Expression of recombinant proteins across a range of different host organisms is often necessary in metabolic engineering applications. Doing so can be facilitated by the use of vectors having origins of replication with a broad-host-range, the option for antibiotic resistance cassettes that are compatible with a particular host, in addition to sequences allowing for the effective transcription and translation of target proteins. We have created a modular 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 P(BAD) promoter and are available with six different antibiotic resistance cassettes. We have demonstrated the use of these vectors in Escherichia coli, Pseudomonas putida, and Burkholderia cepacia.

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4'-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.