Biofilm-based continuous secretion of human lysozyme through cell-surface display of FimH on Saccharomyces cerevisiae

Biofilms enhance microbial tolerance to harsh environments while preserving cellular activity over prolonged periods. Although biofilm-based continuous fermentation is widely applied for production of small-molecule chemicals, its application to macromolecular protein production has been rarely reported. Here, FimH, an adhesin from <i>E. coli</i> fimbriae that is employed for mannose-specific adherence and promotes biofilm formation, was displayed on the surface of <i>S. cerevisiae</i> using the anchoring proteins Sag1C, Sed1, Cwp2 and Ccw12, resulting in an 80-150 % enhancement in biofilm formation. Among them, Sed1 was the most effective, followed by Sag1C. A biofilm-based fermentation system for continuous secretion of human lysozyme (hLYZ) was established via cell-surface display of FimH in <i>S. cerevisiae</i>, achieving stable operation for over 350 h. The engineered strain BY4742-Sag1C-hlyz achieved an average extracellular hLYZ activity of 113.1 U/mL, significantly higher than the control BY4742-hlyz (41.6 U/mL), owing to enhanced cell adhesion that increased the total cell number in biofilm-based fermentation. Its productivity reached 2.36 U/mL/h, representing a 77.4 % increase compared with free-cell fermentation of BY4742-hlyz (1.33 U/mL/h). In conclusion, this study first achieved heterologous expression of a bacterial biofilm-forming gene in <i>S. cerevisiae</i>, enhancing biofilm formation and providing a reference for future expression of bacterial biofilm-related genes in yeast. Furthermore, biofilm-based fermentation enabled continuous secretion of hLYZ, highlighting a promising strategy for continuous production of recombinant proteins.