Mutants of Streptomyces clavuligerus with Disruptions in Different Genes for Clavulanic Acid Biosynthesis Produce Large Amounts of Holomycin: Possible Cross-Regulation of Two Unrelated Secondary Metabolic Pathways

Abstract

A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a ␤-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.Streptomyces clavuligerus produces several secondary metabolites with interesting pharmacological activities. It synthesizes the ␤-lactam antibiotic cephamycin C, the ␤-lactamase inhibitor clavulanic acid, and several antifungal compounds with a clavam structure (for reviews, see references 3 and 15). The clavulanic acid biosynthesis pathway has several steps in common with the pathway for clavam biosynthesis (18,19). In addition to the compounds indicated above, S. clavuligerus produces the antibiotics holomycin and tunicamycin (10). Holomycin is a compound with pyrrothine structure, while tunicamycin is a glucosamine-containing antibiotic. This wealth of genetic information for the biosynthesis of secondary metabolites is characteristic of some Streptomyces species (4, 22).S. clavuligerus is an excellent model for the study of the relationships between the regulatory mechanisms controlling the biosynthesis of the different secondary metabolites produced by these microorganisms. Formation of clavulanic acid is controlled by a LysR-type regulatory protein encoded by the claR gene. Formation of both clavulanic acid and cephamycin C in S. clavuligerus is controlled by the positive autoregulatory protein CcaR (25, 32). Mutant strains with disruption in ccaR do not express the claR gene (26), although this control is not exerted directly by the CcaR regulatory protein and appears to involve a cascade mechanism (32). The control of the formation of cephamycin C and/or clavulanic acid by CcaR or ClaR is exerted at the transcription level (1, 25).However, the ccaR::aph S....

Induction of Holomycin Production and Complex Metabolic Changes by the argR Mutation in Streptomyces clavuligerus NP1

“…The purified holomycin was assayed for activity against E. coli and Bacillus subtilis, examined by thin-layer chromatography (TLC), and analyzed by UV spectrum, electrospray ionization-mass spectrometry (ESI-MS), and tandem mass spectrometry (MS-MS). HPLC was run on a reverse-phase column (DikmaDiamonsil-C 18 ; 5 m; 250 by 4.6 mm) with 0% to 40% acetonitrile as the mobile phase; the flow rate was 1 ml/min, and the detection wavelength was 360 nm (11). Under these conditions, holomycin was detected at 18.3 min.…”

“…The UV spectrum of the purified compound in methanol showed peaks at 244, 300, and 388 nm (Fig. 3B), which correspond to the characteristic peaks of the pyrroline ring (11). The mass spectrum of the purified compound gave an m/z peak of 215 (M ϩ H) ϩ (Fig.…”

“…The complexity of CA-clavam biosynthesis is also reflected at the level of regulation, and at least 6 regulatory genes (ccaR [45,52], claR [43], cvm7P [59], and orf21 to orf23 [25,55]) were identified among the three gene clusters. Intricate cross-regulation between the arginine and CA (12,51), cephamycin C and CA (44,45), and CA and holomycin (11) pathways were also reported. In the case of arginine-CA cross-regulation, the oat genes (homologous to the arginine biosynthetic gene argJ, encoding ornithine acetyltransferase) were found in both the CA (oat2) and the paralog (oat1) clusters, and the oat2 mutant showed interesting changes in CA production levels, depending on the arginine concentrations, indicating it plays a role in controlling the flux between arginine and CA (12).…”

“…It is synthesized from one acetyl-coenzyme A (acetyl-CoA) and two cysteines (11,31). The gene cluster responsible for holomycin biosynthesis was recently identified and characterized.…”

High-Throughput Screening for Streptomyces Antibiotic Biosynthesis Activators

“…2b) were identical to those of a holomycin standard sample. Holomycin is a dithiolopyrrolone antibacterial and anticancer antibiotic produced by a special mutant of S. clavuligerus which was defective in late steps of clavulanic acid biosynthesis, but only minute quantities are synthesized by the wildtype strain (5). This indicated that increasing the copy number of afsRS cla in S. clavuligerus also activated the normally silent holomycin biosynthetic gene cluster.…”