Appl Microbiol Biotechnol (2014) 98:2223–2229 DOI 10.1007/s00253-013-5487-4
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans Jonathan Williams & Anke Trautwein-Schult & Dagmara Jankowska & Gotthard Kunze & Marie A. Squire & Keith Baronian
Received: 30 July 2013 / Revised: 12 December 2013 / Accepted: 15 December 2013 / Published online: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014
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 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, 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 highresolution 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 purinefree minimal media with an inorganic nitrogen source J. Williams : K. Baronian (*) School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand e-mail: [email protected] M. A. Squire Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand A. Trautwein-Schult : D. Jankowska : G. Kunze Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr.3, 06466 Gatersleben, Germany
suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway. Keywords Yeast . Secretion of uric acid . Purine degradation pathway . Identification of uric acid
Introduction We are investigating the use of yeast as the catalytic component of a microbial fuel cell (MFC). Eukaryote cells are not ideal candidates for a biological catalyst in MFCs because the electrons from catabolism are produced in the cytoplasm and mitochondria, and accessing these electrons is usually only possible using lipophilic mediators that can shuttle electrons across the cell membrane. Some electrons are transported across the cell membrane via trans-plasma membrane transport proteins and can be also harvested using a mediator. Recently, it has been demonstrated that electrons are transported across the cell wall in Saccharomyces cerevisiae (Rawson et al. 2012) and are available to an external electrode. However, the currents produced by direct electron transfer are so small that they are of no practical value in a MFC. The transfer of electrons from a cell to an electrode may also be self-mediated, that is a reduced molecule is exported from the cell and can transfer its electron(s) to an electrode. Haslett et al. (2011) reported the presence of an electro-active molecule in the extracellular environment of the yeast Arxula adeninivorans. They reported that cyclic voltammograms (CVs) of A. adeninivorans cell creams and supernatant derived from them had an irreversible oxidation peak at +0.42 V and demonstrated that the peak was generated by a solution species that accumulated rapidly in the extracellular buffer. They also reported that the peak potential was independent of pH in the pH range of 4–12 but would shift positively at a
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lower pH value. In contrast, a very small oxidation peak at the same potential was only detectable in CVs of S. cerevisiae supernatant after several days of incubation. A. adeninivorans is in many respects an ideal MFC biocatalyst; it has a very broad substrate range and is able to grow at up to 48 °C (Wartman et al. 1995) and 20 % salinity. The finding that it exports a reduced molecule makes it possible to transfer more electrons to an electrode than those available through direct electron transfer from the external surface of the cell wall. Additionally, it may be possible to increase the wildtype levels of the redox molecule by genetically modifying the pathway that produces the molecule. This paper reports on the optimisation of the synthesis and accumulation of uric acid A. adeninivorans, its identification, identification of the pathway involved in synthesis by gene manipulation and identification of the source of the molecule.
Methods and materials Yeast strains, media and growth conditions Details of the six A. adeninivorans strains used in this investigation are given in Table 1. A. adeninivorans LS3 and the mutant strains 135, G1212 (aleu2 atrp1::ALEU2), G1212/ YRC102, G1212/YRC102-AYNI1-AUOR and G1224 used in this study were deposited in the strain collection of the Department of Biology, University of Greifswald (LS3 as A. adeninivorans SBUG 724; all other strains as listed in the table). Other yeast species used in this study were Candida albicans, clinical isolate and S. cerevisiae diploid strain α/a F832×Y383 both held in the UC School of Biological Sciences culture collection; Candida parapsilosis, CDC MCC499; and Candida lambica, ICMP 16880. Cells were grown in yeast extract peptone dextrose (YEPD) broth [neutralised soya peptone 20 g L−1 (Oxoid, LP0044, Oxoid Limited, Basingstoke, Hampshire RG24
8PW, UK), yeast extract 10 g L−1 (Oxoid, LP0021, Oxoid Limited, Basingstoke, Hampshire RG24 8PW, UK), D-glucose 20 g L−1 (LabServ, AR, Thermo Fisher Scientific Australia Pty Ltd., Scoresby, Vic 3179, Australia)] sterilised at 121 °C. Yeast nitrogen base without amino acids and ammonium sulphate (YNB without aa and N; Difco, Franklin Lakes, NJ, USA) was used in the determination of the source of purines required for uric acid synthesis. The medium was prepared as a 10-times concentrate [17 g L−1 YNB with a carbon source (dextrose or glycerol 50 g L−1) and a nitrogen source (ammonium sulphate) added to the concentrate]. The 10-times concentrate with C and N sources was sterilised by filtration (0.45 μm) and added to sterile deionised water in 250-mL indented culture flasks to make a total volume of 100 mL. All yeast strains were cultured in two stages. First, a preculture was prepared by inoculating 100 mL YNB medium with a single colony from plate cultures and incubating for 20 h at 37 °C, 180 rpm, in a shaking incubator. 1 millilitre of the pre-culture was used to inoculate duplicate 100 mL medium contained in 250-mL indented culture flasks. These were incubated in a shaking incubator for 20 h, 180 rpm at a temperature of 37 or 47 °C. Extracellular uric acid accumulation Cells were harvested from the cultures by centrifugation at 8,228g, at a temperature of 4 °C for 10 min. The supernatant was discarded, and the cells were washed (twice) by resuspending the pellet in 18.2 MΩ water and centrifuging. The wet weight of the pellet was determined and sterile phosphate-buffered saline (PBS, 0.05 M K2HPO4/KH2PO4, 0.1 M KCl) was added, 1:1 v/w. The airspace above the cell suspension was flushed with N2 and the cells were incubated for 20 h at 37 or 47 °C. Cells were then removed from the fluid by centrifugation for 10 min at 2,870g, at a temperature of 4 °C, and the supernatant was removed and filtered (0.45 μm). Supernatant samples that were not used immediately were sparged for 10 min with N2 gas and stored at 4 °C.
