Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans

Abstract

The yeast Arxula adeninivorans has been previously shown to secrete a large amount of an electro-active molecule. The molecule was produced by cells that had been cultivated in a rich medium, harvested, washed and then suspended in phosphate-buffered saline (PBS). The molecule was easily detectable after 60min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3days. The peak anodic potential of the oxidation peak was 0.42V, and it was shown to be a solution species rather than a cell-attached species. We have optimised the production of the molecule, identified it by high-pressure liquid chromatography (HPLC) fractionation and high-resolution mass spectrometric analysis and determined the pathway involved in its synthesis. It has a mass/charge ratio that corresponds to uric acid, and this identification was supported by comparing UV spectra and cyclic voltammograms of the samples to those of uric acid. An A. adeninivorans xanthine oxidase gene disruption mutant failed to produce uric acid, which added further validity to this identification. It also demonstrated that the purine catabolism pathway is involved in its production. A transgenic A. adeninivorans strain with a switchable urate oxidase gene (AUOX) accumulated uric acid when the gene was switched off but did not when the gene was switched on. Cultivation of cells on amino acid and purine-free minimal media with an inorganic nitrogen source suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway.