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Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in  Panax ginseng  and a long cultivation time (4–6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in  Saccharomyces cerevisiae  and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in  S. cerevisiae . Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in  S. cerevisiae . With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in  S. cerevisiae , we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.

CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae