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Heterologous Expression of Microbial Genes for Drug Metabolism in Gram Negative Bacteria

Jamie E. Prior, Chemical and Biological Engineering, University of Colorado, 1111 Engineering Drive, Campus Box 424, Boulder, CO 80309, Tobin Strom, Anesthesiology, University of Colorado Health Sciences Center, Campus Box B113, Denver, CO, Uwe Christians, Anethesiology, University of Colorado Health Sciences Center, Campus Box B113, Denver, CO, and Ryan T. Gill, Department of Chemical and Biological Engineering, University of Colorado, ECCH 111 Campus Box 424, Boulder, CO 80309.

The FDA receives over 350,000 reports of adverse drug effects each year, making adverse effects “the single leading contributor to preventable patient injury”. One of the primary factors in adverse drug effects is drug metabolism where drug compounds are modified to derivatives that may have differing levels of efficacy or toxicity from the parent drug. Many times these metabolites will be more toxic than the parent drug, as with acetaminophen, and other times the metabolite will be more effective than the parent, as with prodrugs. Thus, toxicity and efficacy studies require not only the parent drug but also the relevant human-like metabolites. Human drug metabolism is primarily accomplished by cytochrome P450 enzymes (P450s) in the liver. Microbial models for human drug metabolism are attractive alternatives to the more expensive conventional methods such as liver microsomes, purified human enzymes, and animal models. Actinoplanes sp. ATCC 53771 is currently being used to produce metabolites of the macrolide immunosuppressant sirolimus. This bacterium is not well characterized and has poor growth conditions, including slow growth, and is difficult to engineer for the production of large quantities of metabolites. Our efforts are directed at (a) identifying P450s from Actinoplanes sp., (b) expressing and identifying P450 activity in an industrially relevant host, and (c) optimizing expression to create a controllable system for in vivo drug metabolism.

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.