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

Abstract

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.

Engineering Critical Amino Acid Residues of Lanosterol Synthase to Improve the Production of Triterpenoids in Saccharomyces cerevisiae

“…Nevertheless, the strain accumulated more unconverted 2,3-oxidosqualene instead of the triterpenoids, which hindered the carbon flux toward the triterpenoid biosynthesis . Similarly, this phenomenon has been observed when the CRISPR/dCas9-based technology was used to decrease the transcription level of the ERG7 gene . Other strategies, such as the TetR-TetO-based gene regulation system, and the N-degron-dependent protein degradation, had a slight effect on the improved production of various triterpenoids. , In addition to S.…”

“…69 Similarly, this phenomenon has been observed when the CRISPR/dCas9-based technology was used to decrease the transcription level of the ERG7 gene. 70 Other strategies, such as the TetR-TetO-based gene regulation system, and the N-degron-dependent protein degradation, had a slight effect on the improved production of various triterpenoids. 71,72 In addition to S. cerevisiae, Arnesen et al truncated the native promoter of the ERG7 gene of Y. lipolytica by 100 bp at its N-terminus for repressing its transcription level.…”

Triterpenoid production with a minimally engineered Saccharomyces cerevisiae chassis

“…Strategies ranged from full deletion to defined control of the transcription rate using repressible promoters for ERG7 transcription or CRISPR interference. 13,[27][28][29][30][31][32] Again, while the relative improvements compared to the reference were always impressive, the absolute amount of triterpenoids produced was most often modest.…”

Recent Advances in Yeast Recombinant Biosynthesis of the Triterpenoid Protopanaxadiol and Glycosylated Derivatives Thereof

“…cerevisiae. The final PPD production of the engineered strain was 295 mg/L, 5.7-fold higher when compared to the reference cultivated in fed-batch fermentation . Because ergosterol is an important component of the plasma membrane, a full deletion of ERG7 will stop ergosterol synthesis, which is an infeasible strategy for PPD synthesis .…”

“…The idea to improve triterpenoid synthesis by reducing sterol synthesis was followed in many studies. The strategies range from deleting ERG7 encoding 2,3-oxidosqualene utilizing lanosterol synthase to advanced transcriptional control and translational control of Erg7p activity . Recently, a degron tag fused to Erg7p was reported, decreasing the carbon flux into the sterol synthesis pathway while improving the precursor supply for triterpenoid synthesis .…”