RESUMEN

Chassis strains, derived from Streptomyces coelicolor M145, deleted for one or more of its four main specialized metabolites biosynthetic pathways (CPK, CDA, RED and ACT), in various combinations, were constructed for the heterologous expression of specialized metabolites biosynthetic pathways of various types and origins. To determine consequences of these deletions on the metabolism of the deleted strains comparative lipidomic and metabolomic analyses of these strains and of the original strain were carried out. These studies unexpectedly revealed that the deletion of the peptidic clusters, RED and/or CDA, in a strain deleted for the ACT cluster, resulted into a great increase in the triacylglycerol (TAG) content, whereas the deletion of polyketide clusters, ACT and CPK had no impact on TAG content. Low or high TAG content of the deleted strains was correlated with abundance or paucity in amino acids, respectively, reflecting high or low activity of oxidative metabolism. Hypotheses based on what is known on the bio-activity and the nature of the precursors of these specialized metabolites are proposed to explain the unexpected consequences of the deletion of these pathways on the metabolism of the bacteria and on the efficiency of the deleted strains as chassis strains.

Asunto(s)

Vías Biosintéticas , Eliminación de Gen , Metaboloma , Streptomyces coelicolor , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Vías Biosintéticas/genética , Lipidómica , Triglicéridos/metabolismo , Triglicéridos/biosíntesis

RESUMEN

BACKGROUND: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor. RESULTS: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869. CONCLUSION: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

Asunto(s)

Antraquinonas , Antibacterianos , Proteínas Bacterianas , Ferritinas , Hierro , Streptomyces coelicolor , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrollo , Antibacterianos/biosíntesis , Antibacterianos/metabolismo , Ferritinas/metabolismo , Ferritinas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hierro/metabolismo , Antraquinonas/metabolismo , Grupo Citocromo b/metabolismo , Grupo Citocromo b/genética , Regulación Bacteriana de la Expresión Génica , Prodigiosina/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Proteómica , Benzoisocromanquinonas

RESUMEN

The biosynthetic pathway of actinorhodin in Streptomyces coelicolor A3(2) has been studied for decades as a model system of type II polyketide biosynthesis. The actinorhodin biosynthetic gene cluster includes a gene, actVI-orfA, that encodes a protein that belongs to the nuclear transport factor-2-like (NTF-2-like) superfamily. The function of this ActVI-ORFA protein has been a long-standing question in this field. Several hypothetical functions, including pyran ring cyclase, enzyme complex stability enhancer, and gene transcription regulator, have been proposed for ActVI-ORFA in previous studies. However, although the recent structural analysis of ActVI-ORFA revealed a solvent-accessible cavity, the protein displayed structural differences to the well-characterized cyclase SnoaL and did not possess a DNA-binding domain. The obtained crystal structure facilitates an inspection of the previous hypotheses regarding the function of ActVI-ORFA. In the present study, we investigated the effects of a series of actVI-orfA test plasmids with different mutations in an established vector/host system. Time-course analysis of dynamic metabolism profiles demonstrated that ActVI-ORFA prevented formation of shunt metabolites and may have a metabolic flux directing function, which shepherds the flux of unstable intermediates towards actinorhodin. The expression studies resulted in the isolation and structure elucidation of two new shunt metabolites from the actinorhodin pathway. Next, we utilized computational modeling to probe the active site of ActVI-ORFA and confirmed the importance of residues R76 and H78 in the flux directing functionality by expression studies. This is the first time such a function has been observed for a member of NTF-2-like superfamily in Streptomyces secondary metabolism.

Asunto(s)

Antraquinonas , Proteínas Bacterianas , Streptomyces coelicolor , Antraquinonas/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Familia de Multigenes , Mutación , Benzoisocromanquinonas

RESUMEN

BACKGROUND: Streptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential. RESULTS: We engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host. CONCLUSION: Our engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Asunto(s)

Productos Biológicos , Ingeniería Metabólica , Familia de Multigenes , Policétidos , Streptomyces , Streptomyces/genética , Streptomyces/metabolismo , Productos Biológicos/metabolismo , Policétidos/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , Vías Biosintéticas/genética

RESUMEN

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Asunto(s)

ADN de Cadena Simple , Proteínas de Unión al ADN , Unión Proteica , Streptomyces coelicolor , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN de Cadena Simple/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominios Proteicos , Mutación , Fenómenos Biofísicos , Termodinámica

RESUMEN

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Asunto(s)

Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Streptomyces coelicolor , Antraquinonas/metabolismo , Antibacterianos/biosíntesis , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Benzoisocromanquinonas , Represión Catabólica , Glucosa/metabolismo , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Prodigiosina/metabolismo , Metabolismo Secundario/genética , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

