Sequence analysis and gene amplification study of the penicillin biosynthesis gene cluster from different strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Industrial strains of Penicillium chrysogenum possess many genomic changes leading to higher levels of penicillin. In this work several production and wild-type strains of Penicillium chrysogenum were used in comparative nucleotide sequence analysis of the biosynthesis cluster. The alignments confirmed sequence conservation not only in promoter regions of the biosynthesis genes but also throughout the entire 44.7-kbp genomic fragment comprising the whole biosynthesis cluster with 15.5-kbp and 13.1-kbp flanking regions. As another titre-enhancing mechanism we subsequently examined gene dosage in two production strains introduced here, NMU2/40 and B14. Quantitative real-time PCR and Southern blot analysis showed the amplification of the biosynthesis genes in both these strains. Through the real-time PCR method the exact copy number was estimated for each of the pcbAB, pcbC and penDE genes. The equal pool of all three genes per genome was confirmed for the both production strains indicating that in these strains the entire penicillin cluster has been amplified as an intact element. Penicillium chrysogenum NMU2/40 was found to carry four copies of the cluster, while six copies were estimated for B14. This also proves the contribution of the additional titre-enhancing mechanisms in both strains, since the industrial data referred much higher production of these strains compared with the single copy reference strain NRRL 1951.     

Correction to: Increased heterologous production of the antitumoral polyketide mithramycin a by engineered Streptomyces lividans TK24 strains

Applied Microbiology and Biotechnology - The original publication contains error error in the Materials and Methods section and in the acknowledgement section.     

Stress-response sigma factor σΗ is essential for morphological differentiation of Streptomyces coelicolor A3(2)

We previously cloned the sigH gene encoding a stress-response sigma factor sigma(H) in Streptomyces coelicolor A3(2), located in an operon with the gene encoding proposed anti-sigma factor UshX. To clarify the in vivo function of sigma(H), a stable null mutant of sigH was prepared by homologous recombination. This mutation appeared to have no obvious effect on vegetative growth, but dramatically affected morphological differentiation. Microscopy showed that the sigH mutant produced undifferentiated hyphae with rare spore chains, giving the colony a pale gray color compared to the dark gray wild-type spores. The sigH mutation partially affected growth under conditions of high osmolarity. Expression of the sigH operon was investigated in the S. coelicolor sigH mutant. Out of four promoters directing expression of the sigH operon, the sigH-P2 promoter--the only promoter preferentially induced by salt-stress conditions--was inactive in the sigH mutant. The results indicated that the sigH-P2 promoter is dependent (directly or indirectly) upon sigma(H) and that the operon is autocatalytically activated. We propose that in S. coelicolor sigma(H) has a dual role, regulating the osmotic response and morphological differentiation.     

Osmoregulation in Streptomyces coelicolor: modulation of SigB activity by OsaC

As free-living non-motile saprophytes, Streptomyces need to adapt to a wide range of environmental conditions and this is reflected by an enormous diversity of regulatory proteins encoded by, for example, the genome of the model streptomycete Streptomyces coelicolor . In this organism, we have identified a new osmoregulation gene, osaC , encoding a member of a novel family of regulatory proteins. Members of the family have a predicted domain composition consisting of an N-terminal kinase domain related to anti-sigma factors, sensory Pas and Gaf domains, and a C-terminal phosphatase domain. osaC is linked to the response regulator gene osaB ; expression analysis of the latter revealed that it is induced after osmotic stress in a σ^B-dependent manner. OsaC is required to return osaB and sigB expression back to constitutive levels after osmotic stress. From analysis of the activities of OsaC_ΔPho, lacking the C-terminal phosphatase domain, and OsaC_N92A, with a substitution of a critical asparagine residue in the kinase domain, we infer that this N-terminal domain functions as a σ^B anti-sigma factor. Indeed, co-purification experiments indicate association of OsaC and σ^B. These results support a model for post-osmotic stress modulation of σ^B activity by OsaC.     

Streptomyces aureofaciens strains as hosts for cloning of genes affecting antibiotic production

Summary Several mutants ofStreptomyces aureofaciens strain were used for protoplast regeneration and plasmid transformation. All tested mutants (excepting R 8/26) were transformable by number of plasmids and shuttle vectors. The transformation of the CTC production strains by plasmid containing cloned CTC resistance gene resulted in 1,1–4 times higher antibiotic production. From the restriction analysis of plasmid, phage and chromosomal DNAs it was estimated, that all tested mutants normally contain the modification system analogous toNae I (Roberts, 1987). Mutant R 8/26 expresses not only complete restriction-modification system mentioned above but also potential second system restricting several actinophages.