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Casey, Eoin
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Casey, Eoin
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Casey, Eoin
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- PublicationFilamentous fungal biofilm for production of human drug metabolites(2013-07)
; ; In drug development, access to drug metabolites is essential for assessment of toxicity and pharmacokinetic studies. Metabolites are usually acquired via chemical synthesis, although biological production is potentially more efficient with fewer waste management issues. A significant problem with the biological approach is the effective half-life of the biocatalyst, which can be resolved by immobilisation. The fungus Cunninghamella elegans is well established as a model of mammalian metabolism, although it has not yet been used to produce metabolites on a large scale. Here, we describe immobilisation of C. elegans as a biofilm, which can transform drugs to important human metabolites. The biofilm was cultivated on hydrophilic microtiter plates and in shake flasks containing a steel spring in contact with the glass. Fluorescence and confocal scanning laser microscopy revealed that the biofilm was composed of a dense network of hyphae, and biochemical analysis demonstrated that the matrix was predominantly polysaccharide. The medium composition was crucial for both biofilm formation and biotransformation of flurbiprofen. In shake flasks, the biofilm transformed 86% of the flurbiprofen added to hydroxylated metabolites within 24 h, which was slightly more than planktonic cultures (76%). The biofilm had a longer effective lifetime than the planktonic cells, which underwent lysis after 2×72 h cycles, and diluting the Sabouraud dextrose broth enabled the thickness of the biofilm to be controlled while retaining transformation efficiency. Thus, C. elegans biofilm has the potential to be applied as a robust biocatalyst for the production of human drug metabolites required for drug development.683Scopus© Citations 28 - PublicationProduction of drug metabolites by immobilised Cunninghamella elegans: from screening to scale-upCunninghamella elegans is a fungus that has been used extensively as a microbial model of mammalian drug metabolism, whilst its potential as a biocatalyst for the preparative production of human drug metabolites has been often proposed, little effort has been made to enable this. Here, we describe a workflow for the application of C. elegans for the production of drug metabolites, starting from well-plate screening assays leading to the preparative production of drug metabolites using fungus immobilised either in alginate or as a biofilm. Using 12- and 96-well plates, the simultaneous screening of several drug biotransformations was achieved. To scale up the biotransformation, both modes of immobilisation enabled semi-continuous production of hydroxylated drug metabolites through repeated addition of drug and rejuvenation of the fungus. It was possible to improve the productivity in the biofilm culture for the production of 4′-hydroxydiclofenac from 1 mg/l h to over 4 mg/l h by reducing the incubation time for biotransformation and the number of rejuvenation steps.
493Scopus© Citations 24