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1.
Artigo em Inglês | MEDLINE | ID: mdl-36232200

RESUMO

This study compared the physiological effects at a metabolomics level with autonomic nervous system responses in adults during soil mixing activities, based on the presence or absence of Streptomyces rimosus in the soil. Thirty adult participants performed soil mixing activities for 5 min using sterilized soil with culture media and Streptomyces rimosus, respectively. Blood samples were drawn twice from each participant after each activity. Electroencephalograms were measured during the activity. Serum metabolites underwent metabolite profiling by gas chromatography, followed by multivariate analyses. Serum brain-derived neurotrophic factor and C-reactive protein levels were measured by Enzyme-Linked Immunosorbent Assay. Soil-emitted volatile organic compounds were identified via solid-phase microextraction and gas chromatography-mass spectroscopy, followed by multivariate analyses. The volatile compound analysis revealed that the terpenoid and benzoid compounds, geosmin, and 2-methylisoborneol were greater in soil with Streptomyces rimosus. Serum metabolomics revealed that the treatment group (soil inoculated with Streptomyces rimosus) possessed relatively higher levels of serotonin compared to the control group (soil mixed with culture media), and serum C-reactive protein levels were significantly lower in the treatment group. In the treatment group, the electroencephalogram revealed that alpha band activity of the occipital lobe increased. This study concludes that Streptomyces rimosus soil contact can positively affect human metabolic and autonomic reactions. Therefore, this pilot study confirmed the possible role of soil microorganisms in horticultural activities for psychophysiological effects in humans.


Assuntos
Streptomyces rimosus , Compostos Orgânicos Voláteis , Adulto , Fator Neurotrófico Derivado do Encéfalo , Proteína C-Reativa , Meios de Cultura , Horticultura , Humanos , Metabolômica , Projetos Piloto , Serotonina , Solo
2.
Angew Chem Int Ed Engl ; 61(39): e202208573, 2022 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-35903822

RESUMO

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.


Assuntos
Produtos Biológicos , Oxitetraciclina , Streptomyces rimosus , Aminoácidos/metabolismo , Produtos Biológicos/metabolismo , Família Multigênica , Oxitetraciclina/metabolismo , Peptídeos Cíclicos/metabolismo , Preparações Farmacêuticas/metabolismo , Streptomyces rimosus/genética , Streptomyces rimosus/metabolismo
3.
Microbiol Spectr ; 10(2): e0243421, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35377231

RESUMO

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus. The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.


Assuntos
Sequenciamento por Nanoporos , Oxitetraciclina , Streptomyces rimosus , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Oxitetraciclina/metabolismo , Plasmídeos/genética , Streptomyces rimosus/genética , Streptomyces rimosus/metabolismo , Transposases/genética , Transposases/metabolismo
4.
Curr Microbiol ; 79(6): 174, 2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35488939

RESUMO

Precursor engineering is an effective strategy for the overproduction of secondary metabolites. The polyene macrolide rimocidin, which is produced by Streptomyces rimosus M527, exhibits a potent activity against a broad range of phytopathogenic fungi. It has been predicted that malonyl-CoA is used as extender units for rimocidin biosynthesis. Based on a systematic analysis of three sets of time-series transcriptome microarray data of S. rimosus M527 fermented in different conditions, the differentially expressed accsr gene that encodes acetyl-CoA carboxylase (ACC) was found. To understand how the formation of rimocidin is being influenced by the expression of the accsr gene and by the concentration of malonyl-CoA, the accsr gene was cloned and over-expressed in the wild-type strain S. rimosus M527 in this study. The recombinant strain S. rimosus M527-ACC harboring the over-expressed accsr gene exhibited better performances based on the enzymatic activity of ACC, intracellular malonyl-CoA concentrations, and rimocidin production compared to S. rimosus M527 throughout the fermentation process. The enzymatic activity of ACC and intracellular concentration of malonyl-CoA of S. rimosus M527-ACC were 1.0- and 1.5-fold higher than those of S. rimosus M527, respectively. Finally, the yield of rimocidin produced by S. rimosus M527-ACC reached 320.7 mg/L, which was 34.0% higher than that of S. rimosus M527. These results confirmed that malonyl-CoA is an important precursor for rimocidin biosynthesis and suggested that an adequate supply of malonyl-CoA caused by accsr gene over-expression led to the improvement in rimocidin production.


