MDG1-mediated transcriptional reprogramming enhances cellulase production and alters thermal activity in recombinant Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is one of the most widely used microbial cell factories for heterologous protein and enzyme production. However, improving production efficiency and tailoring enzyme properties remain a major challenge. Here we identified MDG1, a gene involved in the pheromone signaling pathway, as a previously unrecognized regulator that significantly enhances cellulase production in recombinant yeast. Overexpression of MDG1 significantly increased the extracellular activities of β-glucosidase I (BGLI), cellobiohydrolase I (CBHI), and endo-glycosidase II (EGII). Intriguingly, MDG1 overexpression also altered the thermal activity profile of BGLI, shifting its peak activity from 50 °C to 37 °C-an inversion relative to the parental strain. Integrated transcriptome analyses revealed that MDG1 regulates the expression of genes involved in the cell cycle and protein folding. Targeted modulation of key cell cycle regulators (CLN1, PCL1, SWI5) further improved BGLI activity, confirming their functional involvement. Secretome analysis and functional assays identified the disulfide isomerase Pdi1p as a key contributor to the enhanced enzyme performance at 37 °C. Our study reveals a novel role of MDG1 in coordinating gene networks to improve enzyme activities and reshape enzymatic properties.