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Applied and Environmental Microbiology, June 2005, p. 3276-3284, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3276-3284.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae

Zeynep Vuralhan,¹ Marijke A. H. Luttik,¹ Siew Leng Tai,¹ Viktor M. Boer,¹ Marcos A. Morais,² Dick Schipper,³ Marinka J. H. Almering,¹ Peter Kötter,⁴ J. Richard Dickinson,⁵ Jean-Marc Daran,^1* and Jack T. Pronk¹

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands,¹ Setor de Biologia Molecular/LIKA and Departamento de Genética, Universidade Federal de Pernambuco, Av. Moraes Rego, s/n, CEP 50670-901, Recife, P.E., Brasil,² Beijerinck Laboratory, DSM Research, Delft, The Netherlands,³ Institut für Mikrobiologie, J. W. Goethe Universität Frankfurt, Biozentrum N250, 60439 Frankfurt, Germany,⁴ Cardiff School of Biosciences, Cardiff University, CF10 3TL Cardiff, United Kingdom⁵

Received 20 October 2004/ Accepted 22 December 2004

Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

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* Corresponding author. Mailing address: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. Phone: 31152782412. Fax: 31152782355. E-mail: j.m.daran{at}tnw.tudelft.nl.

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Applied and Environmental Microbiology, June 2005, p. 3276-3284, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.3276-3284.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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