Metabolic Engineering of <i>Saccharomyces cerevisiae</i> for High-Titer Ginsenoside Rh2 Production from Glycerol

As a byproduct of biodiesel production, glycerol is considered as a promising alternative feedstock for microbial fermentation processes. In this study, Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as the carbon source for the efficient production of ginsenoside Rh2 (Rh2), isolated from Panax ginseng that exhibits significant antitumor and antioxidant activities. First, the biosynthesis of 124.67 mg/L protopanaxadiol (PPD), which serves as the aglycone of Rh2, from glycerol was achieved by introducing the heterologous glycerol utilization pathway (″DHA″ pathway) and PPD biosynthesis pathway into S. cerevisiae BY4741. Subsequently, to enhance acetyl-CoA supply from glycerol, we engineered the ″pyruvate dehydrogenase bypass″ and the heterologous citrate cleavage pathway. While both resulting strains showed similar PPD production, metabolomic analysis indicated higher uridine diphosphate glucose accumulation, which serves as the glycosyl donor for Rh2 in the strain incorporating the citrate cleavage pathway. Therefore, the heterologous citrate cleavage pathway-engineered strain was selected, and the mevalonate pathway and PPD biosynthesis pathway in this strain were synergistically enhanced, resulting in a PPD production of 598.28 mg/L. Finally, by overexpressing glycosyltransferase and knocking out EGH1, a high-yielding Rh2 strain was constructed, achieving 700.53 mg/L in flasks and 2.88 g/L via fed-batch fermentation in a 5 L bioreactor, the highest reported titer to date. This study successfully demonstrates the metabolic engineering of yeast cell factories for high-efficiency biosynthesis of plant-derived saponin using glycerol as a nonfermentable carbon substrate.