Genes Specific for the Biosynthesis of Clavam Metabolites Antipodal to Clavulanic Acid Are Clustered with the Gene for Clavaminate Synthase 1 in Streptomyces clavuligerus

Abstract

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames,cvm4 and cvm5, located directly downstream ofcas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 andcas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.

Biosynthesis of clavam metabolites

“…Since the genes for clavulanic acid production are clustered together around cas2, genes specifically involved in 5S clavam biosynthesis were sought by sequencing the region around cas1. Mosher et al [49] found a number of new genes (cvm1, cvm2, cvm3, cvm4, cvm5, and cvm6) in what is now called the clavam gene cluster (Fig. 4).…”

“…4) and shows strong similarity to genes encoding aldo-keto reductases. Mutation of cvm1 has no effect on clavulanic acid production but eliminates clavam-2-carboxylate, 2-hydroxymethylclavam, and alanylclavam production [49]. cvm2 encodes a predicted protein with limited similarity to isomerases, and mutants in cvm2 were severely compromised in 5S clavam production [49,70].…”

“…Mutation of cvm1 has no effect on clavulanic acid production but eliminates clavam-2-carboxylate, 2-hydroxymethylclavam, and alanylclavam production [49]. cvm2 encodes a predicted protein with limited similarity to isomerases, and mutants in cvm2 were severely compromised in 5S clavam production [49,70]. cvm3 encodes a putative flavin reductase predicted to provide reduced flavin for the flavin-dependent mono-oxygenase encoded by cvm5 [49,70].…”

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus

“…3). Mutants disrupted in cvm1 produce undetectable levels of clavam-2-carboxylate, clavaminic acid or 2-hydroxymethylclavam [46]. The function of Cvm1, Cvm2 or Cvm5 in clavam biosynthesis is still unclear.…”

“…The clavulanic acid cluster (already mentioned) was initially isolated by hybridization with the cas2 gene, encoding clavaminate synthase. A second cluster, named the clavam gene cluster, was isolated by using the cas1 gene, a duplicate gene encoding a clavaminate synthase isoenzyme, as probe [46]. This gene cluster contains genes cvm1 to cvm13, cvmG and cvmP ( Fig.…”