D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217

The complete nucleotide sequence of the DAO1 gene encoding D-amino-acid oxidase (DAAO) in the yeast Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217 has been determined. The primary structure of DAAO was deduced from the nucleotide sequence of a cDNA clone that covered the entire amino acid coding sequence. Comparison of cDNA and genomic sequences of DAO1 revealed the presence of five introns. Because this is the first gene of strain ATCC 26217 that has been cloned so far, the nucleotide sequences of these introns were compared to those from other fungi. Upstream of the structural gene there was a stretch of C + T-rich DNA similar to that found in the promoter region of a number of yeast genes. The cDNA gene, which encoded a protein of 368 amino acids (molecular mass 40 kDa), was overexpressed in Escherichia coli under the control of the strong lipoprotein promoter. Interestingly, a significant fraction (13-62%) of the total DAAO activity was recovered in its apoenzyme form, the percentage depending on the culture conditions. This fact allowed a rapid purification of the recombinant DAAO by affinity chromatography. The high level of expression achieved in E. coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.