Research Article J Mol Microbiol Biotechnol 2014;24:67–81 Received: June 25, 2013 DOI: 10.1159/000357674 Accepted: November 29, 2013 Published online: January 24, 2014 Arxula adeninivorans Recombinant Guanine Deaminase and Its Application in the Production of Food with Low Purine Content a a b Anke Trautwein-Schult Dagmara Jankowska Arno Cordes b b a a Petra Hoferichter Christina Klein Andrea Matros Hans-Peter Mock d c a Keith Baronian Rüdiger Bode Gotthard Kunze a b Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben , ASA Spezialenzyme GmbH, c d Wolfenbüttel , and Institute of Microbiology, University of Greifswald, Greifswald , Germany; School of Biological Sciences, University of Canterbury, Christchurch , New Zealand Key Words ole of yeast. The enzyme was demonstrated to reduce levels Arxula adeninivorans · Hyperuricemia · Recombinant of guanine in food. A mixture of guanine deaminase and oth- guanine deaminase · Treatment of food er purine degradation enzymes will allow the reduction of purines in purine-rich foods. © 2014 S. Karger AG, Basel Abstract Purines of exogenous and endogenous sources are degrad- ed to uric acid in human beings. Concentrations >6.8 mg uric Introduction acid/dl serum cause hyperuricemia and its symptoms. Phar- maceuticals and the reduction of the intake of purine-rich Humans absorb different amounts of RNA, DNA, nu- food are used to control uric acid levels. A novel approach to cleotides, nucleosides and free purine bases from food of the latter proposition is the enzymatic reduction of the pu- animal and vegetable origin [Wolfram and Colling, 1987]. rine content of food by purine-degrading enzymes. Here we Purine degradation leads ultimately to CO 2 and ammo- describe the production of recombinant guanine deaminase nia, although in some organisms, intermediate molecules by the yeast Arxula adeninivorans LS3 and its application in accumulate [Hayashi et al., 2000; Keilin, 1959; Lehninger, food. In media supplemented with nitrogen sources hypo- 2004]. In humans, purines from exogenous sources (food) xanthine or adenine, guanine deaminase ( AGDA ) gene ex- and purines from cell turnover are both degraded to uric pression is induced and intracellular accumulation of gua- acid [Wolfram, 1992; Wolfram and Colling, 1987]. nine deaminase (Agdap) protein occurs. The characteristics Reduced renal uric acid excretion combined with in- of the guanine deaminase isolated from wild-type strain LS3 creased uric acid synthesis from endogenous and/or ex- and a transgenic strain expressing the AGDA gene under ogenous sources can cause an increase in serum uric acid control of the strong constitutive TEF1 promoter were deter- concentration in some individuals [Kutzing and Fireste- mined and compared. Both enzymes were dimeric and had in, 2008; Saag and Choi, 2006; Tausche et al., 2009]. A temperature optima of 55 ° C with high substrate specificity serum concentration >6.8 mg uric acid/dl serum is above for guanine and localisation in both the cytoplasm and vacu- the physiological saturation level of uric acid and is de- © 2014 S. Karger AG, Basel G. Kunze 1464–1801/14/0242–0067$39.50/0 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Corrensstrasse 3 E-Mail [email protected] DE–06466 Gatersleben (Germany) www.karger.com/mmb E-Mail kunzeg @ ipk-gatersleben.de fined as hyperuricemia [Terkeltaub, 2010]. A minority of brain of higher eukaryotes [Berger et al., 1985; Kumar and patients with hyperuricemia develop microscopic and Rathi, 1980; Miyamoto et al., 1982; Rossi et al., 1978] macroscopic monosodium urate crystals, which cause where it plays an important role in the branching of den- swelling, inflammation and/or pain in joints and results drites [Fernández et al., 2009]. in painful gout attacks [Sorbera et al., 2010; Terkeltaub, In this study the guanine deaminase ( AGDA ) gene was 2010]. Between 1 and 2% of the population in industrial isolated from the non-conventional, arthroconidial, xe- countries are affected by this disease [Burns and Wort- rotolerant, anamorphic, non-pathogenic yeast, Arxula mann, 2011; Eggebeen, 2007; Sorbera et al., 2010]. adeninivorans LS3 [van der Walt et al., 1990]. The yeast Patients with acute gout attacks are treated with is able to assimilate and use a broad range of substrates, NSAIDs (non-steroidal anti-inflammatory drugs), col- including purines, n -alkanes and starch as sole energy chicine or in rare cases, with corticosteroids or interleukin- and carbon sources and nitrate as a sole nitrogen source 1β inhibitors [Groff et al., 1990; Jordan, 2012; Tausche et [Böer et al., 2009c; Gienow et al., 1990; Middelhoven et al., 2009; Terkeltaub et al., 2010]. In symptomless inter- al., 1984]. It can be cultured at up to 48 ° C and 20% NaCl vals between attacks, uricostatic agents (allopurinol or fe- [Wartmann et al., 1995; Yang et al., 2000]. Above 42 ° C buxostat) which reduce uric acid synthesis