Asunto(s)

Antibiosis , Proteínas Bacterianas , Toxinas Bacterianas , Streptomyces , Antibacterianos/biosíntesis , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antibiosis/efectos de los fármacos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/ultraestructura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Microscopía por Crioelectrón , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Lectinas/ultraestructura , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/genética , Streptomyces/crecimiento & desarrollo , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces griseus/efectos de los fármacos , Streptomyces griseus/genética , Streptomyces griseus/crecimiento & desarrollo , Streptomyces griseus/metabolismo

RESUMEN

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Asunto(s)

Antraciclinas , Sintasas Poliquetidas , Streptomyces coelicolor , Sintasas Poliquetidas/metabolismo , Sintasas Poliquetidas/genética , Antraciclinas/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Streptomyces/metabolismo , Streptomyces/genética , Vías Biosintéticas/genética , Hidroxilación , Antibacterianos/biosíntesis , Antibacterianos/metabolismo , Antibacterianos/química

RESUMEN

Malachite green (MG) poses a formidable threat to ecosystems and human health. Laccase emerges as a promising candidate for MG degradation, prompting an investigation into the catalytic activity modulation of a small laccase (SLAC) from Streptomyces coelicolor, with a focus on amino acid position 228. Through saturation mutagenesis, five mutants with a 50% increase in the specific activity were generated. Characterization revealed notable properties, Km of E228F was 8.8% of the wild type (WT), and E288T exhibited a 133% kcat compared to WT. Structural analyses indicated improved hydrophobicity and electrostatic potential on the mutants' surfaces, with the stable E228F-ABTS complex exhibiting reduced flexibility, possibly contributing to the observed decrease in turnover rate. Mutants demonstrated enhanced MG decolorization, particularly E228G. Site 228 acts as a crucial functional control switch, suggesting its potential role in SLAC engineering. This study provides insights into laccase modulation and offers promising avenues for enzymatic bioremediation applications.

Asunto(s)

Lacasa , Streptomyces coelicolor , Humanos , Lacasa/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Ecosistema , Biodegradación Ambiental

RESUMEN

(-)-Geosmin has high demand in perfumes and cosmetic products for its earthy congenial aroma. The current production of (-)-geosmin is either by distillation of sun-baked soil or by inefficient chemical synthesis because of the presence of multiple chiral centers. Fermentation processes are not viable as the titers of the Streptomyces sp. based processes are low. This work presents an alternative route by the heterologous synthesis of (-)-geosmin in Saccharomyces cerevisiae. The enzyme involved is the bifunctional geosmin synthase that catalyzes the conversion of farnesyl diphosphate to germacradienol and germacradienol to geosmin. This study evaluated the activity of many orthologs of geosmin synthase when expressed heterologously in S. cerevisiae. When the well-characterized CAB41566 from Streptomyces coelicolor origin was tested, germacradienol and germacrene D were detected but no geosmin. Bioinformatic analysis based on high/low identities to N-terminal and C-terminal domains of CAB41566 was carried out to identify different orthologs of geosmin synthase proteins from different bacterial and fungal origins. ADO68918 of Stigmatella aurantiaca origin showed the best activity among the tested orthologs, not only in terms of geosmin production but also an order of magnitude higher total abundance of the products of geosmin synthase as compared to CAB41566. This study successfully demonstrated the production of (-)-geosmin in S. cerevisiae and offers an alternative, sustainable and environment-friendly approach to producing (-)-geosmin.

Asunto(s)

Streptomyces coelicolor , Streptomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Streptomyces/metabolismo , Streptomyces coelicolor/metabolismo , Naftoles/química , Naftoles/metabolismo

RESUMEN

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues. However, no specific sequences of these promoters were found. One of these promoters, ssgBp, was selected to examine this cross-recognition in the native host. It controls the expression of the sporulation-specific gene ssgB. Using a luciferase reporter, the activity of this promoter in S. coelicolor and nine mutant strains lacking individual sigB homologous genes showed that sgBp is dependent on three sigma factors, SigH, SigN, and SigI. To determine which nucleotides in the-10 region are responsible for the selection of a specific SigB homologue, promoters mutated at the last three nucleotide positions were tested in the two-plasmid system. Some mutant promoters were specifically recognized by a distinct set of SigB homologues. Analysis of these mutant promoters in the native host showed the role of these nucleotides. A conserved nucleotide A at position 5 was essential for promoter activity, and two variable nucleotides at positions 4 and 6 were responsible for the partial selectivity of promoter recognition by SigB homologues.

