title: The NADP-dependent glutamate dehydrogenase gene from the astaxanthin producer Xanthophyllomyces dendrorhous: use of Its promoter for controlled gene expression.
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 (hygR) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 microg/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.
authors.affiliation: R&D Biology, Antibióticos S.A, Avenida de Antibióticos 59-61, 24009, León, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Rodríguez-Sáiz
authors.forename: Ramiro P
authors.initials: RP
authors.lastname: Godio
authors.forename: Vanessa
authors.initials: V
authors.lastname: Alvarez
authors.forename: Juan Luis
authors.initials: JL
authors.lastname: de la Fuente
authors.forename: Juan F
authors.initials: JF
authors.lastname: Martín
authors.forename: José Luis
authors.initials: JL
authors.lastname: Barredo
mesh_terms: D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D018086:Glutamate Dehydrogenase (NADP+) / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D017385:Sequence Homology; D024341:Xanthophylls / Q000378:metabolism
chemical_list: D024341:Xanthophylls; C005948:astaxanthine; D018086:Glutamate Dehydrogenase (NADP+)