To date, we have identified two full P450 gene sequences from Actinoplanes sp., along with at least ten other partial sequences for future examination. Gene expression of the P450s in E. coli presents several challenges, including vastly differing codon bias, and the lack of required accessory proteins for P450 activity. Pseudomonas aeruginosa is a Gram negative bacterium that has similar codon bias to Actinoplanes sp., and also contains endogenous P450 systems that are potentially compatible with the cloned genes. A broad-host-range expression vector has been created for the controllable expression of heterologous proteins in different Gram negative bacteria, including both E. coli and Pseudomonas strains. The two P450s from Actinoplanes sp., CYP PA1 and CYP PA2, have been expressed with a polyhistidine tag for detection in Pseudomonas aeruginosa for the in vivo metabolism of sirolimus. Future works includes identification of metabolites from both identified P450s along with identification and characterization of other P450s in Actinoplanes sp., and finally, work to optimize both expression and metabolism.