Table 1 A. adeninivorans strains used in this study Strain
Description
Reference
LS3
A dimorphic wild-type strain with yeast morphology up to 42 °C and a filamentous morphology at higher temperatures (CBS 8244) A mutant strain that is filamentous at all temperatures
Gienow et al. (1990)
A Δatrp1 gene disruption mutant derived from LS3 G1212 transformant containing the empty YRC102 vector without insertions and was used as a control to exclude vector-only effects on the cell G1212/YRC102 with inserted AYNI1 promoter–AUOR gene-PHO5 terminator expression module for a strong NaNO3-inducible expression of the AUOR gene A Δaxor gene disruption mutant preventing production of uric acid via purine catabolism
Steinborn et al. (2007) Trautwein-Schult et al. (2013) Trautwein-Schult et al. (2013) Jankowska et al. (2013a)
135 G1212 (aleu2 atrp1::ALEU2) G1212/YRC102 G1212/YRC102-AYNI1-AUOR39 G1224 (aleu2 atrp1::ALEU2 axor::ATRP1)
Wartmann et al. (2000)
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Cyclic voltammetry Cyclic voltammetry (CV) was performed using the ADInstruments PowerLab 2/20 with ML160 Potentiostat, using eDAQ EChem v2.5.4 data logging software (both eDAQ Pty Ltd., Denistone East, Australia). A threeelectrode configuration comprising a 2-mm DIA glassy carbon working electrode (Tianjin Aida Hengsheng Technology Co. Ltd., Nankai, Tianjin, China), platinum counter electrode and a Ag/AgCl reference electrode (3 M KCl) (G-Glass, Victoria, Australia) was used for voltammetry. The glassy carbon electrode was polished between each scan using aluminium oxide powder (0.3 μm, LECO) on a Lecloth (LECO, St Joseph, MI, USA). All CVs were performed in a Faraday cage and were scanned from +0.2 to +0.8 V at 100 mV/s with three replicate CVs recorded for each sample. The area under voltammogram peak was integrated to give an estimate of the charge transferred by uric acid in the sample.
HPLC isolation and MS analysis HPLC was used to isolate the electro-active molecule from the supernatant solution; 100 μL of sample was separated on a 250 mm×4.60 mm Phenomenex Jupiter C18 column (Torrance, CA, USA) at 20±3 °C at a flow rate of 1.0 mL/min. The separation protocol used to isolate and collect the molecule was 0 % acetonitrile/100 % water for 7 min, ramping to 30 % acetonitrile/70 % water over 4 min, holding for 2 min, increasing to 100 % acetonitrile over 2 min, holding for 2 min, decreasing to 0 % acetonitrile/100 % water over 2 min and reequilibrating for 5 min. Peaks at 210 nm were detected using a Dionex UltiMate 3000 Diode Array Detector and collected using a Dionex ASI-100 Autosampler (Sunnyvale, CA, USA). High-resolution mass spectrometric data was collected on a maXis 3G UHR-Qq-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) coupled to a Dionex UltiMate 3000 LC system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Five microlitres of sample was injected into a flow of 50:50 water (0.5 % formic acid)/ acetonitrile at 0.2 mL/min. Five microlitres of ESI-L Low Concentration Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) was injected after each sample to calibrate the system.
Results Electrochemistry of the electro-active molecule CVs of the supernatant from cells incubated in PBS for 20 h at 37 or 47 °C were performed. Typical CVs for the supernatant are shown in Fig. 1. The peak anodic potentials for the supernatant of A. adeninivorans LS3, 135 and G1212 at 37 °C are all +0.39 V. Haslett et al. (2011) reported a peak potential for LS3 supernatant of +0.42. All strains used in this study showed lower peak potentials, although in early trials, peak potentials were generally higher (+0.43 to +0.46 V). In these trials, the supernatant was not filtered, and electrode fouling may account for this difference. Furthermore, the pH of the supernatant samples was not controlled, as the bulk volume of PBS was expected to absorb any small perturbations in pH; however, the peak anodic potential of uric acid is affected by pH, tending to become more negative as the solution becomes more acidic (Zhang and Lin 2001). Expansion of the current axis revealed a small reduction peak corresponding to an oxidation peak, indicating that the oxidation process of uric acid is electrochemically slightly reversible (data not shown). In all three strains, supernatant from cells incubated at 47 °C produced larger oxidation peaks than those incubated at 37 °C (Fig. 1). Integration of the peaks for strains LS3, 135 and G1212 showed that there was up to a twofold increase in charge transfer in LS3 and 135 and a little less in strain G1212 (Table 2). Furthermore, the results showed a little difference between concentrations of uric acid in supernatant derived from LS3 (yeast at 37 °C) and 135 (filamentous at 37 °C), suggesting that the morphology of the cells does not cause a change in the production rate of the molecule. All further incubations of the cells were at 47 °C.
37°C incubation
30
47°C incubation 25 20 Current [ A]
Samples for high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) analysis were from cells incubated in 18.2 MΩ H2O for 3 days to increase the concentration of the unidentified molecule and to reduce the salt content of the supernatant.
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15 10 5 0 0 -5
0.1
0.2
0.3
0.4 0.5 Potential [V]
0.6
0.7
0.8
0.9
Fig. 1 Typical CVs for A. adeninivorans LS3 incubated in PBS at 37 and 47 °C showing the increase in size of the oxidation peak for cells incubated at 47 °C
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Table 2 The average integral of current with respect to time for A. adeninivorans LS3, 135 and G1212 showing the differences between cells grown at 37 and 47 °C Strain
Current at 37 °C (μA/s)
Current at 47 °C (μA/s)
LS3 135 G1212
3.79 3.71 3.74
8.27 8.67 6.63
Identification of the electro-active molecule Initially, in the HPLC separations, the electro-active molecule was eluted in a broad over-range peak. Modification to the elution gradient, however, separated this peak into three peaks. Samples for voltammetry were pooled matching fractions from multiple runs of the purification method. Because the three peaks have such similar elution times, the small amount of variation in elution and collection times across many runs made it difficult to localise the electrochemical activity to one specific peak. It had been observed that size of the reduction peak in samples of supernatant stored under air would steadily reduce over time with the reduction signal disappearing entirely in a few days. This was presumed to be the result of oxidation by atmospheric oxygen. In order to isolate which peak represented the electrochemically active molecule, a sample of supernatant was stored under air for 3 days before performing HPLC analysis. The chromatogram was then compared to that of the same sample analysed while it was fresh. Matching the chromatogram peaks (Fig. 2) revealed a noticeable decrease in the area of the first peak in the unpreserved sample (a) and a concomitant increase in a minor peak seen in the fresh voltammogram (c). The first peak (a) was thus thought to be the reduced target molecule and the increased peak the
oxidised product of the target molecule (c). Peaks (b and d) appear to be unchanged by exposure to air. The fraction corresponding to peak (a) was analysed by mass spectrometry. It gave a strong signal at m/z 169.0356, which is a high-resolution fit for an empirical formula of C5H5N4O3 (calculated MH+ requires m/z 169.0356). Based on the assumption that one of the hydrogen atoms is the source of the ionisation, the unknown molecule was tentatively identified as uric acid. A 5-μL sample of sodium urate solution was injected into the column to compare its elution time with the supernatant peak. The elution times were not a perfect match but were within the variation previously observed between runs (Fig. 3). Further evidence for the identification of the peak as uric acid is provided by the comparison of its UV spectrum with that of a sodium urate. These are a very close match implying that the two molecules are either the same or extremely closely structurally related (Fig. 4). These spectra differ a little from those typically given in the literature; Zinellu et al. (2004) describes peaks at 197, 233 and 292 nm, while Mei et al. (1996) depict the last two peaks at 238 and 292 nm, and Vasilevskiĭ et al. (2001) give 238 and 293 nm. In contrast, the putative uric acid peak from supernatant samples generally displays maxima at 197, 230 and 285 nm. While these figures tend to indicate maxima at slightly shorter wavelengths, the UV spectrum of uric acid does vary slightly with pH, shifting toward longer wavelengths as it is deprotonated (Stimson and Reuter 1943). Confirmation of production of uric acid by gene manipulation The pathway for the breakdown of purine to uric acid is shown in Fig. 5. The pathway in A. adeninivorans differs slightly from the ‘standard’ pathway in that it lacks adenosine deaminase (Jankowska et al. 2013b). Adenosine is degraded to adenine by purine nucleoside phosphorylase (PNP), and adenine is degraded to hypoxanthine by adenine deaminase.
Absorbance [mAU]
700 Sample Urate
600 500
400 300 200 100 0
5
Fig. 2 Overlaid chromatograms of a fresh supernatant sample and the same sample after being allowed to oxidise. Arrows indicate matching pairs of peaks. Peak a decreases on exposure to air, while peak c increases. Peaks b and d appear to be unchanged by exposure to air
5.5
6 Time [min]
6.5
7
Fig. 3 Overlaid chromatograms of the peaks in a supernatant sample and that of a sodium urate solution. The similar elution times for the sodium urate peak and the putative uric acid peak support the tentative identification
Appl Microbiol Biotechnol (2014) 98:2223–2229
2227
1400 Sample
1200
Urate
Absorbance [mAU]
1000 800 600 400 200 0 190 -200
210
230
250
270
290
310
330
Wavelength [nm]
Fig. 4 The overlaid UV spectra for the putative uric acid peak from a supernatant sample and a sample of sodium urate. The shapes of the spectra are very similar, and the maxima of each curve match each other within 0.5 nm
The mutant A. adeninivorans G1224 is a xanthine oxidase/ dehydrogenase gene disruption mutant that is incapable of converting hypoxanthine to xanthine and xanthine to uric acid (Jankowska et al. 2013a). Supernatant from cultures of this strain does not show a uric acid peak (Fig. 6). This supports the identification of the molecule as uric acid, and furthermore, it can be inferred from this result that the uric acid is the product of purine degradation and not some other pathway.
Fig. 5 Diagram showing the purine degradation pathway for A. adeninivorans. Purines that are not salvaged are degraded to uric acid and further to allantoin and allantoic acid
A transgenic strain, G1212/YRC102-AYNI1-AUOR39, which has urate oxidase linked to a nitrate-inducible AYNI1 promoter, demonstrated that the observed oxidation peak was the result of a molecule that was a substrate for urate oxidase, i.e. uric acid, and provided further evidence for the purine degradation pathway being involved in the high levels of uric acid production. Figure 7 shows the absence of the peak when the cells are incubated in the presence of YNB medium with NaNO3, indicating that urate oxidase is being produced and degrading uric acid, whereas in the absence of NaNO3, uric acid accumulates, as evidenced by the oxidation peak. Determination of the source of the purines, either exogenous or endogenous, was investigated by cultivating A. adeninivorans LS3 on a medium that was free of purines. YNB medium is free of purines and amino acids, with added NH4SO4 being the only significant N source. The medium does contain very small amounts of nitrogen-containing vitamins, but their concentration is less than that required for the amount of N in the uric acid produced. The medium from cells incubated in this medium was analysed by CV, which indicated that the cells were producing uric acid. This suggests that uric acid or its precursors are synthesised by the cell from NH4SO4. Cyclic voltammograms on 24-h supernatants of four species that are related to A. adeninivorans (C. albicans, C. parapsilosis, C. lambica and S. cerevisiae) did not show
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20 G1224
G1212/YR C102
Current [ A]
15
10
5
0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Potential [V]
-5
Fig. 6 CVs for A. adeninivorans G1212/YRC102 and G1224. The G1224 strain has the gene for xanthine oxidase deleted, resulting in the absence of a uric acid oxidation peak
peaks at ~0.5 V. These results suggest that it is a purine in excess of normal cell function that is being degraded in A. adeninivorans to produce uric acid.
