Expression of recombinant proteins across a range of different host organisms is often necessary in metabolic engineering applications, necessitating the use of broad-host-range vectors for protein expression. Several components are required for such vectors including stable replication in multiple species, antibiotic resistance cassettes, effective transcription, and translation of the protein. We have created a set of broad-host-range expression vectors for protein expression in Gram negative bacteria. These vectors use the broad-host-range pBBR1 replicon as well as the arabinose-inducible PBAD promoter and are available with six different antibiotic resistance cassettes. Furthermore, we have examined the effectiveness of different 5' untranslated regions with a set of broad-host-range expression vectors by comparing expression of GFPuv in Escherichia coli and Pseudomonas putida.

These vectors have been used to express cytochrome P450 enzymes identified in the Gram positive strain Actinoplanes sp. in both E. coli and P. putida. The Actinoplanes strain has been used to produce human-like drug metabolites of several macrolide immunosuppressants in much larger quantities than can be produced using other in vitro or in vivo drug metabolism systems. One or more cytochrome P450 enzymes are responsible for the observed biotransformation, but the strain is uncharacterized and difficult to work with. We are working to identify the specific P450 enzymes responsible for the biotransformation and use metabolic engineering with a more industrially relevant host to perform in vivo drug metabolism.