  Beta-lactamase expression in Streptomyces cacaoi; ; et al in Journal of Bacteriology (1990), 172(11), 6427-6434 Plasmids were prepared by inserting genomic DNA fragments from Streptomyces cacaoi within the mel gene of plasmid pIJ702. The inserted DNA fragments contain the beta-lactamase-encoding bla gene and ... [more ▼] Plasmids were prepared by inserting genomic DNA fragments from Streptomyces cacaoi within the mel gene of plasmid pIJ702. The inserted DNA fragments contain the beta-lactamase-encoding bla gene and upstream nucleotide sequences of various lengths. The transcription start point of bla was identified by nuclease S1 mapping. Upstream nucleotide sequences of sufficient lengths had an enhancing effect on beta-lactamase production by the Streptomyces host. The dot blot hybridization assay revealed that this effect was exerted at the transcriptional level. Experimental evidence strongly suggests that the underlying mechanism involves, at least in part, one or several trans-acting elements. In one of the constructs, in which the upstream nucleotide sequence was reduced to 0.3 kb, the bla promoter was present but the bla gene was expressed by readthrough from a promoter, possibly the mel promoter, of the pIJ702 vector. [less ▲] Detailed reference viewed: 8 (3 ULg)Cloning and amplified expression in streptomyces-lividans of the gene encoding the extracellular beta-lactamase from streptomyces-cacaoi; ; et al in Journal of General Microbiology (1987), 133(10), 2915-2920 A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number ... [more ▼] A 19 kb SphI DNA fragment containing the gene for the extracellular active-site serine beta-lactamase of Streptomyces cacaoi KCC-SO352 was cloned in Streptomyces lividans TK24 using the high-copy-number plasmid pIJ702 as vector. A 30-fold higher yield of beta-lactamase was obtained from S. lividans strain ML1, carrying the recombinant plasmid pDML51, than from S. cacaoi grown under optimal production conditions. In all respects (molecular mass, isoelectric point, kinetics of inhibition by beta-iodopenicillanate) the overproduced S. lividans ML1 beta-lactamase was identical to the original S. cacaoi enzyme. A considerable reduction of beta-lactamase production was caused by elimination of a 12.8 kb portion of the 19 kb DNA fragment by cleavage at an internal SphI site located more than 3 kb upstream of the beta-lactamase structural gene. The beta-lactamase gene was located within a 1.8 NcoI-BclI fragment but when this fragment was cloned in S. lividans pIJ702, the resulting strain produced hardly any more beta-lactamase than the original S. cacaoi. [less ▲] Detailed reference viewed: 9 (0 ULg) |
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