Discussion The electrochemical properties of uric acid are well documented as it is a significant analyte for the development of voltammetric biosensors (Zhang and Lin 2001; Wang et al. 2002; Raj and Ohsaka 2003; Ramesh and Sampath 2004). The properties described by these authors, such as oxidation potentials and near-irreversibility of oxidation, parallel the characteristics collected from the supernatant samples from the incubation of A. adeninivorans incubated in PBS. The overproduction of uric acid probably consumes a significant amount of energy. It is possible that a selection 20 G1212/YRC102
18 16
G1212/YRC102-AYNI1AUOR39 with nitrate G1212/YRC102-AYNI1AUOR39 without nitrate
Current [ A]
14 12 10 8 6 4 2 0 -2 -4
0
0.1
0.2
0.3
0.4 0.5 Potential [V]
0.6
0.7
0.8
0.9
Fig. 7 CVs for A. adeninivorans G1212/YRC102 and G1212/YRC102AYNI-AUOR39 with a NO3-inducible expression module that results in the production of urate oxidase when NO3 is present in the medium, resulting in the absence of a uric acid oxidation peak. The peak is visible when the same cells are grown in a NO3-free medium
pressure has given rise to and maintains this phenomenon. Masoud et al. (2012) report that uric acid is weakly antimicrobial; however, they do not give the concentration at which they made this observation. They also show that uric acid complexed with U has increased antimicrobial activity. Some primates, birds, reptiles and insects also excrete uric acid as the final product of nitrogenous catabolism. Ames et al. (1981) hypothesise that the accumulation of uric acid in the blood plasma of humans acts as a potent antioxidant. That the secretion of uric acid into the extracellular environment of Arxula has an antioxidant function that confers an advantage on the cells is, however, not known. Chen and Fink (2006) describe the release of two aromatic alcohols which act as signalling molecules in S. cerevisiae and, in their introduction, discuss the occurrence of other quorum-sensing molecules in fungi. Necrotic mammalian cells release uric acid as a danger signal to activate both innate and adaptive immune effectors including neutrophils, cytotoxic T cells and antibodies (Behrens et al. 2008). It may be that the release of uric acid by A. adeninivorans also acts as a communication mechanism, i.e. quorum-sensing. Whatever the purpose, if there is one, in the release of uric acid by A. adeninivorans, it is interesting because its production is not seen in the three other yeast species that we have tested. We have previously reported that A. adeninivorans lacks an adenosine deaminase which is atypical of the common purine degradation pathway. Instead, it degrades adenosine monophosphate with PNP (Jankowska et al. 2013b). The product adenine is then degraded with adenine deaminase, which is seen in the common purine breakdown pathway. Interestingly, an analysis of the Cryptococcus neoformans genome has found that it has neither the gene for adenosine deaminase or adenine deaminase (Morrow et al. 2012) and degrades adenosine monophosphate using an AMP deaminase to inosine monophosphate and, further, to hypoxanthine, which is the product of adenine deaminase in Arxula. In addition, Cultrone et al. (2005) have reported that xanthine dehydrogenase is also absent in C. neoformans, but a potential equivalent, an α-ketoglutarate-dependent dioxygenase, is present which is also different from Arxula which uses xanthine dehydrogenase (Jankowska 2013a). This variation in these two yeast species adds to the evidence that degradation of purines is, in general, quite variable. Our findings support the notion that the synthesis of purines and their subsequent degradation to uric acid is the source of the extracellular uric acid. There are many pathways that contribute to the accumulation of purines, which can be catabolised to uric acid, and significant overproduction of purine in excess of cell requirements might provide the purine catabolism pathway with the necessary substrate to produce extra uric acid.
Appl Microbiol Biotechnol (2014) 98:2223–2229
A phenotype of excessive purine synthesis that results in elevated uric acid excretion is well documented in humans as the inherited disorder, Lesch-Nyhan syndrome (LNS) (Sculley et al. 1992). This disorder arises as a result of the failure of the purine salvage pathway, more specifically, a deficiency in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) pathway. The absence of HGPRT prevents free purines from being salvaged, and as a result, they are degraded to uric acid. Furthermore, since phosphoribosyl pyrophosphate (PRPP) is not being used in the salvage process, it becomes available to allosterically activate de novo purine production, increasing overall purine production and thus elevating uric acid production further. Cloning a functional copy of the gene for HGPRT, HPT1, into a uric acid-producing strain of A. adeninivorans to determine if high uric acid production is suppressed would confirm the presence of this mutation in Arxula. Alternatively, a mutant strain of S. cerevisiae with HPT1 knocked out should produce high levels of uric acid, which would also provide evidence for this possibility. The use of uric acid as a source of electrons in an A. adeninivorans microbial fuel cell would require a significant increase in its production. We have produced a strain that has the urate oxidase gene disrupted so that uric acid cannot be degraded, thereby increasing its secretion, and we are about to commence testing the strain in a MFC. Acknowledgments This work was funded by Science and Innovation, New Zealand Ministry of Business, Innovation and Employment (contract LVLX0802DET) and the University of Canterbury.
