Efficient production of unspecific Peroxygenases using continuous culture of Komagataella phaffii

• Usage of RAMOS reveals process information in media screen and optimization with respirational data • Modified BSM reduces 57.5% cost and avoids precipitate during continuous culture. • Production rate enhances 107.66% with optimized continuous culture process parameters compared to fed-batch. • Economic efficiency is greatly enhanced with 21.8% lower unit cost of UPO. Unspecific peroxygenases (UPO) utilize hydrogen peroxide as cosubstrate and catalyze a broad range of selective oxidation reactions, offering advantages over complex and costly cytochromes P450 systems. Despite their potential in organic synthesis, the industrial adoption of UPO is hindered by the cost-inefficiency of fed-batch fermentation, which suffers from protracted cultivation timelines and inter-batch variability. This study developed a continuous culture process for UPO production using Komagataella phaffii . Feeding medium was first optimized to be suitable for continuous culture, then an optimal dilution rate was determined using high-throughput parallel bioreactors that give higher UPO productivity. A modified Basal Salt Medium (mBSM) with reduced magnesium sulfate concentrations was finally selected and a dilution rate of 0.03 h -1 was chosen, as these combined conditions gave a volumetric UPO production rate of 5597.93 U/L/h and a specific activity production rate of 85.64 U/gDCW/h, which were 107.66%, and 67.56% higher over the fed-batch process, respectively. In addition, the established continuous process reduced unit cost of UPO by 21.8% for producing the same amount of UPO. These findings highlight the feasibility and economic benefits of continuous culture for UPO production.