Assuntos
Malonil Coenzima A , Streptomyces rimosus , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Malonil Coenzima A/metabolismo , Polienos/metabolismo , Streptomyces rimosus/metabolismo
5.
Biomolecules ; 11(11)2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34827738

RESUMO

The aim of this study was to quantitatively characterize the morphology of the filamentous microorganisms Aspergillus terreus ATCC 20542 and Streptomyces rimosus ATCC 10970, cocultivated in stirred tank bioreactors, and to characterize their mutual influence with the use of quantitative image analysis. Three distinct coculture initiation strategies were applied: preculture versus preculture, spores versus spores and preculture versus preculture with time delay for one of the species. Bioreactor cocultures were accompanied by parallel monoculture controls. The results recorded for the mono- and cocultures were compared in order to investigate the effect of cocultivation on the morphological evolution of A. terreus and S. rimosus. Morphology-related observations were also confronted with the analysis of secondary metabolism. The morphology of the two studied filamentous species strictly depended on the applied coculture initiation strategy. In the cocultures initiated by the simultaneous inoculation, S. rimosus gained domination or advance over A. terreus. The latter microorganism dominated only in these experiments in which S. rimosus was introduced with a delay.


Assuntos
Aspergillus , Streptomyces rimosus , Reatores Biológicos
6.
Molecules ; 26(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34641580

RESUMO

In the present study, Streptomyces rimosus was confronted with Streptomyces noursei, Penicillium rubens, Aspergillus niger, Chaetomium globosum, or Mucor racemosus in two-species submerged co-cultures in shake flasks with the goal of evaluating the oxytetracycline production and morphological development. The co-culture of S. rimosus with S. noursei exhibited stimulation in oxytetracycline biosynthesis compared with the S. rimosus monoculture, whereas the presence of M. racemosus resulted in a delay in antibiotic production. Different strategies of initiating the "S. rimosus + S. noursei" co-cultures were tested. The improvement in terms of oxytetracycline titers was recorded in the cases where S. noursei was co-inoculated with S. rimosus in the form of spores. As the observed morphological changes were not unique to the co-culture involving S. noursei, there was no evidence that the improvement of oxytetracycline levels could be attributed mainly to morphology-related characteristics.


Assuntos
Oxitetraciclina/biossíntese , Streptomyces rimosus/metabolismo , Streptomyces/metabolismo , Antibacterianos/biossíntese , Técnicas de Cocultura , Esporos Bacterianos , Streptomyces/citologia , Streptomyces rimosus/citologia
7.
J Zhejiang Univ Sci B ; 22(5): 383-396, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33973420

RESUMO

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (ΔpyrF) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.


Assuntos
Deleção de Genes , Técnicas de Inativação de Genes/métodos , Orotidina-5'-Fosfato Descarboxilase/genética , Streptomyces rimosus/genética , Teste de Complementação Genética , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Streptomyces rimosus/efeitos dos fármacos
8.
Methods Mol Biol ; 2296: 303-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977456

RESUMO

Streptomyces rimosus is used for production of the broad-spectrum antibiotic oxytetracycline (OTC). S. rimosus belongs to Actinomyces species, a large group of microorganisms that produce diverse set of natural metabolites of high importance in many aspects of our life. In this chapter, we describe specific molecular biology methods and a classical homologous recombination approach for targeted in-frame deletion of a target gene or entire operon in S. rimosus genome. The presented protocols will guide you through the design of experiment and construction of homology arms and their cloning into appropriate vectors, which are suitable for gene-engineering work with S. rimosus. Furthermore, two different protocols for S. rimosus transformation are described including detailed procedure for targeted gene replacement via double crossover recombination event. Gene deletion is confirmed by colony PCR, and colonies are further characterized by cultivation and metabolite analysis. As the final step, we present in trans complementation of the deleted gene, to confirm functionality of the engineering approach achieved by gene disruption. A number of methodological steps and protocols are optimized for S. rimosus strains including the use of the selected reporter genes. Protocols described in this chapter can be applied for studying function of any individual gene product in diverse OTC-producing Streptomyces rimosus strains.


