FUNCTIONAL-ANALYSIS OF THE GENE ENCODING THE CLAVAMINATE SYNTHASE-2 ISOENZYME INVOLVED IN CLAVULANIC ACID BIOSYNTHESIS IN STREPTOMYCES-CLAVULIGERUS

A Streptomyces clavuligerus mutant disrupted in cas2, encoding the cla vaminate synthase (CAS2) isoenzyme, was constructed by a gene replacem ent procedure. The resulting cas2 mutant showed no clavulanic acid pro duction when grown in starch-asparagine medium. However, in soy medium , the cas2 mutant did produce clavulanic acid, although in amounts les s than those produced by wild-type cultures. This medium-dependent lea ky 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 sugges ted that CAS1 and CAS2 both contribute to clavulanic acid production b ut that their production is regulated differently. Under nutritional c onditions 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, c as2 is transcribed both as a 1.2-kb monocistronic transcript and as pa rt of a 5.3-kb polycistronic transcript. High-resolution S1 nuclease a nalysis located the transcription start point of the monocistronic cas 2 transcript at a C residue 103 nucleotides upstream from the cas2 sta rt codon.