Asunto(s)

Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Factor sigma , Esporas Bacterianas , Streptomyces coelicolor , Transcripción Genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Plásmidos/genética , Secuencia de Bases

RESUMEN

In Streptomyces, multiple paralogs of SsgA-like proteins (SALPs) are involved in spore formation from aerial hyphae. However, the functions of SALPs have not yet been elucidated in other actinobacterial genera. Here, we report the primary function of an SsgB ortholog (AmSsgB) in Actinoplanes missouriensis, which develops terminal sporangia on the substrate mycelia via short sporangiophores. Importantly, AmSsgB is the sole SALP in A. missouriensis. The transcription of AmssgB was upregulated during sporangium formation, consistent with our previous findings that AmssgB is a member of the AmBldD regulon. The AmssgB null mutant (ΔAmssgB) strain formed non-globose irregular structures on the substrate mycelium. Transmission electron microscopy revealed that the irregular structures contained abnormally septate hypha-like cells, without an intrasporangial matrix. These phenotypic changes were restored by complementation with AmssgB. Additionally, analysis of the heterologous expression of seven SALP-encoding genes from Streptomyces coelicolor A3(2) (ssgA-G) in the ΔAmssgB strain revealed that only ssgB could compensate for AmSsgB deficiency. This indicated that SsgB of S. coelicolor A3(2) and AmSsgB have comparable functions in A. missouriensis. In contrast to the ΔAmssgB strain, the ftsZ-disrupted strain showed a severe growth defect and produced small sporangium-like structures that swelled to some extent. These findings indicate that AmSsgB is crucial for the early stages of sporangium formation, not for spore septum formation in the late stages. We propose that AmSsgB is involved in sporangium formation by promoting the expansion of the "presporangium" structures formed on the tips of the substrate hyphae. IMPORTANCE: SsgB has been proposed as an archetypical SsgA-like protein with an evolutionarily conserved function in the morphological development of spore-forming actinomycetes. SsgB in Streptomyces coelicolor A3(2) is involved in spore septum formation. However, it is unclear whether this is the primary function of SsgBs in actinobacteria. This study demonstrated that the SsgB ortholog (AmSsgB) in Actinoplanes missouriensis is essential for sporangium expansion, which does not seem to be related to spore septum formation. However, the heterologous expression of ssgB from S. coelicolor A3(2) restored morphological abnormalities in the ΔAmssgB mutant. We propose that the primary function of SsgB is to initiate sporulation in differentiating cells (e.g., aerial hyphae in Streptomyces and "presporangium" cells in A. missouriensis) although its molecular mechanism remains unknown.

Asunto(s)

Actinobacteria , Actinoplanes , Streptomyces coelicolor , Streptomyces , Esporangios/metabolismo , Streptomyces/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Actinobacteria/metabolismo , Proteínas Bacterianas/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo

RESUMEN

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

Asunto(s)

Proteínas Bacterianas , ARN Polimerasas Dirigidas por ADN , ARN Bacteriano , Factor sigma , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN no Traducido , Factor sigma/metabolismo , Factor sigma/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Transcripción Genética

RESUMEN

Zur is a Fur-family metalloregulator that is widely used to control zinc homeostasis in bacteria. In Streptomyces coelicolor, Zur (ScZur) acts as both a repressor for zinc uptake (znuA) gene and an activator for zinc exporter (zitB) gene. Previous structural studies revealed three zinc ions specifically bound per ScZur monomer; a structural one to allow dimeric architecture and two regulatory ones for DNA-binding activity. In this study, we present evidence that Zur contains a fourth specific zinc-binding site with a key histidine residue (H36), widely conserved among actinobacteria, for regulatory function. Biochemical, genetic, and calorimetric data revealed that H36 is critical for hexameric binding of Zur to the zitB zurbox and further binding to its upstream region required for full activation. A comprehensive thermodynamic model demonstrated that the DNA-binding affinity of Zur to both znuA and zitB zurboxes is remarkably enhanced upon saturation of all three regulatory zinc sites. The model also predicts that the strong coupling between zinc binding and DNA binding equilibria of Zur drives a biphasic activation of the zitB gene in response to a wide concentration change of zinc. Similar mechanisms may be pertinent to other metalloproteins, expanding their response spectrum through binding multiple regulatory metals.

Asunto(s)

Proteínas Bacterianas , Streptomyces coelicolor , Zinc , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Regulación Bacteriana de la Expresión Génica , Histidina/metabolismo , Histidina/química , Unión Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Zinc/metabolismo

RESUMEN

In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7 ± 9.58 U mL-1 was obtained in fed-batch fermentation. It's the highest level of CsnA from Streptomyces coelicolor expressed in P. pastoris so far. The hydrolytic process of CsnA showed a time-dependent manner. Chitosan oligosaccharides (COSs) generated by CsnA showed antifungal activity against Fusarium oxysporum sp. cucumerinum (F. oxysporum sp. cucumerinum). The secreted expression and hydrolytic performance make the enzyme a desirable biocatalyst for industrial controllable production of chitooligosaccharides with specific degree of polymerization, which have potential to control fungi that cause important crop diseases.

