PhD Abstract

Fungal biofilm as a novel biocatalyst for the production of important pharmaceutical compound

Laura Quinn, University College Dublin, 2015

This project investigated the potential of the fungus Cunninghamella elegans to produce drug metabolites. It has previously been established that Cunninghamella elegans can be used as a microbial model of mammalian metabolism but has not yet been used to produce drug metabolites on a large scale. The first part of this project focused on developing a workflow for the application of Cunninghamellaelegans for the production of drug metabolites, from well-plate screening assays to the semi-preparative production of drug metabolites using C. elegans alginateimmobilised cells and biofilm. The simultaneous screening of a number of drugs was achieved using 12- and 96-well plates. Alginate-immobilised cells and biofilm both enabled semi-continuous production of hydroxylated metabolites through repetitive additions of drug and rejuvenation of the fungus. The productivity of the biofilm cultures was improved 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.

The effects of physicochemical parameters on drug metabolite production such as temperature, pH, solvents and substrate concentration were examined as there was no previous knowledge on their effects on biotransformation. It was demonstrated that the fungus was temperature sensitive and optimal biotransformations occurred between 23-32 ⁰C and that the greatest amount of biotransformation occurred at pH 7. The scalability of the biofilm-catalysed biotransformation process was investigated and it was observed that rotation speed had an effect on biofilm growth. Cytochrome P450 gene expression of C. elegans biofilm grown in 250 ml flasks and 2 l flasks were examined. An RT-PCR method was developed to study the expression levels of cytochrome P450 monooxygenase genes during biofilm development and after incubation with diclofenac. The gene expression levels of seven putative CYP genes that were previously found in the C. elegans sequence, were examined. The results showed that there was a difference in expression in C. elegans biofilm cultures and the level of expression previously seen in planktonic cells.

The triphenylmethane dye malachite green is used in the textile industry and as a biocide in fish farming. It is toxic to mammalian cells and has been shown to cause cancer in animals. Leucomalachite green is the major metabolite arising from the reduction of malachite green and because of its toxicity, mutagenicity and its relatively higher lipophilicity, it is retained in fish muscle and fat. In this project we investigated the decolourisation of malachite green by Cunninghamella elegans biofilm and planktonic cells and evaluated its reusability. It was demonstrated that complete decolourisation occurred within 15 min of addition of dye followed by a slower degradation of the dye in the biomass. The ability of the biofilm to decolourise the dye in a continuous process was investigated and with regular rejuvenation the biofilm could be used for at least 20 additions of dye over a 3 month period. Decolourisation could be conducted in acidic pH and in the presence of cytochrome P450 inhibitors.