To convert cephalosporm C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21 (DE3)/pET-DAAO, a high DAAO activity of 250 U ml(-1) was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E coli BL21 (DE3)/pET-ACY with a high expression level of 3000 U l(-1). A novel recombinant strain, BL21 (DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml(-1) and GL-7-ACA acylase activity of 950 U l(-1) were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly. (C) 2004 Elsevier Inc. All rights reserved.