De Novo biosynthesis of scopoletin from glucose by engineered Saccharomyces cerevisiae

Scopoletin, a plant-derived coumarin, exhibits diverse pharmacological activities. Nowadays, scopoletin is mainly produced by plant extraction. In this study, a Saccharomyces cerevisiae strain capable of de novo scopoletin biosynthesis was constructed by successively integrating ten heterologous genes into a p-coumaric acid producing strain. The following strategies were employed to improve the yield of scopoletin: (1) A high-yield caffeic acid platform strain (301.98 mg/L) was established by increasing the HpaBC copy number, integration of EcaroL and RgTAL, feedback-resistant mutant expression cassette (ARO3^D154N/ARO4^G141S/ARO7^K229L), and knockout competing pathway genes (ARO10/PDC5); (2) Two distinct pathways from caffeic acid to scopoletin were introduced and selected; (3) Integration of heterologous genes AtHCT and AtC3’H, to construct the pathway from p-coumaroyl-CoA to caffeoyl-CoA; (4) Attenuating PYK1 by introducing the mutant gene of PYK1^D146N to increase PEP supply; (5) Overexpression of cofactor regeneration genes (ADO1, SAH1, and ZWF1) to strengthen the supply of SAM and NADPH; (6) Adjustment of the fermentation medium. The engineered strain produced scopoletin of 55.32 mg/L in shake flasks. To our knowledge, this is the first report of de novo scopoletin biosynthesis in S. cerevisiae. These results establish new insights for microbial production of scopoletin and coumarin derivatives.