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APPLIED MICROBIAL AND CELL PHYSIOLOGY
Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans Jonathan Williams & Anke Trautwein-Schult & Dagmara Jankowska & Gotthard Kunze & Marie A. Squire & Keith Baronian
Received: 30 July 2013 / Revised: 12 December 2013 / Accepted: 15 December 2013 / Published online: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014
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 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, 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 highresolution 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 purinefree minimal media with an inorganic nitrogen source J. Williams : K. Baronian (*) School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand e-mail: [email protected] M. A. Squire Department of Chemistry, University of Canterbury, Private Bag 4800, Christchurch, New Zealand A. Trautwein-Schult : D. Jankowska : G. Kunze Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr.3, 06466 Gatersleben, Germany
suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway. Keywords Yeast . Secretion of uric acid . Purine degradation pathway . Identification of uric acid
Introduction We are investigating the use of yeast as the catalytic component of a microbial fuel cell (MFC). Eukaryote cells are not ideal candidates for a biological catalyst in MFCs because the electrons from catabolism are produced in the cytoplasm and mitochondria, and accessing these electrons is usually only possible using lipophilic mediators that can shuttle electrons across the cell membrane. Some electrons are transported across the cell membrane via trans-plasma membrane transport proteins and can be also harvested using a mediator. Recently, it has been demonstrated that electrons are transported across the cell wall in Saccharomyces cerevisiae (Rawson et al. 2012) and are available to an external electrode. However, the currents produced by direct electron transfer are so small that they are of no practical value in a MFC. The transfer of electrons from a cell to an electrode may also be self-mediated, that is a reduced molecule is exported from the cell and can transfer its electron(s) to an electrode. Haslett et al. (2011) reported the presence of an electro-active molecule in the extracellular environment of the yeast Arxula adeninivorans. They reported that cyclic voltammograms (CVs) of A. adeninivorans cell creams and supernatant derived from them had an irreversible oxidation peak at +0.42 V and demonstrated that the peak was generated by a solution species that accumulated rapidly in the extracellular buffer. They also reported that the peak potential was independent of pH in the pH range of 4–12 but would shift positively at a
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lower pH value. In contrast, a very small oxidation peak at the same potential was only detectable in CVs of S. cerevisiae supernatant after several days of incubation. A. adeninivorans is in many respects an ideal MFC biocatalyst; it has a very broad substrate range and is able to grow at up to 48 °C (Wartman et al. 1995) and 20 % salinity. The finding that it exports a reduced molecule makes it possible to transfer more electrons to an electrode than those available through direct electron transfer from the external surface of the cell wall. Additionally, it may be possible to increase the wildtype levels of the redox molecule by genetically modifying the pathway that produces the molecule. This paper reports on the optimisation of the synthesis and accumulation of uric acid A. adeninivorans, its identification, identification of the pathway involved in synthesis by gene manipulation and identification of the source of the molecule.
Methods and materials Yeast strains, media and growth conditions Details of the six A. adeninivorans strains used in this investigation are given in Table 1. A. adeninivorans LS3 and the mutant strains 135, G1212 (aleu2 atrp1::ALEU2), G1212/ YRC102, G1212/YRC102-AYNI1-AUOR and G1224 used in this study were deposited in the strain collection of the Department of Biology, University of Greifswald (LS3 as A. adeninivorans SBUG 724; all other strains as listed in the table). Other yeast species used in this study were Candida albicans, clinical isolate and S. cerevisiae diploid strain α/a F832×Y383 both held in the UC School of Biological Sciences culture collection; Candida parapsilosis, CDC MCC499; and Candida lambica, ICMP 16880. Cells were grown in yeast extract peptone dextrose (YEPD) broth [neutralised soya peptone 20 g L−1 (Oxoid, LP0044, Oxoid Limited, Basingstoke, Hampshire RG24
8PW, UK), yeast extract 10 g L−1 (Oxoid, LP0021, Oxoid Limited, Basingstoke, Hampshire RG24 8PW, UK), D-glucose 20 g L−1 (LabServ, AR, Thermo Fisher Scientific Australia Pty Ltd., Scoresby, Vic 3179, Australia)] sterilised at 121 °C. Yeast nitrogen base without amino acids and ammonium sulphate (YNB without aa and N; Difco, Franklin Lakes, NJ, USA) was used in the determination of the source of purines required for uric acid synthesis. The medium was prepared as a 10-times concentrate [17 g L−1 YNB with a carbon source (dextrose or glycerol 50 g L−1) and a nitrogen source (ammonium sulphate) added to the concentrate]. The 10-times concentrate with C and N sources was sterilised by filtration (0.45 μm) and added to sterile deionised water in 250-mL indented culture flasks to make a total volume of 100 mL. All yeast strains were cultured in two stages. First, a preculture was prepared by inoculating 100 mL YNB medium with a single colony from plate cultures and incubating for 20 h at 37 °C, 180 rpm, in a shaking incubator. 1 millilitre of the pre-culture was used to inoculate duplicate 100 mL medium contained in 250-mL indented culture flasks. These were incubated in a shaking incubator for 20 h, 180 rpm at a temperature of 37 or 47 °C. Extracellular uric acid accumulation Cells were harvested from the cultures by centrifugation at 8,228g, at a temperature of 4 °C for 10 min. The supernatant was discarded, and the cells were washed (twice) by resuspending the pellet in 18.2 MΩ water and centrifuging. The wet weight of the pellet was determined and sterile phosphate-buffered saline (PBS, 0.05 M K2HPO4/KH2PO4, 0.1 M KCl) was added, 1:1 v/w. The airspace above the cell suspension was flushed with N2 and the cells were incubated for 20 h at 37 or 47 °C. Cells were then removed from the fluid by centrifugation for 10 min at 2,870g, at a temperature of 4 °C, and the supernatant was removed and filtered (0.45 μm). Supernatant samples that were not used immediately were sparged for 10 min with N2 gas and stored at 4 °C.
