Development and optimization of a glycerol-based fed-batch strategy for the production of peroxidases with Komagataella phaffii under the PDF promoter

Abstract Background Unsaturated polyester resins are commonly polymerized using cobalt-based siccatives. However, growing health concerns and increasing demand for cobalt highlight the need for more sustainable alternatives. Heterologously produced peroxidases, produced through secretory expression in Komagataella phaffii ( K. phaffii , previously classified as Pichi a pastoris ), represent a promising substitute for cobalt in such applications. To tailor the bioprocess for the production of peroxidases, dedicated bioprocess development and optimization are required. Rather than relying on methanol induction, the process should aim to achieve production via glycerol derepression under the control of the P DF promoter. This strategy mitigates the safety risks and metabolic stress associated with methanol utilization. Results This study presents the development and optimization of a three-phase bioprocess for peroxidase production in K. phaffii under the P DF promoter, induced via glycerol derepression. The process comprises a batch growth phase, a growth and induction fed-batch phase and a second fed-batch phase for product formation. Exponential feeding at 50% of the maximum specific growth rate (µ max ) was identified as the optimum, balancing growth, induction and productivity. During the production phase, a glycerol feed at 20% of the maximum uptake rate (q Smax ) and a duration of up to 96 h yielded the best results. The final optimized process achieved a titer of 12.36 U*mL −1 , a yield of 46.31 U*g −1 , and a space–time yield of 0.081 U*mL −1 *h −1 , which clearly outcompeted the methanol reference process by a factor of 13.4. Strikingly, the use of crude glycerol further improved performance by approximately 10%, demonstrating process robustness. Overall, this bioprocess enables efficient and sustainable peroxidase production, offering a viable alternative to cobalt-based catalysts in polyester resin curing. Conclusions This work demonstrates the feasibility of a robust, methanol-free bioprocess for peroxidase production in K. phaffii , driven by glycerol derepression under the P DF promoter. The optimized process achieves high titers and yields, maintains performance even with crude glycerol, and could support sustainable replacement of cobalt-based siccatives in industrial polymerization. These findings provide a valuable foundation for advancing biocatalytic solutions in green manufacturing.