Assuntos
Oxitetraciclina/biossíntese , Streptomyces rimosus/genética , Streptomyces rimosus/metabolismo , Antibacterianos/biossíntese , Clonagem Molecular/métodos , Deleção de Genes , Genoma Bacteriano/genética , Recombinação Homóloga/genética , Biologia Molecular
9.
Microb Cell Fact ; 20(1): 47, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33596911

RESUMO

BACKGROUND: Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses. RESULTS: In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites. CONCLUSIONS: This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.


Assuntos
Oxitetraciclina/biossíntese , Streptomyces rimosus/genética , Família Multigênica , Streptomyces rimosus/metabolismo
10.
Sci Rep ; 11(1): 2489, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510321

RESUMO

A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (ß (1-3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.


Assuntos
Celulases , Streptomyces rimosus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Celulases/química , Celulases/isolamento & purificação , Laminaria/química
11.
Appl Microbiol Biotechnol ; 104(23): 10191-10202, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33057790

RESUMO

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, was found to be highly effective against a broad range of fungal plant pathogens. Current understanding of the regulatory mechanism of rimocidin biosynthesis and morphological differentiation in S. rimosus M527 is limited. NsdA is considered a negative regulator involved in morphological differentiation and biosynthesis of secondary metabolites in some Streptomyces species. In this study, nsdAsr was cloned from S. rimosus M527. The role of nsdAsr in rimocidin biosynthesis and morphological differentiation was investigated by gene deletion, complementation, and over-expression. A ΔnsdAsr mutant was obtained using CRISPR/Cas9. The mutant produced more rimocidin (46%) and accelerated morphological differentiation than the wild-type strain. Over-expression of nsdAsr led to a decrease in rimocidin production and impairment of morphological differentiation. Quantitative RT-PCR analysis revealed that transcription of rim genes responsible for rimocidin biosynthesis was upregulated in the ΔnsdAsr mutant but downregulated in the nsdAsr over-expression strain. Similar effects have been described for Streptomyces coelicolor M145 and the industrial toyocamycin-producing strain Streptomyces diastatochromogenes 1628. KEY POINTS: • A negative regulator for sporulation and rimocidin production was identified. • The CRISPR/Cas9 system was used for gene deletion in S. rimosus M527.


Assuntos
Streptomyces rimosus , Streptomyces , Regulação Bacteriana da Expressão Gênica , Polienos , Streptomyces/genética
12.
J Basic Microbiol ; 60(5): 435-443, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32128846

RESUMO

Chemical pesticides or insecticides with complex structures are highly abundant in the biosphere and have inevitable side effects on farmland, natural resources, and human health. Deltamethrin is the most popular and widely used pesticide that disrupts the cellular calcium channels. In the present study, isolated strains of bacteria were examined to determine the ones that were capable of degrading deltamethrin. Different species of bacteria were evaluated in terms of the capability to degrade deltamethrin. It is important to note that Streptomyces rimosus was able to degrade up to 200 mg/L deltamethrin concentration and could be grown in mineral salt medium agar containing deltamethrin to be used as a source of carbon and energy. The results demonstrated that there is a diversity of deltamethrin-degrading bacteria in agricultural soil ecosystems. The application of these bacteria, especially S. rimosus, might be used as a bioremediation technique to decrease pesticide contamination of the ecosystem.