Asunto(s)

Saccharomycetales , Streptomyces coelicolor , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Proteínas Recombinantes/metabolismo , Pichia/genética , Pichia/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo

RESUMEN

S. lividans and S. coelicolor are phylogenetically closely related strains with different abilities to produce the same specialized metabolites. Previous studies revealed that the strong antibiotic producer, S. coelicolor, had a lower ability to assimilate nitrogen and phosphate than the weak producer, Streptomyces lividans, and this resulted into a lower growth rate. A comparative proteomic dataset was used to establish the consequences of these nutritional stresses on the abundance of proteins of the translational apparatus of these strains, grown in low and high phosphate availability. Our study revealed that most proteins of the translational apparatus were less abundant in S. coelicolor than in S. lividans whereas it was the opposite for ET-Tu 3 and a TrmA-like methyltransferase. The expression of the latter being known to be under the positive control of the stringent response whereas that of the other ribosomal proteins is under its negative control, this indicated the occurrence of a strong activation of the stringent response in S. coelicolor. Furthermore, in S. lividans, ribosomal proteins were more abundant in phosphate proficiency than in phosphate limitation suggesting that a limitation in phosphate, that was also shown to trigger RelA expression, contributes to the induction of the stringent response.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Fosfatos , Streptomyces coelicolor , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/crecimiento & desarrollo , Antibacterianos/biosíntesis , Antibacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fosfatos/metabolismo , Streptomyces lividans/metabolismo , Streptomyces lividans/genética , Proteoma , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Biosíntesis de Proteínas , Nitrógeno/metabolismo , Proteómica , Estrés Fisiológico

RESUMEN

IMPORTANCE: Central metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate or glucosamine 6-phosphate is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity." We made use of this concept by selecting for suppressors in genes related to glucosamine toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and major facilitator superfamily transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents the toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 shed new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.

Asunto(s)

Streptomyces coelicolor , Streptomyces , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Glucosamina/metabolismo , Streptomyces/genética , Amino Azúcares/metabolismo , Antibacterianos , Genes Reguladores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica

RESUMEN

Streptomyces produce complex bioactive secondary metabolites with remarkable chemical diversity. Benzoisochromanequinone polyketides actinorhodin and naphthocyclinone are formed through dimerization of half-molecules via single or double carbon-carbon bonds, respectively. Here we sequenced the genome of S. arenae DSM40737 to identify the naphthocyclinone gene cluster and established heterologous production in S. albus J1074 by utilizing direct cluster capture techniques. Comparative sequence analysis uncovered ncnN and ncnM gene products as putative enzymes responsible for dimerization. Inactivation of ncnN that is homologous to atypical co-factor independent oxidases resulted in the accumulation of fogacin, which is likely a reduced shunt product of the true substrate for naphthocyclinone dimerization. In agreement, inactivation of the homologous actVA-3 in S. coelicolor M145 also led to significantly reduced production of actinorhodin. Previous work has identified the NAD(P)H-dependent reductase ActVA-4 as the key enzyme in actinorhodin dimerization, but surprisingly inactivation of the homologous ncnM did not abolish naphthocyclinone formation and the mutation may have been complemented by an endogenous gene product. Our data suggests that dimerization of benzoisochromanequinone polyketides require two-component reductase-oxidase systems.

Asunto(s)

Policétidos , Streptomyces coelicolor , Oxidorreductasas/metabolismo , Antibacterianos/metabolismo , Dimerización , Antraquinonas/metabolismo , Carbono/metabolismo , Policétidos/metabolismo , Streptomyces coelicolor/metabolismo

RESUMEN

The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/I reduction potential (E°'T1Cu ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°'T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°'T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°'T1Cu . While minimal and small E°'T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°'T1Cu increase. Moreover, the influence of these mutations on E°'T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°'T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat /KM ) relative to WT-SLAC.

Asunto(s)

Lacasa , Streptomyces coelicolor , Cobre/química , Lacasa/metabolismo , Mutación , Oxidación-Reducción , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo

RESUMEN

BACKGROUND: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds. RESULTS: Oviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study. CONCLUSIONS: The metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by 'silent' BGCs.

Asunto(s)

Policétidos , Streptomyces coelicolor , Streptomyces , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Ingeniería Metabólica/métodos , Streptomyces/genética , Policétidos/metabolismo , Familia de Multigenes