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.