Assuntos
Nitrilas/metabolismo , Praguicidas/metabolismo , Piretrinas/metabolismo , Poluentes do Solo/metabolismo , Streptomyces rimosus/isolamento & purificação , Streptomyces rimosus/metabolismo , Agricultura , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Biodiversidade , Solo/química , Microbiologia do Solo , Streptomyces rimosus/classificação , Streptomyces rimosus/crescimento & desenvolvimento
13.
Appl Microbiol Biotechnol ; 104(10): 4445-4455, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32221690

RESUMO

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, is highly effective against a broad range of fungal plant pathogens, but at low yields. Elicitation is an effective method of stimulating the yield of bioactive secondary metabolites. In this study, the biomass and filtrate of a culture broth of Escherichia coli JM109, Bacillus subtilis WB600, Saccharomyces cerevisiae, and Fusarium oxysporum f. sp. cucumerinum were employed as elicitors to promote rimocidin production in S. rimosus M527. Adding culture broth and biomass of S. cerevisiae (A3) and F. oxysporum f. sp. cucumerinum (B4) resulted in an increase of rimocidin production by 51.2% and 68.3% respectively compared with the production under normal conditions in 5-l fermentor. In addition, quantitative RT-PCR analysis revealed that the transcriptions of ten genes (rimA to rimK) located in the gene cluster involved in rimocidin biosynthesis in A3 or B4 elicitation experimental group were all higher than those of a control group. Using a ß-glucuronidase (GUS) reporter system, GUS enzyme activity assay, and Western blot analysis, we discovered that elicitation of A3 or B4 increased protein synthesis in S. rimosus M527. These results demonstrate that the addition of elicitors is a useful approach to improve rimocidin production.Key Points • An effective strategy for enhancing rimocidin production in S. rimosus M527 is demonstrated. • Overproduction of rimocidin is a result of higher expressed structural genes followed by an increase in protein synthesis.


Assuntos
Família Multigênica , Streptomyces rimosus/metabolismo , Bacillus subtilis , Biomassa , Vias Biossintéticas , Meios de Cultura/farmacologia , Escherichia coli , Fusarium , Polienos/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae , Metabolismo Secundário/efeitos dos fármacos , Streptomyces rimosus/efeitos dos fármacos
14.
J Biosci Bioeng ; 129(2): 140-145, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31564502

RESUMO

Regulation of secondary metabolism involves complex interactions of both pathway-specific regulators and global regulators, which may trigger or repress the expression of genes involved in antibiotic biosynthesis. Similarly, many of these global regulatory proteins belong to two-component systems. In this study, a new two-component system (TCS) AfrQ1Q2 homologous to AfsQ1Q2 of Streptomyces coelicolor was acquired from the genome sequence of Streptomyces rimosus M4018 by using bioinformatics analysis. RT-PCR results showed co-transcription of afrQ1 (RR) and afrQ2 (HK) in S. rimosus. Consequently, the significant enhancement in oxytetracycline (OTC) yield in afrQ1-disrupted mutant was observed when cultivated in the defined minimal medium (MM) with glycine as the sole nitrogen source. In order to further investigate the regulation mechanism of AfrQ1Q2 in OTC production, the transcriptional levels of five biosynthesis and regulation related genes such as oxyB, otrB, otcG, otcR and otrC were tested by qRT-PCR, which indicated a significantly up-regulatory trend in the afrQ1-disrupted mutant. Meanwhile, a down-regulatory trend of each gene was tested in the complementary mutant as compared to wild type M4018. Moreover, these selected five genes were positively correlated with OTC production. Conclusively, these findings suggested that the TCS AfrQ1Q2 could be one of the global regulators, which negatively regulates OTC production via activating pathway specific regulators in S. rimosus M4018.


Assuntos
Antibacterianos/biossíntese , Oxitetraciclina/biossíntese , Streptomyces rimosus/metabolismo , Sequência de Bases , Genoma Bacteriano , Mutação , Streptomyces rimosus/genética
15.
Microb Cell Fact ; 18(1): 196, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699090

RESUMO

BACKGROUND: The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. RESULTS: We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. CONCLUSION: Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.


Assuntos
Aeropyrum/enzimologia , Proteínas de Bactérias , Endopeptidases , Microrganismos Geneticamente Modificados , Proteínas Recombinantes , Streptomyces rimosus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces rimosus/genética , Streptomyces rimosus/metabolismo
16.
J Zhejiang Univ Sci B ; 20(11): 891-900, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31595725

RESUMO

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in ß-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.


