Thinking big in small scale: characterization of peroxidase production in Komagataella phaffii using two-compartment scale-down system

The replacement of cobalt-based siccatives in unsaturated polyester resin curing requires sustainable and scalable biocatalytic alternatives, with fungal peroxidases representing a promising option. For industrial implementation, however, robust large-scale production processes are essential. In this study, we investigated the robustness of a glycerol-based peroxidase production process in K. phaffii driven by the glycerol-derepressible P DF promoter, focusing on industrially relevant bioreactor inhomogeneities. A two-compartment scale-down system was used to mimic substrate and oxygen gradients arising from insufficient mixing and mass transfer at large scale. Substrate inhomogeneity alone led to a moderate reduction in peroxidase titers (approx. 23%), indicating partial sensitivity to localized substrate oversupply. In contrast, simultaneous substrate and oxygen inhomogeneity resulted in a counterintuitive increase in peroxidase titers of approx. 13% compared to homogeneous reference conditions. This suggests that dynamic transitions between substrate limitation and surplus can positively affect peroxidase formation under P DF promoter control. Based on this observation, a modified process strategy incorporating glycerol pulses during the depression phase was evaluated. While this approach demonstrated high robustness, it did not enhance productivity. Overall, the results demonstrate that the developed K. phaffii process is resilient to large-scale inhomogeneities and can even benefit from specific dynamic cultivation conditions.