Reengineering of ergosterol biosynthesis in Saccharomyces cerevisiae and its sustainable application in citrus peel processing industry

Ergosterol is an important sterol primarily found in fungi, yeast, and certain plants, with particularly high concentrations in yeast and fungus. As the precursor to vitamin D₂, it is converted into vitamin D₂ upon exposure to ultraviolet (UV) radiation. In this study, we successfully reengineered the ergosterol biosynthesis in Saccharomyces cerevisiae through a systematic synthetic biology strategy, and explored its sustainable application in citrus peel processing industry by using the citrus peel hydrolysate as a fermentation substrate. Through the MVA pathway module, the post-squalene pathway module, and the ergosterol biosynthesis module strategies, the titer of ergosterol was significantly increased to 23.38 mg/g DCW, which was nearly 19.15-fold higher than that of the original strain. The engineered strain was capable of producing 2.35 and 5.65 g/L of ethanol at 24 h in shake flasks and 5-L bioreactor fermentation by using the citrus peel hydrolysate as a fermentation substrate, respectively. The conversion rate of ethanol from citrus peel powder was approximately 3.3 %. To the best of our knowledge, this is the first report on the reengineering of ergosterol biosynthesis in Saccharomyces cerevisiae for resisting the stress of D-limonene, implying the engineered strain offers a promising avenue for the development of sustainable and eco-friendly bioenergy solutions in citrus peel processing industry.