Transcriptional analysis of the DD-peptidase/penicillin-binding protein-encoding dac gene of Streptomyces R61: use of the promoter and signal sequences in a secretion vector.

The promoter region of the gene encoding the extracellular DD-peptidase/penicillin-binding protein of Streptomyces R61 has been identified by in vivo promoter probing and S1 mapping. A secretion vector, pDML116, was constructed by inserting into the multicopy Streptomyces plasmid pIJ702, a 247 bp DNA sequence that contained the transcriptional, translational and secretory signals and the 12 amino acid N-terminal region-encoding sequence of the mature Streptomyces DD-peptidase/penicillin-binding protein. Insertion, downstream of this 247 bp segment, of the Streptomyces R61 DD-peptidase-encoding gene or the Escherichia coli R-TEM beta-lactamase-encoding gene yielded plasmids pDML120 and pDML128, respectively, which allowed expression and secretion of the relevant enzymes by Streptomyces lividans. The maximal secretion levels obtained were 42 mg protein/ml for the autologous Streptomyces DD-peptidase and 0.9 mg protein/ml for the heterologous E. coli beta-lactamase.