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

• Published in: International journal of molecular sciences Vol. 22; no. 21; p. 11836
• Main Authors: Lim, Soo-Hwan, Baek, Jong-In, Jeon, Byeong-Min, Seo, Jung-Woo, Kim, Min-Sung, Byun, Ji-Young, Park, Soo-Hoon, Kim, Su-Jin, Lee, Ju-Young, Lee, Jun-Hyoung, Kim, Sun-Chang
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 31.10.2021; MDPI
• Subjects: Biosynthesis; Carbon sources; Cell growth; CRISPR; CRISPR interference; CRISPR-Cas Systems; Cytochrome; Fermentation; Fungi; Ginseng; Ginsenosides; Lanosterol; Lanosterol synthase; Mass production; Metabolic Engineering; Metabolic flux; Metabolic Networks and Pathways; Metabolic pathways; Metabolism; Metabolites; Proteins; protopanaxadiol; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sapogenins - metabolism; Sterols; triterpenes; Yeast; CRISPR interference; protopanaxadiol; triterpenes; ginsenosides; metabolic engineering; Saccharomyces cerevisiae; lanosterol
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms222111836

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 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 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 . 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 . With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in , we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.