Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichiapastorisis presented. Making use of the P. pastorisAOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg·L1 in the whole broth and a biomass concentration of 150 g·L1 cell dry weight, thus comprising a space-time yield of 5.2 mg·L1·h1. Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased space-time yield of 25 mg·L1·h1 while reducing the cell dry weight concentration from 150 g·L1 in fed-batch to 65 g·L1 in continuous cultivation.