Table 1 A. adeninivorans strains used in this study Strain
Description
Reference
LS3
A dimorphic wild-type strain with yeast morphology up to 42 °C and a filamentous morphology at higher temperatures (CBS 8244) A mutant strain that is filamentous at all temperatures
Gienow et al. (1990)
A Δatrp1 gene disruption mutant derived from LS3 G1212 transformant containing the empty YRC102 vector without insertions and was used as a control to exclude vector-only effects on the cell G1212/YRC102 with inserted AYNI1 promoter–AUOR gene-PHO5 terminator expression module for a strong NaNO3-inducible expression of the AUOR gene A Δaxor gene disruption mutant preventing production of uric acid via purine catabolism
Steinborn et al. (2007) Trautwein-Schult et al. (2013) Trautwein-Schult et al. (2013) Jankowska et al. (2013a)
135 G1212 (aleu2 atrp1::ALEU2) G1212/YRC102 G1212/YRC102-AYNI1-AUOR39 G1224 (aleu2 atrp1::ALEU2 axor::ATRP1)
Wartmann et al. (2000)
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Cyclic voltammetry Cyclic voltammetry (CV) was performed using the ADInstruments PowerLab 2/20 with ML160 Potentiostat, using eDAQ EChem v2.5.4 data logging software (both eDAQ Pty Ltd., Denistone East, Australia). A threeelectrode configuration comprising a 2-mm DIA glassy carbon working electrode (Tianjin Aida Hengsheng Technology Co. Ltd., Nankai, Tianjin, China), platinum counter electrode and a Ag/AgCl reference electrode (3 M KCl) (G-Glass, Victoria, Australia) was used for voltammetry. The glassy carbon electrode was polished between each scan using aluminium oxide powder (0.3 μm, LECO) on a Lecloth (LECO, St Joseph, MI, USA). All CVs were performed in a Faraday cage and were scanned from +0.2 to +0.8 V at 100 mV/s with three replicate CVs recorded for each sample. The area under voltammogram peak was integrated to give an estimate of the charge transferred by uric acid in the sample.
HPLC isolation and MS analysis HPLC was used to isolate the electro-active molecule from the supernatant solution; 100 μL of sample was separated on a 250 mm×4.60 mm Phenomenex Jupiter C18 column (Torrance, CA, USA) at 20±3 °C at a flow rate of 1.0 mL/min. The separation protocol used to isolate and collect the molecule was 0 % acetonitrile/100 % water for 7 min, ramping to 30 % acetonitrile/70 % water over 4 min, holding for 2 min, increasing to 100 % acetonitrile over 2 min, holding for 2 min, decreasing to 0 % acetonitrile/100 % water over 2 min and reequilibrating for 5 min. Peaks at 210 nm were detected using a Dionex UltiMate 3000 Diode Array Detector and collected using a Dionex ASI-100 Autosampler (Sunnyvale, CA, USA). High-resolution mass spectrometric data was collected on a maXis 3G UHR-Qq-TOF mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) coupled to a Dionex UltiMate 3000 LC system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Five microlitres of sample was injected into a flow of 50:50 water (0.5 % formic acid)/ acetonitrile at 0.2 mL/min. Five microlitres of ESI-L Low Concentration Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) was injected after each sample to calibrate the system.
Results Electrochemistry of the electro-active molecule CVs of the supernatant from cells incubated in PBS for 20 h at 37 or 47 °C were performed. Typical CVs for the supernatant are shown in Fig. 1. The peak anodic potentials for the supernatant of A. adeninivorans LS3, 135 and G1212 at 37 °C are all +0.39 V. Haslett et al. (2011) reported a peak potential for LS3 supernatant of +0.42. All strains used in this study showed lower peak potentials, although in early trials, peak potentials were generally higher (+0.43 to +0.46 V). In these trials, the supernatant was not filtered, and electrode fouling may account for this difference. Furthermore, the pH of the supernatant samples was not controlled, as the bulk volume of PBS was expected to absorb any small perturbations in pH; however, the peak anodic potential of uric acid is affected by pH, tending to become more negative as the solution becomes more acidic (Zhang and Lin 2001). Expansion of the current axis revealed a small reduction peak corresponding to an oxidation peak, indicating that the oxidation process of uric acid is electrochemically slightly reversible (data not shown). In all three strains, supernatant from cells incubated at 47 °C produced larger oxidation peaks than those incubated at 37 °C (Fig. 1). Integration of the peaks for strains LS3, 135 and G1212 showed that there was up to a twofold increase in charge transfer in LS3 and 135 and a little less in strain G1212 (Table 2). Furthermore, the results showed a little difference between concentrations of uric acid in supernatant derived from LS3 (yeast at 37 °C) and 135 (filamentous at 37 °C), suggesting that the morphology of the cells does not cause a change in the production rate of the molecule. All further incubations of the cells were at 47 °C.
37°C incubation
30
47°C incubation 25 20 Current [ A]
Samples for high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) analysis were from cells incubated in 18.2 MΩ H2O for 3 days to increase the concentration of the unidentified molecule and to reduce the salt content of the supernatant.
