초록
<P>Ginsenosides, the predominant bioactive components of <I>Panax</I> 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 <I>Panax</I> species. Herein, we used two crude recombinant UDP-glycosyltransferases (UGTs), PgUGT74AE2 and UGTPg1 from <I>Panax ginseng</I>, to catalyze the glycosylation of DM with UDP-glucose (UDPG) as the sugar donor to produce DM glucosides 3-<I>O</I>-β-<SMALL>D</SMALL>-glucopyranosyl-dammar-24-ene-3β,20<I>S</I>-diol (3β-<I>O</I>-Glc-DM) and 20-<I>O</I>-β-<SMALL>D</SMALL>-glucopyranosyl-dammar-24-ene-3β,20<I>S</I>-diol (20<I>S-O</I>-Glc-DM), respectively. The <I>in vitro</I> and <I>in vivo</I> assays demonstrated that both 3β-<I>O</I>-Glc-DM and 20<I>S-O</I>-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-<I>O</I>-Glc-DM and 20<I>S-O</I>-Glc-DM by introducing the codon-optimized genes encoding DM synthase (DS) together with PgUGT74AE2 or UGTPg1 into <I>Saccharomyces cerevisiae</I>, respectively. Furthermore, multistep metabolic engineering strategies were applied, including optimization of chassis cell, multi-copy integration of heterologous genes <I>via</I> 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<SUP>−1</SUP> 3β-<I>O</I>-Glc-DM and 5.6 g L<SUP>−1</SUP> 20<I>S-O</I>-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 <I>de novo</I> 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.</P><P>Graphic Abstract</P><P>A green and sustainable approach is established by metabolic engineering for industrial production of bioactive dammarenediol-II glucosides.<BR/><IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c8gc04066d'/><BR/></P>