Polyketomycin is a tetracyclic quinone glycoside produced by Streptomyces diastatochromogenes T?6028. It shows cytotoxic and antibiotic activity, in particular against Gram-positive multi-drug-resistant strains (for example, MRSA). The polyketomycin biosynthetic gene cluster has been sequenced and characterised. Its identity was proven by inactivation of a alpha-ketoacyl synthase gene (pokP1) of the "minimal polyketide synthase II" system. In order to obtain valuable information about tailoring steps, we performed further gene-inactivation experiments. The generation of mutants with deletions in oxygenase genes (pokO1, pokO2, both in parallel and pokO4) and methyltransferase genes (pokMT1, pokMT2 and pokMT3) resulted in new polyketomycin derivatives, and provided information about the organisation of the biosynthetic pathway.

Ribosome recycling factor (RRF), a product of the frr gene, is responsible for the dissociation of ribosomes from messenger RNA after the termination of translation. In order to overexpress frr gene in the toyocamycin (TM) producer Streptomyces diastatochromogenes 1628, we cloned and placed the gene under the control of the constitutive promoter PermE(*). The resulting plasmid pIB139-frr was integrated into the chromosome of S. diastatochromogenes 1628 by conducting intergeneric conjugation. The strain S. diastatochromogenes 1628 containing pIB139-frr (1628-FRR) showed a 33.3 % increase in cell growth and a 46 % increase in TM production compared to wild-type strain 1628 when cultivated in a 7 l fermentor. In addition, it was possible to shorten the fermentation time from 84 to 72 h. Furthermore, by conducting reverse transcription polymerase chain reaction (RT-PCR) analysis, we discovered that the transcriptional levels of regulatory gene adpA-sd, toyF, and toyG involved in TM biosynthesis were enhanced in S. diastatochromogenes 1628-FRR compared to S. diastatochromogenes 1628. In addition, by using a fluorescent intensity reporter system, which is based on the green fluorescent protein (GFP), and by using Western blot analysis, we revealed that overexpression of frr also strongly promoted protein biosynthesis in late growth phase. These findings confirmed that by increasing copy number of frr gene, it is a useful approach to improve antibiotic production.

An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate. The purified enzyme had a molecular mass of 30,862 +/- 5.8 Da, as determined by electrospray mass spectrometry. The esterase-encoding gene was cloned on a 5.1-kb MboI fragment from S. diastatochromogenes genomic DNA into Streptomyces lividans TK23 by using plasmid vector pIJ702. Nucleotide sequence analysis predicted a 978-bp open reading frame, estA, encoding a protein of 326 amino acids, a potential ribosome binding site, and a putative 35- or 36-residue signal peptide for secretion in S. lividans or S. diastatochromogenes, respectively. The transcriptional initiation site was mapped 29 nucleotides upstream from the predicted translational start codon of estA in S. diastatochromogenes. The protein sequence deduced from the estA gene was similar to that of the esterase from the plant pathogen Streptomyces scabies. Both enzymes lacked the conserved motif GXSXG carrying the active-site serine of hydrolytic enzymes. A serine modified by [1,3-3H]diisopropyl fluorophosphate was located at position 11 of the mature enzyme in the sequence GDSYT. This finding and results obtained by site-directed mutagenesis studies indicate that serine 11 may be the active-site nucleophile.