Cloning and sequencing of the alcohol oxidase-encoding gene (AOD1) from the formaldehyde-producing asporogeneous methylotrophic yeast, Candida boidinii S2

Abstract

Alcohol oxidase (AOD) is the first key enzyme for methanol metabolism in methylotrophic yeasts. AOD activity is strictly regulated by carbon source. The AOD1 gene was cloned from a gene library of the asporogenous formaldehyde-producing methylotrophic yeast, Candida boidinii S2. The complete nucleotide sequence of the gene and its 5'- and 3'-flanking regions (4174 bp) were determined. To identify the conserved and divergent sequences of the AOD1 gene and its 5'-flanking sequences among different species of methylotrophic yeasts, the AOD-encoding genes from C. boidinii S2 (AOD1), Hansenula polymorpha (MOX) and Pichia pastoris (AOX1 and AOX2) were compared. In addition to conserved amino acid sequences, several DNA segments in the G+C-rich region of 5'-flanking sequences were also found to be conserved. Northern analysis showed that the AOD1 gene transcript was induced by methanol, but was not detected when cells were grown on ethanol or glucose. Thus, as in ascosporogenous methylotrophic yeasts, AOD1 gene expression in C. boidinii appears to be controlled at the RNA level.