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15 10 5 0 0 -5
0.1
0.2
0.3
0.4 0.5 Potential [V]
0.6
0.7
0.8
0.9
Fig. 1 Typical CVs for A. adeninivorans LS3 incubated in PBS at 37 and 47 °C showing the increase in size of the oxidation peak for cells incubated at 47 °C
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Table 2 The average integral of current with respect to time for A. adeninivorans LS3, 135 and G1212 showing the differences between cells grown at 37 and 47 °C Strain
Current at 37 °C (μA/s)
Current at 47 °C (μA/s)
LS3 135 G1212
3.79 3.71 3.74
8.27 8.67 6.63
Identification of the electro-active molecule Initially, in the HPLC separations, the electro-active molecule was eluted in a broad over-range peak. Modification to the elution gradient, however, separated this peak into three peaks. Samples for voltammetry were pooled matching fractions from multiple runs of the purification method. Because the three peaks have such similar elution times, the small amount of variation in elution and collection times across many runs made it difficult to localise the electrochemical activity to one specific peak. It had been observed that size of the reduction peak in samples of supernatant stored under air would steadily reduce over time with the reduction signal disappearing entirely in a few days. This was presumed to be the result of oxidation by atmospheric oxygen. In order to isolate which peak represented the electrochemically active molecule, a sample of supernatant was stored under air for 3 days before performing HPLC analysis. The chromatogram was then compared to that of the same sample analysed while it was fresh. Matching the chromatogram peaks (Fig. 2) revealed a noticeable decrease in the area of the first peak in the unpreserved sample (a) and a concomitant increase in a minor peak seen in the fresh voltammogram (c). The first peak (a) was thus thought to be the reduced target molecule and the increased peak the
oxidised product of the target molecule (c). Peaks (b and d) appear to be unchanged by exposure to air. The fraction corresponding to peak (a) was analysed by mass spectrometry. It gave a strong signal at m/z 169.0356, which is a high-resolution fit for an empirical formula of C5H5N4O3 (calculated MH+ requires m/z 169.0356). Based on the assumption that one of the hydrogen atoms is the source of the ionisation, the unknown molecule was tentatively identified as uric acid. A 5-μL sample of sodium urate solution was injected into the column to compare its elution time with the supernatant peak. The elution times were not a perfect match but were within the variation previously observed between runs (Fig. 3). Further evidence for the identification of the peak as uric acid is provided by the comparison of its UV spectrum with that of a sodium urate. These are a very close match implying that the two molecules are either the same or extremely closely structurally related (Fig. 4). These spectra differ a little from those typically given in the literature; Zinellu et al. (2004) describes peaks at 197, 233 and 292 nm, while Mei et al. (1996) depict the last two peaks at 238 and 292 nm, and Vasilevskiĭ et al. (2001) give 238 and 293 nm. In contrast, the putative uric acid peak from supernatant samples generally displays maxima at 197, 230 and 285 nm. While these figures tend to indicate maxima at slightly shorter wavelengths, the UV spectrum of uric acid does vary slightly with pH, shifting toward longer wavelengths as it is deprotonated (Stimson and Reuter 1943). Confirmation of production of uric acid by gene manipulation The pathway for the breakdown of purine to uric acid is shown in Fig. 5. The pathway in A. adeninivorans differs slightly from the ‘standard’ pathway in that it lacks adenosine deaminase (Jankowska et al. 2013b). Adenosine is degraded to adenine by purine nucleoside phosphorylase (PNP), and adenine is degraded to hypoxanthine by adenine deaminase.
Absorbance [mAU]
700 Sample Urate
600 500
400 300 200 100 0
5
Fig. 2 Overlaid chromatograms of a fresh supernatant sample and the same sample after being allowed to oxidise. Arrows indicate matching pairs of peaks. Peak a decreases on exposure to air, while peak c increases. Peaks b and d appear to be unchanged by exposure to air
5.5
6 Time [min]
6.5
7
Fig. 3 Overlaid chromatograms of the peaks in a supernatant sample and that of a sodium urate solution. The similar elution times for the sodium urate peak and the putative uric acid peak support the tentative identification
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2227
1400 Sample
1200
Urate
Absorbance [mAU]
1000 800 600 400 200 0 190 -200
210
230
250
270
290
310
330
Wavelength [nm]
Fig. 4 The overlaid UV spectra for the putative uric acid peak from a supernatant sample and a sample of sodium urate. The shapes of the spectra are very similar, and the maxima of each curve match each other within 0.5 nm
The mutant A. adeninivorans G1224 is a xanthine oxidase/ dehydrogenase gene disruption mutant that is incapable of converting hypoxanthine to xanthine and xanthine to uric acid (Jankowska et al. 2013a). Supernatant from cultures of this strain does not show a uric acid peak (Fig. 6). This supports the identification of the molecule as uric acid, and furthermore, it can be inferred from this result that the uric acid is the product of purine degradation and not some other pathway.
Fig. 5 Diagram showing the purine degradation pathway for A. adeninivorans. Purines that are not salvaged are degraded to uric acid and further to allantoin and allantoic acid
A transgenic strain, G1212/YRC102-AYNI1-AUOR39, which has urate oxidase linked to a nitrate-inducible AYNI1 promoter, demonstrated that the observed oxidation peak was the result of a molecule that was a substrate for urate oxidase, i.e. uric acid, and provided further evidence for the purine degradation pathway being involved in the high levels of uric acid production. Figure 7 shows the absence of the peak when the cells are incubated in the presence of YNB medium with NaNO3, indicating that urate oxidase is being produced and degrading uric acid, whereas in the absence of NaNO3, uric acid accumulates, as evidenced by the oxidation peak. Determination of the source of the purines, either exogenous or endogenous, was investigated by cultivating A. adeninivorans LS3 on a medium that was free of purines. YNB medium is free of purines and amino acids, with added NH4SO4 being the only significant N source. The medium does contain very small amounts of nitrogen-containing vitamins, but their concentration is less than that required for the amount of N in the uric acid produced. The medium from cells incubated in this medium was analysed by CV, which indicated that the cells were producing uric acid. This suggests that uric acid or its precursors are synthesised by the cell from NH4SO4. Cyclic voltammograms on 24-h supernatants of four species that are related to A. adeninivorans (C. albicans, C. parapsilosis, C. lambica and S. cerevisiae) did not show
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20 G1224
G1212/YR C102
Current [ A]
15
10
5
0 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Potential [V]
-5
Fig. 6 CVs for A. adeninivorans G1212/YRC102 and G1224. The G1224 strain has the gene for xanthine oxidase deleted, resulting in the absence of a uric acid oxidation peak
peaks at ~0.5 V. These results suggest that it is a purine in excess of normal cell function that is being degraded in A. adeninivorans to produce uric acid.
