Construction of an efficient enzyme-cell@material biocatalyst through the biofilm immobilization of Komagataella phaffii for continuous biocatalysis.

The ever-growing demand for cost-effective and green biocatalytic transformations has prompted the rational design and development of robust biocatalytic tools. However, transformations are hindered by limited continuous process and enzymatic instability. Here, 10 Flo family related genes in Komagataella phaffii were systematically evaluated to assess their adhesive properties. For the first time, we identified the KpFlo11C domain of BSC1p as facilitating cell aggregation on carriers, thereby enhancing the biofilm immobilization process. Furthermore, an engineered K. phaffii strain, fixing β-galactosidase on the cell surface, was constructed by optimizing the signal peptide and gene dosage, for enhancing the efficiency of enzyme targeting and anchoring, as well as the proportion of cells displaying the enzyme. Finally, the KpFlo11C domain was overexpressed in this K. phaffii cell display system to construct the enzyme-cell@material biocatalyst, which exhibited robust continuous production of galacto-oligosaccharides (GOS) at a rate of 8.16 g/L/h in a 6-L fermenter. The development of this enzyme-cell@material biocatalyst, which offers a highly efficient, stable, low-cost, and simplified biocatalytic process, provides a direction for the application of other yeasts in large-scale industrial continuous production.