Deletion of scbA enhances antibiotic production in Streptomyces lividans

Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.     

Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

Purification and structural determination of SCB1, a gamma-butyrolactone that elicits antibiotic production in Streptomyces coelicolor A3(2)

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.     

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor

γ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.     

Streptomyces coelicolor A3(2) produces a new yellow pigment associated with the polyketide synthase Cpk

Streptomyces coelicolor A3(2) is an extensively studied model organism for the genetic studies of Streptomycetes - a genus known for the production of a vast number of bioactive compounds and complex regulatory networks controlling morphological differentiation and secondary metabolites production. We present the discovery of a presumptive product of the Cpk polyketide synthase. We have found that on the rich medium without glucose S. coelicolor A3(2) produces a yellow compound secreted into the medium. We have proved by complementation that production of the observed yellow pigment is dependent on cpk gene cluster previously described as cryptic type I polyketide synthase cluster. The pigment production depends on the medium composition, does not occur in the presence of glucose, and requires high density of spore suspension used for inoculation.     

Biosynthes of polyketide antibiotics by various actinomycin producing Streptomyces species]

A collection of actinomycin-producing Streptomyces strains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned act gene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolor revealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between biosynthetic pathways of actinomycins and polyketides is discussed.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

Biosynthesis and combinatorial biosynthesis of pikromycin-related macrolides in Streptomyces venezuelae

Pikromycin-related macrolides have recently attracted significant research interest because they are structurally related to the semisynthetic ketolide antibiotics that have demonstrated promising potential in combating multi-drug-resistant respiratory pathogens. Cloning and in-depth studies of the pikromycin biosynthetic gene cluster from Streptomyces venezuelae have led to new avenues in modular polyketide synthases, deoxysugar biosynthesis, cytochrome P450 hydroxylase, secondary metabolite gene regulation, and antibiotic resistance. Moreover, the knowledge and tools used for these studies are proving to be valuable in the development of advanced technologies for combinatorial biosynthesis of new macrolide antibiotics. This review summarizes these new developments and introduces S. venezuelae as a powerful new system for secondary metabolite pathway engineering from bench-top genetic manipulation to product fermentation.     

IM-2, a butyrolactone autoregulator,induces production of several nucleoside antibiotics in Streptomyces sp. FRI-5

IM-2 is one of the butyrolactone autoregulators of Streptomyces, which triggers production of a blue pigment in Streptomyces sp. FRI-5 at a concentration of 0.6 ng/ml. In the absence of IM-2, Streptomyces sp. FRI-5 was found to produce d-cycloserine. However, the addition of IM-2 at 5-h cultivation stopped both growth and d-cycloserine production, and instead induced production of several different antibiotics. The IM-2-induced antibiotics were isolated from the culture broth, and assigned as the nucleoside antibiotics, showdomycin and minimycin. Therefore, IM-2 was concluded to be a global regulator of a secondary metabolism, which not only induced the production of nucleoside antibiotics but also suppressed d-cycloserine production.     

Identification of glucose kinase‐dependent and ‐independent pathways for carbon control of primary metabolism,development and antibiotic production in Streptomyces coelicolor by quantitative proteomics

Members of the soil‐dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient availability is a major determinant for the switch to development and antibiotic production in streptomycetes. Carbon catabolite repression (CCR), a main signalling pathway underlying this phenomenon, was so far considered fully dependent on the glycolytic enzyme glucose kinase (Glk). Here we provide evidence of a novel Glk‐independent pathway in Streptomyces coelicolor, using advanced proteomics that allowed the comparison of the expression of some 2000 proteins, including virtually all enzymes for central metabolism. While CCR and inducer exclusion of enzymes for primary and secondary metabolism and precursor supply for natural products is mostly mediated via Glk, enzymes for the urea cycle, as well as for biosynthesis of the γ‐butyrolactone Scb1 and the responsive cryptic polyketide Cpk are subject to Glk‐independent CCR. Deletion of glkA led to strong downregulation of biosynthetic proteins for prodigionins and calcium‐dependent antibiotic (CDA) in mannitol‐grown cultures. Repression of bldB, bldN, and its target bldM may explain the poor development of S. coelicolor on solid‐grown cultures containing glucose. A new model for carbon catabolite repression in streptomycetes is presented.