High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

Background: Ginsenosides are Panax plant-derived triterpenoid with wide applications in cardiovascular protection and immunity-boosting. However, the saponins content of Panax plants is fairly low, making it time-consuming and unsustainable by direct extraction. Protopanaxadiol (PPD) is a common precursor of dammarane-type saponins, and its sufficient supply is necessary for the efficient synthesis of ginsenoside. Results: In this study, a combinational strategy was used for the construction of an efficient yeast cell factory for PPD production. Firstly, a PPD-producing strain was successfully constructed by modular engineering in Saccharomyces cerevisiae BY4742 at the multi-copy sites. Then, the INO2 gene, encoding a transcriptional activator of the phospholipid biosynthesis, was fine-tuned to promote the endoplasmic reticulum (ER) proliferation and improve the catalytic efficiency of ER-localized enzymes. To increase the metabolic flux of PPD, dynamic control, based on a carbon-source regulated promoter P-HXT1, was introduced to repress the competition of sterols. Furthermore, the global transcription factor UPC2-1 was introduced to sterol homeostasis and up-regulate the MVA pathway, and the resulting strain BY-V achieved a PPD production of 78.13 +/- 0.38 mg/g DCW (563.60 +/- 1.65 mg/L). Finally, sugarcane molasses was used as an inexpensive substrate for the first time in PPD synthesis. The PPD titers reached 1.55 +/- 0.02 and 15.88 +/- 0.65 g/L in shake flasks and a 5-L bioreactor, respectively. To the best of our knowledge, these results were new records on PPD production. Conclusion: The high-level of PPD production in this study and the successful comprehensive utilization of low-cost carbon source-sugarcane molassesindicate that the constructed yeast cell factory is an excellent candidate strain for the production of high-value-added PPD and its derivativeswith great industrial potential.