Production of Recombinant Human Aldehyde Oxidase in Escherichia coli and Optimization of Its Application for the Preparative Synthesis of Oxidized Drug Metabolites

Abstract

Recombinant human aldehyde oxidase (AO) was expressed in Escherichia coli. Different cell disruption methods and conditions of cell culture in shake flasks and bioreactors and of biotransformation on an analytical scale were tested to optimize the synthesis of oxidized AO drug metabolites. The volumetric productivity was increased 24‐fold by optimizing the cell culture conditions. The highest yield was achieved in a 25 L stirred tank bioreactor under non‐oxygen‐limited conditions and high lactose feed rate. Suspensions of highly concentrated and well‐aerated whole cells at neutral pH and relatively low temperatures led to the best conversion. The solvent for the substrate and the buffering agent for the biotransformation had an important effect. In a biotransformation with AO, 210 mg of famciclovir was converted to diacetyl penciclovir a yield of 82 %. The optimized protocol represents a viable method for the preparative synthesis of oxidized AO metabolites of drugs.

Metabolism by Aldehyde Oxidase: Drug Design and Complementary Approaches to Challenges in Drug Discovery

“…14 Use of cellular or subcellular systems to provide a translatable profile of AO-mediated metabolite generation can be difficult due to the chemo-and regio-specificity of the enzyme, coupled with the variability in the expression of the enzyme in the different systems. Use of a MoCo producing E. coli-based bacterial system, ecoAO, 213,233 can provide a means by which AO metabolites can be generated in sufficient quantities for structural identification by NMR and for use as an authentic standard in any kinetic profile assay where substrate depletion alone will not provide all the relevant data.…”

“…Use of a MoCo producing E. coli-based bacterial system, ecoAO, , can provide a means by which AO metabolites can be generated in sufficient quantities for structural identification by NMR and for use as an authentic standard in any kinetic profile assay where substrate depletion alone will not provide all the relevant data.…”

The Development of Biocatalysis as a Tool for Drug Discovery

“…[41] served as a model reaction to demonstrate the applicability of this biocatalyst for preparative metabolite synthesis. [51] 3.6 UDP-glucuronosyltransferases (UGTs) UDP-glucuronosyltransferases (UGTs) are important enzymes of the phase II metabolism of xenobiotics. They are responsible for the glucuronidation reaction, which transforms for example potentially harmful lipophilic into hydrophilic compounds.…”

“…[46,48] The active metabolite penciclovir itself is used in other drugs for the local treatment of herpes simplex virus. [49] Human AOX1 was expressed in E. coli in catalytically active [50,51] The oxidation of famciclovir to diacetylpenciclovir under whole-cell biocatalysis on 200 mg scale…”