This work aims at elucidating the interrelationship between the cellular physiology and the target enzyme, as well as the development of a whole cell biocatalyst for production of trans-4-hydroxyproline. Critical parameters from molecular level to process relevant level were investigated, such as kinetics of the isolated enzymes, biocatalytic capacity of the host and its performance at process conditions. The diversity of the target enzyme was increased by random mutagenesis and sequence based homology modeling. A chromatographic procedure was developed for the purification of muteins and homologs, which showed different properties in vitro and in vivo. The performance of the whole cell biocatalysts were compared and optimized, whereas the biocatalytic capacity was determined to be 48 U/gCDW by metabolic flux balance analysis. Based on these results, a biotransformation approach using in situ generated recombinant resting cells was established, and has been successfully transferred to industry for further applications. Finally, a roadmap for process oriented optimization of whole cell biocatalysts carrying similar enzymes was proposed.