Role of GntR family regulatory gene SCO1678 in gluconate metabolism in streptomyces coelicolor M145

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed

SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors

Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed

Pleiotropic regulatory genes bldA, adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNALeuUAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining

Revealing Genome-Based Biosynthetic Potential of Streptomyces sp. BR123 Isolated from Sunflower Rhizosphere with Broad Spectrum Antimicrobial Activity

Actinomycetes, most notably the genus Streptomyces, have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genomebased evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesis

Revealing genome-based biosynthetic potential of Streptomyces sp. BR123 isolated from sunflower rhizosphere with broad spectrum antimicrobial activity

Actinomycetes, most notably the genus Streptomyces, have great importance due to their role in the discovery of new natural products, especially for finding antimicrobial secondary metabolites that are useful in the medicinal science and biotechnology industries. In the current study, a genome-based evaluation of Streptomyces sp. isolate BR123 was analyzed to determine its biosynthetic potential, based on its in vitro antimicrobial activity against a broad range of microbial pathogens, including gram-positive and gram-negative bacteria and fungi. A draft genome sequence of 8.15 Mb of Streptomyces sp. isolate BR123 was attained, containing a GC content of 72.63% and 8103 protein coding genes. Many antimicrobial, antiparasitic, and anticancerous compounds were detected by the presence of multiple biosynthetic gene clusters, which was predicted by in silico analysis. A novel metabolite with a molecular mass of 1271.7773 in positive ion mode was detected through a high-performance liquid chromatography linked with mass spectrometry (HPLC-MS) analysis. In addition, another compound, meridamycin, was also identified through a HPLC-MS analysis. The current study reveals the biosynthetic potential of Streptomyces sp. isolate BR123, with respect to the synthesis of bioactive secondary metabolites through genomic and spectrometric analysis. Moreover, the comparative genome study compared the isolate BR123 with other Streptomyces strains, which may expand the knowledge concerning the mechanism involved in novel antimicrobial metabolite synthesi

Challenging old microbiological treasures for natural compound biosynthesis capacity

Strain collections are a treasure chest of numerous valuable and taxonomically validated bioresources. The Leibniz Institute DSMZ is one of the largest and most diverse microbial strain collections worldwide, with a long tradition of actinomycetes research. Actinomycetes, especially the genus Streptomyces, are renowned as prolific producers of antibiotics and many other bioactive natural products. In light of this, five Streptomyces strains, DSM 40971T, DSM 40484T, DSM 40713T, DSM 40976T, and DSM 40907T, which had been deposited a long time ago without comprehensive characterization, were the subject of polyphasic taxonomic studies and genome mining for natural compounds based on in vitro and in silico analyses. Phenotypic, genetic, and phylogenomic studies distinguished the strains from their closely related neighbors. The digital DNA–DNA hybridization and average nucleotide identity values between the five strains and their close, validly named species were below the threshold of 70% and 95%–96%, respectively, determined for prokaryotic species demarcation. Therefore, the five strains merit being considered as novel Streptomyces species, for which the names Streptomyces kutzneri sp. nov., Streptomyces stackebrandtii sp. nov., Streptomyces zähneri sp. nov., Streptomyces winkii sp. nov., and Streptomyces kroppenstedtii sp. nov. are proposed. Bioinformatics analysis of the genome sequences of the five strains revealed their genetic potential for the production of secondary metabolites, which helped identify the natural compounds cinerubin B from strain DSM 40484T and the phosphonate antibiotic phosphonoalamide from strain DSM 40907T and highlighted strain DSM 40976T as a candidate for regulator-guided gene cluster activation due to the abundance of numerous “Streptomyces antibiotic regulatory protein” (SARP) genes

Genetic Engineering of Streptomyces ghanaensis ATCC14672 for Improved Production of Moenomycins

Streptomycetes are soil-dwelling multicellular microorganisms famous for their unprecedented ability to synthesize numerous bioactive natural products (NPs). In addition to their rich arsenal of secondary metabolites, Streptomyces are characterized by complex morphological differentiation. Mostly, industrial production of NPs is done by submerged fermentation, where streptomycetes grow as a vegetative mycelium forming pellets. Often, suboptimal growth peculiarities are the major bottleneck for industrial exploitation. In this work, we employed genetic engineering approaches to improve the production of moenomycins (Mm) in Streptomyces ghanaensis, the only known natural direct inhibitors of bacterial peptidoglycan glycosyltransferses. We showed that in vivo elimination of binding sites for the pleiotropic regulator AdpA in the oriC region strongly influences growth and positively correlates with Mm accumulation. Additionally, a marker- and “scar”-less deletion of moeH5, encoding an amidotransferase from the Mm gene cluster, significantly narrows down the Mm production spectrum. Strikingly, antibiotic titers were strongly enhanced by the elimination of the pleiotropic regulatory gene wblA, involved in the late steps of morphogenesis. Altogether, we generated Mm overproducers with optimized growth parameters, which are useful for further genome engineering and chemoenzymatic generation of novel Mm derivatives. Analogously, such a scheme can be applied to other Streptomyces spp

Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from <i>Streptomyces ghanaensis</i> ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3′→5′) GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis

Regulatory Control of Rishirilide(s) Biosynthesis in <i>Streptomyces bottropensis</i>

Streptomycetes are well-known producers of numerous bioactive secondary metabolites widely used in medicine, agriculture, and veterinary. Usually, their genomes encode 20–30 clusters for the biosynthesis of natural products. Generally, the onset and production of these compounds are tightly coordinated at multiple regulatory levels, including cluster-situated transcriptional factors. Rishirilides are biologically active type II polyketides produced by Streptomyces bottropensis. The complex regulation of rishirilides biosynthesis includes the interplay of four regulatory proteins encoded by the rsl-gene cluster: three SARP family regulators (RslR1-R3) and one MarR-type transcriptional factor (RslR4). In this work, employing gene deletion and overexpression experiments we revealed RslR1-R3 to be positive regulators of the biosynthetic pathway. Additionally, transcriptional analysis indicated that rslR2 is regulated by RslR1 and RslR3. Furthermore, RslR3 directly activates the transcription of rslR2, which stems from binding of RslR3 to the rslR2 promoter. Genetic and biochemical analyses demonstrated that RslR4 represses the transcription of the MFS transporter rslT4 and of its own gene. Moreover, DNA-binding affinity of RslR4 is strictly controlled by specific interaction with rishirilides and some of their biosynthetic precursors. Altogether, our findings revealed the intricate regulatory network of teamworking cluster-situated regulators governing the biosynthesis of rishirilides and strain self-immunity

ORIGINAL Open Access SimReg1 is a master switch for biosynthesis and export of simocyclinone D8 and its precursors

Analysis of the simocyclinone biosynthesis (sim) gene cluster of Streptomyces antibioticus Tü6040 led to the identification of a putative pathway specific regulatory gene simReg1. In silico analysis places the SimReg1 protein in the OmpR-PhoB subfamily of response regulators. Gene replacement of simReg1 from the S. antibioticus chromosome completely abolishes simocyclinone production indicating that SimReg1 is a key regulator of simocyclinone biosynthesis. Results of the DNA-shift assays and reporter gene expression analysis are consistent with the idea that SimReg1 activates transcription of simocyclinone biosynthesis, transporter genes, regulatory gene simReg3 and his own transcription. The presence of extracts (simocyclinone) from S. antibioticus Tü6040 × pSSimR1-1 could dissociate SimReg1 from promoter regions. A preliminary model for regulation of simocyclinone biosynthesis and export is discussed