Metabolic engineering of Saccharomyces cerevisiae for de novo biosynthesis of aesculin and cichoriin

Coumarin glycosides are plant-derived polyphenols with diverse pharmacological activities and broad applications in the pharmaceutical, food and cosmetic industries. The low catalytic activity of 4CL and C2’H/F6’H, along with the poor stability of acyl-CoA and hydroxylated acyl-CoA intermediates, constitutes the major bottleneck for the conversion of phenolic acids to simple coumarins and limits yield improvement. Therefore, screening high-efficiency enzyme variants is critical for enhancing coumarin biosynthesis. In this study, we obtained the optimal combination Ptr4CL5‑AcF6’H1‑AtCOSY by screening esculetin synthases, which markedly enhanced the conversion efficiency of caffeic acid to esculetin, and we constructed a de novo biosynthetic pathway for esculetin. Multi-copy integration strengthened pathway flux and increased esculetin production by 87.67%. Heterologous expression of AcUGT92G7 and TOGT enabled the de novo biosynthesis of aesculin and cichoriin. Fine-tuning at the transcriptional and translational levels further optimized their titers, which reached 12.63 ± 0.31 mg/L and 8.51 ± 0.32 mg/L for aesculin and cichoriin, respectively. Meanwhile, pilot‑scale tests verified the feasibility of this strategy. This study established de novo biosynthetic pathways for the two coumarin glycosides in S. cerevisiae , providing reliable strategies for efficient green biomanufacturing of coumarins and glycoside derivatives.