<jats:title>ABSTRACT</jats:title> <jats:p> <jats:named-content content-type="genus-species">Streptomyces clavuligerus</jats:named-content> <jats:italic>claR</jats:italic> :: <jats:italic>aph</jats:italic> is a <jats:italic>claR</jats:italic> -defective mutant, but in addition to its <jats:italic>claR</jats:italic> defect it also carries fewer copies of the resident linear plasmids pSCL2 and pSCL4 (on the order of 4 × 10 <jats:sup>5</jats:sup> -fold lower than the wild-type strain), as shown by qPCR. To determine the function of ClaR without potential interference due to plasmid copy number, a new strain, <jats:named-content content-type="genus-species">S. clavuligerus</jats:named-content> Δ <jats:italic>claR</jats:italic> :: <jats:italic>aac</jats:italic> , with <jats:italic>claR</jats:italic> deleted and carrying the wild-type level of plasmids, was constructed. Transcriptomic analyses were performed in <jats:named-content content-type="genus-species">S. clavuligerus</jats:named-content> Δ <jats:italic>claR</jats:italic> :: <jats:italic>aac</jats:italic> and <jats:named-content content-type="genus-species">S. clavuligerus</jats:named-content> ATCC 27064 as the control strain. The new Δ <jats:italic>claR</jats:italic> mutant did not produce clavulanic acid (CA) and showed a partial expression of genes for the early steps of the CA biosynthesis pathway and a very poor expression (1 to 8%) of the genes for the late steps of the CA pathway. Genes for cephamycin C biosynthesis were weakly upregulated (1.7-fold at 22.5 h of culture) in the Δ <jats:italic>claR</jats:italic> mutant, but genes for holomycin biosynthesis were expressed at levels from 3- to 572-fold higher than in the wild-type strain, supporting the observed overproduction of holomycin by <jats:named-content content-type="genus-species">S. clavuligerus</jats:named-content> Δ <jats:italic>claR</jats:italic> :: <jats:italic>aac</jats:italic> . Interestingly, three secondary metabolites produced by gene clusters SMCp20, SMCp22, and SMCp24, encoding still-cryptic compounds, had partially or totally downregulated their genes in the mutant, suggesting a regulatory role for ClaR wider than previously reported. In addition, the <jats:italic>amfR</jats:italic> gene was downregulated, and consequently, the mutant did not produce aerial mycelium. Expression levels of about 100 genes in the genome were partially up- or downregulated in the Δ <jats:italic>claR</jats:italic> mutant, many of them related to the upregulation of the sigma factor-encoding <jats:italic>rpoE</jats:italic> gene.