Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus

Abstract

A Streptomyces clavuligerus mutant disrupted in cas2, encoding the clavaminate synthase (CAS2) isoenzyme, was constructed by a gene replacement procedure. The resulting cas2 mutant showed no clavulanic acid production when grown in starch-asparagine medium. However, in soy medium, the cas2 mutant did produce clavulanic acid, although in amounts less than those produced by wild-type cultures. This medium-dependent leaky phenotype correlated well with the presence of the cas1 transcript, encoding the CAS1 isoenzyme, in cultures grown in soy medium and with its absence from those grown in starch-asparagine medium. This suggested that CAS1 and CAS2 both contribute to clavulanic acid production but that their production is regulated differently. Under nutritional conditions in which cas1 expression is blocked, cas2 becomes essential for clavulanic acid production. Northern (RNA) analysis revealed that while cas1 is transcribed as a 1.4-kb monocistronic transcript only, cas2 is transcribed both as a 1.2-kb monocistronic transcript and as part of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease analysis located the transcription start point of the monocistronic cas2 transcript at a C residue 103 nucleotides upstream from the cas2 start codon.

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation

“…S. venezuelae genes initially cloned in pUC18 or pBluescript II (SK+) were recloned in the conjugal E. coli plasmid pJV326 before insertion of the 1?5 kb apramycin resistance (Am R ) cassette from pUC120A (Paradkar & Jensen, 1995) or pJV225 (Chang et al, 2001) at a site near the centre of the cloned fragment. To disrupt cmlK (ORF11), conjugal plasmid pJV526 was linearized with NotI and ligated with Am R from pJV225 to give pJV527.…”

p-Aminobenzoic acid and chloramphenicol biosynthesis in Streptomyces venezuelae: gene sets for a key enzyme, 4-amino-4-deoxychorismate synthase The GenBank accession number for the sequence reported in this paper is AF189258.

“…The vector used to disrupt pabAB has been described by Brown et al (1996). To disrupt the putative pabB in pJV305, a 1n5 kb DNA fragment containing an apramycin resistance (Am R ) gene (Paradkar & Jensen, 1995) was inserted into the Eco72I site of ORF1 to give pJV307. The disrupted ORF1 in pJV307 was then recloned as pJV323 in pHJL400.…”

Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species

“…In addition, the production of both cephamycin and clavulanic acid in S. clavuligerus is controlled principally by the ccaR (dclX) gene, which codes for an OmpR-like transcriptional regulator, and is located within the cephamycin cluster (21,22). In contrast, the clavam cluster is at least 20-30 kb away from the cephamycin͞clavulanic acid clusters on the S. clavuligerus chromosome, and the regulation of clavam production is distinct from the coregulated production of cephamycins and clavulanic acid (23)(24)(25). These observations suggest that clavulanic acid production in a clavam-producing ancestor, which acquired the ability to produce cephamycin, could have arisen via chromosomal duplication of the clavam cluster followed by subsequent acquisition of the late steps in the clavulanic acid pathway, which involve the stereochemical inversions that are key to ␤-lactamase activity.…”