by inhibition the morphology changes from budding cells to mycelia of xanthine oxidoreductase [Eggebeen, 2007; Hu and [Wartmann et al., 1995]. Tomlinson, 2008; Pascual et al., 2009; Wittköpper et al., The ADGA gene was overexpressed to produce high 2011], uricosuric agents (probenecid or benzbromarone) concentrations of recombinant guanine deaminase pro- which promote uric acid excretion [Eggebeen, 2007; Em- tein. Additionally, the application of the enzyme in a pro- merson, 1996] or recombinant urate oxidase (rasburicase cess to reduce purines in food was investigated. or pegloticase) which enzymatically degrade uric acid [Leinmüller, 2001; Schlesinger et al., 2011; Sharma and Verma, 2004; Sundy et al., 2007] are used. R e s u l t s The uptake of exogenous purines can be reduced by decreasing the intake of purine-rich food such as meat Identification and Characterisation of the AGDA (especially muscle tissue, bowel and skin), fish (herring Gene and shrimps) and some plants (soy and other legumes) The putative nucleotide sequence of the AGDA gene [Wolfram, 1992; Wolfram and Colling, 1987]. Addition- was determined from the complete A. adeninivorans se- ally, avoidance of alcohol and loss of weight are useful in quence and annotated genome [manuscript in progress] the prevention of gout attacks [Wolfram, 1992]. and the AGDA gene was located as single copy on chro- Our approach to prevent hyperuricemia involves the mosome 4. The ORF of the AGDA gene comprises 1,428 addition of a mixture of different purine-degrading en- bp which encodes a protein with 475 amino acids (mo- zymes to the food, either during production or during lecular mass 53 kDa; isoelectric point 5.7). Agdap has ingestion. This study characterises one of those enzymes, identity with other fungal guanine deaminases, including guanine deaminase, and investigates its performance in a Ogataea parapolymorpha DL-1 (accession: EFW97840 – purine-rich food. 55%), Komagataella pastoris GS115 (XP_002490656 – Guanine deaminase (EC 3.5.4.3, guanine aminohy- 51%), Saccharomyces cerevisiae strains (CAY79050, drolase), is a metal-dependent enzyme that is highly con- EDN60126 and EEU08967 – 49–50%), Ajellomyces der- served, and was first described in 1932 [Schmidt, 1932]. matitidis strains (EEQ86158 and XP_002620893 – 45%), It catalyses the irreversible deamination of guanine to Schizosaccharomyces pombe 972h- (NP_587874 – 48%) xanthine and ammonia [Fernández et al., 2010; Glantz and Paracoccidioides brasiliensis Pb03 (EEH21407 – and Lewis, 1978; Lehninger, 2004; Maynes et al., 2000]. It 44%). is responsible for purine degradation in bacteria [Liaw et al., 2004; Maynes et al., 2000], lower eukaryotes [Nolan, Nitrogen Source-Dependent Expression of the AGDA 1984; Saint-Marc and Daignan-Fornier, 2004; Shavlov- Gene skii and Kuznetsova, 1974], plants [Barankiewicz and The analysis of AGDA transcription regulation was Paszkowski, 1980; Negishi et al., 1994] and is present in done with qRT-PCR and the Arxula genes AHBS4 , ALG9 , the liver [Gupta and Glantz, 1985; Lewis and Glantz, TEF1 and TFIID as stably expressed endogenous refer- 1974] and kidney [Kubo et al., 2006] of higher eukaryotes. ences. The wild-type strain was cultured in YMM-glucose Guanine deaminase was also found to be active in the supplemented with 43.5 m M NH4 H2 PO4 as nitrogen 68 J Mol Microbiol Biotechnol 2014;24:67–81 Trautwein-Schult et al. DOI: 10.1159/000357674 90 Adenine 0.4 Hypoxanthine 80 Uric acid 70 Guanine 0.3 60 Nitrate Ammonium 50 0.2 40 30 Activity (U/mg) 20 0.1 10 Fig. 1. Influence of the nitrogen source on quantity (AU) Normalized relative AGDA transcript accumulation (a , c ) and 0 0 0 5 10 15 20 25 0 1020304050 Agdap activity ( b , d ). Normalised relative a Time (h) b Time (h) quantity of AGDA transcript level was ana- lysed with qRT-PCR and calculated ac- 200 0.4 cording to Hellemans et al. [2007] with 180 Arxula genes AHBS4 , ALG9 , TEF1 and 160 TFIID used as stably expressed endogenous 0.3 references. A. adeninivorans LS3 was cul- 140 tured 24 h at 30 ° C in YMM-glucose- 120 NH 4 H2 PO4 (43.5 m M ) before being shifted 100 0.2 to YMM-glucose supplemented with dif- 80 ferent nitrogen sources ( a ) 5 m M adenine, 60 Activity (U/mg) b M 0.1 hypoxanthine or NaNO 3 for 24 h, ( ) 5 m 40 adenine, hypoxanthine, uric acid, NaNO , 3 20 NH4 H2 PO4 or 0.2 m M guanine for 48 h, ( c ) quantity (AU) Normalized relative 0 M 0 0.5, 1, 3, 4 and 5 m adenine, hypoxanthine 0246 0246 or NaNO for 2 h, and ( d) 0.25, 0.5, 1, 2.5 3 cdInducer concentration (mM) Inducer concentration (mM) and 5 m M adenine, hypoxanthine, uric acid, NH4 H2 PO4 or 0.2 m M guanine for 4 h. source (N-source) for 24 h at 30 ° C before the washed cells where the activity increase was minimal ( fig. 1 b). The were shifted to YMM-glucose containing 5 m M adenine, negative controls (YMM-glucose-NH4 H2 PO4 or YMM- 5 m M hypoxanthine or 5 mM NaNO3 as nitrogen source.