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Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans
Authors:Erik Böer  Anja Schröter  Rüdiger Bode  Michael Piontek  Gotthard Kunze
Affiliation:1. Leibniz‐Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D‐06466 Gatersleben, Germany;2. Institut für Mikrobiologie, Universit?t Greifswald, Jahnstrasse 15a, D‐17487 Greifswald, Germany;3. ARTES Biotechnology GmbH, Elisabeth‐Selbert‐Strasse 9, D‐40764 Langenfeld, Germany
Abstract:In Arxula adeninivorans nitrate assimilation is mediated by the combined actions of a nitrate transporter, a nitrate reductase and a nitrite reductase. Single‐copy genes for these activities (AYNT1, AYNR1, AYNI1, respectively) form a 9103 bp gene cluster localized on chromosome 2. The 3210 bp AYNI1 ORF codes for a protein of 1070 amino acids, which exhibits a high degree of identity to nitrite reductases from the yeasts Pichia anomala (58%), Hansenula polymorpha (58%) and Dekkera bruxellensis (54%). The second ORF (AYNR1, 2535 bp) encodes a nitrate reductase of 845 residues that shows significant (51%) identity to nitrate reductases of P. anomala and H. polymorpha. The third ORF in the cluster (AYNT1, 1518 bp) specifies a nitrate transporter with 506 amino acids, which is 46% identical to that of H. polymorpha. The three genes are independently expressed upon induction with NaNO3. We quantitatively analysed the promoter activities by qRT–PCR and after fusing individual promoter fragments to the phytase (phyK) gene from Klebsiella sp. ASR1. The AYNI1 promoter was found to exhibit the highest activity, followed by the AYNT1 and AYNR1 elements. Direct measurements of nitrate and nitrite reductase activities performed after induction with NaNO3 are compatible with these results. Both enzymes show optimal activity at around 42°C and near‐neutral pH, and require FAD as a co‐factor and NADPH as electron donor. Copyright © 2009 John Wiley & Sons, Ltd.
Keywords:Arxula adeninivorans  nitrate transporter  nitrate reductase  nitrite reductase  nitrate assimilation  yeast
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