Construction and optimization of microbial cell factories for sustainable production of bioactive dammarenediol-II glucosides

Ginsenosides, the predominant bioactive components of Panax species, are biosynthesized by glycosylation at C3–OH and/or C20–OH of protopanaxadiol (PPD), and C6–OH and/or C20–OH of protopanaxatriol (PPT). Dammarenediol-II (DM), the direct precursor of PPD, has two hydroxyls at C3 and C20 positions, but DM glucosides have scarcely been identified from Panax species. Herein, we used two crude recombinant UDP-glycosyltransferases (UGTs), PgUGT74AE2 and UGTPg1 from Panax ginseng, to catalyze the glycosylation of DM with UDP-glucose (UDPG) as the sugar donor to produce DM glucosides 3-O-β-D-glucopyranosyl-dammar-24-ene-3β,20S-diol (3β-O-Glc-DM) and 20-O-β-D-glucopyranosyl-dammar-24-ene-3β,20S-diol (20S-O-Glc-DM), respectively. The in vitro and in vivo assays demonstrated that both 3β-O-Glc-DM and 20S-O-Glc-DM exhibited higher anti-colon cancer activities than natural ginsenosides. In order to produce DM glucosides in an economical, efficient and convenient way, we refactored the complete biosynthetic pathways of 3β-O-Glc-DM and 20S-O-Glc-DM by introducing the codon-optimized genes encoding DM synthase (DS) together with PgUGT74AE2 or UGTPg1 into Saccharomyces cerevisiae, respectively. Furthermore, multistep metabolic engineering strategies were applied, including optimization of chassis cell, multi-copy integration of heterologous genes via the CRISPR/Cas9 system, increase of precursor supply by overexpressing rate-limiting enzymes, down-regulation of the competitive pathway to redirect the metabolic flux towards the target products, and overexpression of the transcriptional activator. Finally, the titers of 2.4 g L−1 3β-O-Glc-DM and 5.6 g L−1 20S-O-Glc-DM were achieved through fed-batch fermentation in a 3 L bioreactor. This is the first study to demonstrate the anti-colon cancer activities of DM glucosides and to achieve the de novo biosynthesis of DM glucosides with high titers in microbial cell factories. This study has established a green and sustainable approach for the industrial production of DM glucosides, which provides promising candidates for new drug research and development.