Characterization and complementation of a cephalosporin-deficient mutant of Streptomyces clavuligerus NRRL 3585

Abstract

We have characterized a mutant of Streptomyces clavuligerus NRRL 3585 which is almost completely blocked in cephalosporin biosynthesis and exhibits depressed activities of both the delta(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase and cyclase enzymes of the cephalosporin pathway. A wild-type DNA region was cloned which partially restores antibiotic production, ACV synthetase and cyclase activities to this mutant. The recombinant plasmid exhibits a variable copy number in different transformants. Hybridization experiments indicate that sequences homologous to the cloned region are present in various beta-lactam-producing Streptomyces spp. but absent in species which are not known to produce this class of antibiotics. Furthermore, the chromosomal copy of the cloned region lies in close proximity to a gene coding for the isopenicillin N synthase gene of the cephalosphorin pathway.

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli

“…While further sequence, transcriptional, and biochemical analyses are needed to establish whether lat has a role in the regulation of antibiotic synthesis, cloning lat on a multicopy plasmid without supplying exogenous lysine to the host may lead to lysine auxotrophy (22) resulting from excessive lysine catabolism. This may explain the anomalous plasmid copy number effect seen with pNBR1 reported by Piret et al (27).…”

“…Piret et al (27) reported that S. clavuligerus NP1, a mutant severely impaired in cephalosporin synthesis and with lower (but not absent) ACVS and IPNS activity, was complemented by DNA that mapped close to pcbC. Interestingly, although pcbC from Streptomyces griseus (8) and S. clavuligerus (13) is transcribed as a monocistronic message, IPNS is down-regulated in the mutant and activity is only partially restored in the transformants.…”

“…Other activities, epimerase and expandase, are subsequently produced and sustained during antibiotic production. As pointed out by Piret et al (27), ACVS is a major target for nutritional control of cephalosporin biosynthesis, but it now seems that regulation of LAT and the possible coordinated regulation of LAT and ACVS should be considered in the overall control system.…”

Precursor flux control through targeted chromosomal insertion of the lysine epsilon-aminotransferase (lat) gene in cephamycin C biosynthesis

“…Analysis of the DNA sequence in this region showed that an inverted repeat (28), corresponding to a potential transcriptional terminator, separates the two genes. The significance of this repeat has not been determined, and there is contradictory evidence as to whether lat/pcbAB comprises a single polycistronic message (10,25). This was potentially significant to our expression strategy because transcriptional activation of a lat/pcbAB polycistronic message could have led to the concomitant increase in both LAT and ACV synthetase activities.…”

“…Twentyfive milliliters of culture was collected by centrifugation at 3,000 x g for 15 min and washed twice with 10.3% sucrose. The pellet was resuspended in 2 ml of 1-mg/ml lysozyme in P buffer containing 0.3 M sucrose, 0.57 mM KSO4, 25 After trituration three times with a pipette before and after dilution with 2.5 ml of P buffer, the protoplast suspension was filtered through a sterile cotton plug. The filtered protoplasts were centrifuged at 1,000 x g for 10 min, washed three times with P buffer, and counted in a hemacytometer.…”