Cloning A Streptomyces clavuligerus Genetic Locus Involved in Clavulanic Acid Biosynthesis

Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by Streptomyces clavuligerus

“…Bailey et al first described the cloning of a genetic locus involved in clavulanic acid production by complementation of a nonproducing mutant (11). Later, Jensen et al reported a 12-kb EcoRI fragment from the genome of S. clavuligerus which contained all the genetic information required for the production of clavulanic acid in the heterologous host, Streptomyces lividans (31).…”

“…A further suggestion that the clavulanic acid biosynthetic cluster might be larger than previously appreciated could be found in the reported dclC locus, which mapped to ca. 4 kb downstream of orf9 (61) and complemented a clavulanic acid nonproducing mutant (11).…”

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

“…One milligram dry cell weight (DCW) of seed culture was used to inoculate 50 ml fermentation medium (per litre): 30g tryptone soya broth (TSB) (Oxoid), 10 g soluble starch, 20.9 g MOPS buffer, pH adjusted to 7.0 with KOH (modified from Jensen et al 1986) in a 500-ml unbaffied flask and the flask was incubated at 30°C and 250 rpm. S. clavuligerus protoplasts were prepared, transformed and regenerated as described by Bailey et al (1984). Escheriehia coli ESS, a/3-1actam-supersensitive strain, was grown in Luria broth and stored in 20% glycerol at -8 0°C .…”

“…many Streptornyees host species the ability to produce the pigment melanin. However, since tyrosinase is very poorly expressed in S. elavuligerus carrying pIJ702 (Bailey et al 1984; our own observations), insert frequencies in the ligation reactions were assessed in S. lividans 1326 by scoring white colonies. Ligation reactions with insert frequencies of 75%-80% were used.…”

“…Mutant NP1 was transformed with the ligation mixture and transformants were screened for cephalosporin production in the plate bioassay. The transformation protocol used (Bailey et al 1984) gave frequencies of only 80-500 transformants per Ixg vector D N A in the ligation mixture. Among about 4000 thiostrepton-resistant colonies tested, six produced small inhibition zones.…”