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.
Piron-Fraipont
C
C
Departement de Microbiologie, Université de Liège, Institut de Chimie, Belgium.
Lenzini
M V
MV
Dusart
J
J
Ghuysen
J M
JM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004251:DNA Transposable Elements; D005798:Genes, Bacterial; D006602:Hexosyltransferases; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010458:Peptidyl Transferases; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase