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

Abstract

The complete nucleotide sequence of the DA07 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 DA07 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 i t s 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 f . coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.

Fusion protein of Vitreoscilla hemoglobin with D‐amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C

“…DAO converts CPC to ␣-ketoadipyl-7-aminocephalosporanic acid (AKA-7-ACA), which is further decarboxylated by the hydrogen peroxide produced in the reaction to give rise to 7-␤-(4-carboxybutanamido)cephalosporanic acid (glutaryl-7-ACA). In cooperation with glutaryl acylase, these two enzymes can convert CPC to 7-aminocephalosporanic acid (7-ACA), a starting compound for the production of semisynthetic ␤-lactam antibiotics (Alonso et al, 1998). By three consecutive bioconversion processes with DAO, glutaryl acylase, and penicillin acylase, a chemoenzymatic synthesis of cephamandole was possible from CPC without the purification of intermediates (Terreni et al, 2001).…”

Overproduction and characterization of a recombinant D-amino acid oxidase from Arthrobacter protophormiae

“…This behavior differs from other D-AAOs that were expressed in E. coli. Up to 62% of the R. gracilisD-AAO were expressed as apoenzyme in E. coli (Alonso et al 1998), and the activity of recombinant T. variabilis D-AAO could be increased by 33% when FAD was added to the enzyme assay .…”

“…D-AAOs from fungi, fish, birds, reptiles and different mammalian tissues have been discovered during the last decades (Konno and Yasumura 1992). The best explored D-AAOs originate from pig kidney (Curti et al 1973;Fukui et al 1987;Watanabe et al 1989) and the yeasts Rhodotorula gracilis (Alonso et al 1998;Casalin et al 1991;Harris et al 1999;Molla et al 1998;Pilone et al 1989a;Pilone et al 1987;Ramon et al 1998) and Trigonopsis variabilis (Berg and Rodden 1976;Gonzalez et al 1997;Isoai et al 2002;Szwajcer and Mosbach 1985;Yu et al 2002). The crystal structures of the D-AAOs from R. gracilis and pig kidney were resolved (Mattevi et al 1996;Miura et al 1997;Umhau et al 2000), and different approaches of enzyme engineering were performed to better understand their catalytic, structural and biotechnological properties (Boselli et al 2004;Caldinelli et al 2006;Ju et al 2000;Piubelli et al 2002;Sacchi et al 2002).…”

Biosafety considerations for selectable and scorable markers used in cassava (Manihot esculentaCrantz) biotechnology

“…Some D forms of amino acids such as D-alanine and D-serine are toxic to some plants. Introducing either of the genes, dao1 from Rhodotorula gracilis (Alonso et al, 1998) (Erikson et al, 2003). The unique feature of this system is that it can be used as a positive or negative selection system depending on the additives to the medium.…”