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DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking. [ABSTRACT FROM AUTHOR]

A 135,638 bp DNA region that encompasses the leinamycin (LNM) biosynthetic gene cluster was sequenced from Streptomyces atroolivaceus S-140. The boundaries of the lnm cluster were defined by systematic inactivation of open reading frames within the sequenced region. The lnm cluster spans 61.3 kb of DNA and consists of 27 genes encoding nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), hybrid NRPS-PKS, resistance, regulatory, and tailoring enzymes, as well as proteins of unknown function. A model for LNM biosynthesis is proposed, central to which is the LNM hybrid NRPS-PKS megasynthetase consisting of discrete (LnmQ and LnmP) and modular (LnmI) NRPS, acyltransferase-less PKS (LnmG, LnmI, and LnmJ), and PKS modules with unusual domain organization. These studies unveil an unprecedented architectural complexity for the LNM hybrid NRPS-PKS megasynthetase and set the stage to investigate the molecular basis for LNM biosynthesis. [Copyright &y& Elsevier]

In genetic crosses, HC-toxin production in the filamentous fungus Cochliobolus carbonum appears to be controlled by a single locus, TOX2. At the molecular level, TOX2 is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. All copies of four of the TOX2 genes were previously mapped within a 540-kb stretch of DNA in strain SB111. Subsequently, an additional three TOX2 genes, TOXE, TOXF, and TOXG, have been discovered. In this paper we have mapped all copies of the new genes, a total of seven, and show that except for one of the two copies of TOXE, which was previously shown to be on a chromosome of 0.7 Mb in strain SB111, they are all linked to the previously known TOX2 genes within ∼600 kb of each other on a chromosome of 3.5 Mb. We show here that this chromosome also contains at least one non-TOX2 gene, EXG2, which encodes an exo-β1,3-glucanase. EXG2 is still present in strains that have undergone spontaneous deletion of up to ∼1.4 Mb of the 3.5-Mb chromosome. The results contribute to our understanding of the complex organization of the genes involved in HC-toxin biosynthesis and are consistent with the hypothesis that a reciprocal chromosomal translocation accounts for the pattern of distribution of the TOX2 genes in different C. carbonum isolates. [Copyright &y& Elsevier]

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard., Chemistry and Chemical Biology