microbiology, Vol 144, 1095-1101, Copyright © 1998 by Society for General Microbiology
D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217
J Alonso, JL Barredo, B Diez, E Mellado, F Salto, JL Garcia and E Cortes
Department of Molecular Microbiology, Centro de Investigaciones Biologicas (CSIC), Madrid, Spain.
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.
