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The NADP-dependent Glutamate Dehydrogenase Gene from the Astaxanthin Producer Xanthophyllomyces dendrorhous: Use of Its Promoter for Controlled Gene Expression

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Abstract

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase (GDH) activity from Xanthophyllomyces dendrorhous has been cloned and characterized, and its promoter used for controlled gene expression in this red-pigmented heterobasidiomycetous yeast. We determined the nucleotide sequence of a 4701 bp DNA genomic fragment, showing an open reading frame of 1871 bp interrupted by five introns with fungal consensus splice-site junctions. The predicted protein (455 amino acids; 49 kDa) revealed high identity to GDHs, especially to those from the fungi Cryptococcus neoformans (70%), Sclerotinia sclerotiorum (66%), and several species of Aspergillus (66–67%). Gene phylogenies support the grouping of X. dendrorhous GDH close to those from the majority of the filamentous fungi. The promoter region of the gdhA gene (PgdhA) contains a TATA-like box and two large pyrimidine stretches. The use of PgdhA for gene expression was validated by electrotransformation of X. dendrorhous using an in-frame fusion with the hygromycin resistance gene (hyg R) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 μg/ml hygromycin, but gene expression in Czapek medium was repressed when using ammonium acetate as a nitrogen source. PgdhA is a valuable tool for controlled gene expression in Basidiomycetes.

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Acknowledgments

This study was supported in part by the PROFIT project FIT-010000-2006-20 granted to Antibióticos S.A. and INBIOTEC. The authors thank P. Merino, C. Flórez, A. Morán, M. Sandoval, and C. Cañón for their excellent technical assistance. V. Álvarez was supported by a fellowship “Acción MIT-F2” from the Spanish Ministry of Science and Technology.

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Authors and Affiliations

  1. R&D Biology, Antibióticos S.A, Avenida de Antibióticos 59-61, 24009, León, Spain

    Marta Rodríguez-Sáiz, Vanessa Álvarez, Juan Luis de la Fuente & José Luis Barredo

  2. Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León Av. Real 1, 24006, León, Spain

    Ramiro P. Godio & Juan F. Martín

Authors
  1. Marta Rodríguez-Sáiz
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  2. Ramiro P. Godio
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  3. Vanessa Álvarez
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Correspondence to José Luis Barredo.

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Rodríguez-Sáiz, M., Godio, R.P., Álvarez, V. et al. The NADP-dependent Glutamate Dehydrogenase Gene from the Astaxanthin Producer Xanthophyllomyces dendrorhous: Use of Its Promoter for Controlled Gene Expression. Mol Biotechnol 41, 165–172 (2009). https://doi.org/10.1007/s12033-008-9123-y

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