Discussion The electrochemical properties of uric acid are well documented as it is a significant analyte for the development of voltammetric biosensors (Zhang and Lin 2001; Wang et al. 2002; Raj and Ohsaka 2003; Ramesh and Sampath 2004). The properties described by these authors, such as oxidation potentials and near-irreversibility of oxidation, parallel the characteristics collected from the supernatant samples from the incubation of A. adeninivorans incubated in PBS. The overproduction of uric acid probably consumes a significant amount of energy. It is possible that a selection 20 G1212/YRC102
18 16
G1212/YRC102-AYNI1AUOR39 with nitrate G1212/YRC102-AYNI1AUOR39 without nitrate
Current [ A]
14 12 10 8 6 4 2 0 -2 -4
0
0.1
0.2
0.3
0.4 0.5 Potential [V]
0.6
0.7
0.8
0.9
Fig. 7 CVs for A. adeninivorans G1212/YRC102 and G1212/YRC102AYNI-AUOR39 with a NO3-inducible expression module that results in the production of urate oxidase when NO3 is present in the medium, resulting in the absence of a uric acid oxidation peak. The peak is visible when the same cells are grown in a NO3-free medium
pressure has given rise to and maintains this phenomenon. Masoud et al. (2012) report that uric acid is weakly antimicrobial; however, they do not give the concentration at which they made this observation. They also show that uric acid complexed with U has increased antimicrobial activity. Some primates, birds, reptiles and insects also excrete uric acid as the final product of nitrogenous catabolism. Ames et al. (1981) hypothesise that the accumulation of uric acid in the blood plasma of humans acts as a potent antioxidant. That the secretion of uric acid into the extracellular environment of Arxula has an antioxidant function that confers an advantage on the cells is, however, not known. Chen and Fink (2006) describe the release of two aromatic alcohols which act as signalling molecules in S. cerevisiae and, in their introduction, discuss the occurrence of other quorum-sensing molecules in fungi. Necrotic mammalian cells release uric acid as a danger signal to activate both innate and adaptive immune effectors including neutrophils, cytotoxic T cells and antibodies (Behrens et al. 2008). It may be that the release of uric acid by A. adeninivorans also acts as a communication mechanism, i.e. quorum-sensing. Whatever the purpose, if there is one, in the release of uric acid by A. adeninivorans, it is interesting because its production is not seen in the three other yeast species that we have tested. We have previously reported that A. adeninivorans lacks an adenosine deaminase which is atypical of the common purine degradation pathway. Instead, it degrades adenosine monophosphate with PNP (Jankowska et al. 2013b). The product adenine is then degraded with adenine deaminase, which is seen in the common purine breakdown pathway. Interestingly, an analysis of the Cryptococcus neoformans genome has found that it has neither the gene for adenosine deaminase or adenine deaminase (Morrow et al. 2012) and degrades adenosine monophosphate using an AMP deaminase to inosine monophosphate and, further, to hypoxanthine, which is the product of adenine deaminase in Arxula. In addition, Cultrone et al. (2005) have reported that xanthine dehydrogenase is also absent in C. neoformans, but a potential equivalent, an α-ketoglutarate-dependent dioxygenase, is present which is also different from Arxula which uses xanthine dehydrogenase (Jankowska 2013a). This variation in these two yeast species adds to the evidence that degradation of purines is, in general, quite variable. Our findings support the notion that the synthesis of purines and their subsequent degradation to uric acid is the source of the extracellular uric acid. There are many pathways that contribute to the accumulation of purines, which can be catabolised to uric acid, and significant overproduction of purine in excess of cell requirements might provide the purine catabolism pathway with the necessary substrate to produce extra uric acid.
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A phenotype of excessive purine synthesis that results in elevated uric acid excretion is well documented in humans as the inherited disorder, Lesch-Nyhan syndrome (LNS) (Sculley et al. 1992). This disorder arises as a result of the failure of the purine salvage pathway, more specifically, a deficiency in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) pathway. The absence of HGPRT prevents free purines from being salvaged, and as a result, they are degraded to uric acid. Furthermore, since phosphoribosyl pyrophosphate (PRPP) is not being used in the salvage process, it becomes available to allosterically activate de novo purine production, increasing overall purine production and thus elevating uric acid production further. Cloning a functional copy of the gene for HGPRT, HPT1, into a uric acid-producing strain of A. adeninivorans to determine if high uric acid production is suppressed would confirm the presence of this mutation in Arxula. Alternatively, a mutant strain of S. cerevisiae with HPT1 knocked out should produce high levels of uric acid, which would also provide evidence for this possibility. The use of uric acid as a source of electrons in an A. adeninivorans microbial fuel cell would require a significant increase in its production. We have produced a strain that has the urate oxidase gene disrupted so that uric acid cannot be degraded, thereby increasing its secretion, and we are about to commence testing the strain in a MFC. Acknowledgments This work was funded by Science and Innovation, New Zealand Ministry of Business, Innovation and Employment (contract LVLX0802DET) and the University of Canterbury.
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