Highly Efficient Production of Ergothioneine from Glycerol through Gene Mining and CRISPR-Cas9 Synthetic Biology Technology in <i>Saccharomyces cerevisiae</i>

Glycerol, a byproduct of biodiesel produced from waste cooking oil, is a renewable carbon source. Moreover, applying glycerol in Saccharomyces cerevisiae bioconversion to value-added products would not induce the Crabtree effect. Therefore, in this study, the engineered S. cerevisiae IMX581 was genetically modified using CRISPR-Cas9 to enhance ergothioneine (EGT) production, an antioxidant known for its antiaging and anti-inflammatory effects, with glycerol utilized as the main carbon source. Through the optimization of EGT biosynthesis overexpression cassettes and the enhancement of glycerol utilization and upstream metabolism, results showed more than a 10-fold increase in EGT production compared to using glucose in shake-flask fermentation. However, glycerol utilization faced challenges in supporting yeast growth, hindering large-scale fermentation. To address this, supplementing with small amounts of ethanol and acetic acid proved more beneficial for yeast growth than using glycerol alone. By applying the above strategies, a 10-day fed-batch fermentation without amino acid supplementation resulted in a high yeast cell density and an EGT yield of 1.14 g/L, the highest conversion rate from glycerol to date. The engineered S. cerevisiae developed in this study provided an efficient method for the synthesis of EGT from glycerol and an alternative for bioconversion without inducing the Crabtree effect.