Summary auto-generated
This study describes the cloning, sequencing, and expression of the DAO1 gene from the yeast Rhodotorula gracilis, which encodes D-amino-acid oxidase (DAAO), an FAD-containing flavoenzyme. The researchers obtained both genomic and cDNA sequences, discovering that the genomic gene contains five introns—an unusually high number for yeast genes. The cDNA encodes a 368-amino-acid protein with a molecular mass of 40 kDa. The gene was successfully overexpressed in Escherichia coli using a strong lipoprotein promoter, yielding approximately 20-fold higher enzyme activity than the yeast source organism. Notably, 13-62% of the recombinant DAAO was recovered in an inactive apoenzyme form lacking FAD cofactor, depending on culture conditions. This apoenzyme form enabled rapid purification via affinity chromatography. The purified recombinant enzyme displayed identical biochemical properties to the native yeast enzyme, with specific activity of 194 units per milligram protein on D-alanine substrate. DAAO has significant biotechnological importance as a biocatalyst for producing 7-aminocephalosporanic acid, an intermediate in cephalosporin antibiotic synthesis. This work establishes E. coli as a suitable heterologous host for industrial-scale DAAO production and provides a platform for protein engineering studies.
Key findings
- The DAO1 gene from R. gracilis contains five introns with sequences similar to related fungal genes, an unusually high number for yeast genes
- Recombinant DAAO production in E. coli achieved 20-fold higher specific activity than the native yeast source organism
- Between 13-62% of recombinant DAAO was recovered as enzymatically inactive apoenzyme, enabling rapid purification by affinity chromatography
- Purified recombinant DAAO displayed identical biochemical properties to the native yeast enzyme with specific activity of 194 U/mg protein on D-alanine
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Abstract
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.