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Background  Understanding the bioprocess limitations is critical for the efficient design of biocatalysts to facilitate process feasibility and improve process economics. In this study, a proline hydroxylation process with recombinant  Escherichia coli  expressing  l -proline  cis -4-hydroxylase (SmP4H) was investigated. The factors that influencing the metabolism of microbial hosts and process economics were focused on for the optimization of  cis -4-hydroxy- l -proline (CHOP) production.    Results  In recombinant  E. coli , SmP4H synthesis limitation was observed. After the optimization of expression system, CHOP production was improved in accordance with the enhanced SmP4H synthesis. Furthermore, the effects of the regulation of proline uptake and metabolism on whole-cell catalytic activity were investigated. The improved CHOP production by repressing  putA  gene responsible for  l -proline degradation or overexpressing  l -proline transporter  putP  on CHOP production suggested the important role of substrate uptake and metabolism on the whole-cell biocatalyst efficiency. Through genetically modifying these factors, the biocatalyst activity was significantly improved, and CHOP production was increased by twofold. Meanwhile, to further improve process economics, a two-strain coupling whole-cell system was established to supply co-substrate (α-ketoglutarate, α-KG) with a cheaper chemical  l -glutamate as a starting material, and 13.5 g/L of CHOP was successfully produced.    Conclusions  In this study, SmP4H expression, and  l -proline uptake and degradation, were uncovered as the hurdles for microbial production of CHOP. Accordingly, the whole-cell biocatalysts were metabolically engineered for enhancing CHOP production. Meanwhile, a two-strain biotransformation system for CHOP biosynthesis was developed aiming at supplying α-KG more economically. Our work provided valuable insights into the design of recombinant microorganism to improve the biotransformation efficiency that catalyzed by Fe(II)/α-KG-dependent dioxygenase.    Electronic supplementary material  The online version of this article (10.1186/s12934-017-0821-7) contains supplementary material, which is available to authorized users.

Process optimization for enhancing production of cis-4-hydroxy-l-proline by engineered Escherichia coli