Assuntos
Conjugação Genética , Regiões Promotoras Genéticas , Streptomyces rimosus/genética , Glucuronidase/genética
17.
Int J Syst Evol Microbiol ; 69(8): 2577-2583, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31225792

RESUMO

Streptomyces rimosus is currently composed of two subspecies: Streptomyces rimosus subsp. rimosus and Streptomyces rimosus subsp. paromomycinus. The 16S rRNA gene similarity between type strains of these two subspecies is 99.03 %, whereas that between S. rimosus subsp. paromomycinus and Streptomyces chrestomyceticusis 100 %. To assess the taxonomic status of S. rimosus subsp. paromomycinus, genome sequencing was performed on the type strains of S. rimosus subsp. paromomycinus and S. chrestomyceticus. Digital DNA-DNA hybridization values between S. rimosus subsp. paromomycinus NBRC 15454T and S. rimosus subsp. rimosus ATCC 10970T and between S. rimosus subsp. paromomycinus NBRC 15454T and S. chrestomyceticus NBRC 13444T were 35.4 and 59.9 %, respectively, which are less than the thresholds for bacterial species delineation and indicate that S. rimosus subsp. paromomycinus is not S. rimosus, but an independent species different from S. rimosus and S. chrestomyceticus. In addition, phenotypic data also support that S. rimosus subsp. paromomycinus is distinct from S. chrestomyceticus. Therefore, S. rimosus subsp. paromomycinus should be reclassified as a novel species, for which we propose the name Streptomyces paromomycinus sp. nov. The type strain is NBRC 15454T (=ATCC 14827T=DSM 41429T=JCM 4541T=JCM 4871T=NRRL 2455T=VKM Ac-605T).


Assuntos
Filogenia , Streptomyces/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Streptomyces rimosus/classificação
18.
Appl Microbiol Biotechnol ; 103(16): 6645-6655, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240365

RESUMO

High-yielding industrial Streptomyces producer is usually obtained by multiple rounds of random mutagenesis and screening. These strains have great potential to be developed as the versatile chassis for the discovery and titer improvement of desired heterologous products. Here, the industrial strain Streptomyces rimosus 461, which is a high producer of oxytetracycline, has been engineered as a robust host for heterologous expression of chlortetracycline (CTC) biosynthetic gene cluster. First, the industrial chassis strain SR0 was constructed by deleting the whole oxytetracycline gene cluster of S. rimosus 461. Then, the biosynthetic gene cluster ctc of Streptomyces aureofaciens ATCC 10762 was integrated into the chromosome of SR0. With an additional constitutively expressed cluster-situated activator gene ctcB, the CTC titer of the engineering strain SRC1 immediately reached 1.51 g/L in shaking flask. Then, the CTC titers were upgraded to 2.15 and 3.27 g/L, respectively, in the engineering strains SRC2 and SRC3 with the enhanced ctcB expression. Further, two cluster-situated resistance genes were co-overexpressed with ctcB. The resultant strain produced CTC up to 3.80 g/L in shaking flask fermentation, which represents 38 times increase in comparison with that of the original producer. Overall, SR0 presented in this study have great potential to be used for heterologous production of tetracyclines and other type II polyketides.


Assuntos
Anti-Infecciosos/metabolismo , Vias Biossintéticas/genética , Clortetraciclina/biossíntese , Engenharia Metabólica/métodos , Streptomyces rimosus/metabolismo , Clonagem Molecular , Deleção de Genes , Família Multigênica , Recombinação Genética , Streptomyces aureofaciens/genética , Streptomyces rimosus/genética
19.
Arch Biochem Biophys ; 671: 111-122, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31251922

RESUMO

In this study, we identified a new gene (aph(3″)-Id) coding for a streptomycin phosphotransferase by using phylogenetic comparative analysis of the genome of the oxytetracycline-producing strain Streptomyces rimosus ATCC 10970. Cloning the aph(3″)-Id gene in E.coli and inducing its expression led to an increase in the minimum inhibitory concentration of the recombinant E.coli strain to streptomycin reaching 350 µg/ml. To evaluate the phosphotransferase activity of the recombinant protein APH(3″)-Id we carried out thin-layer chromatography of the putative 32P-labeled streptomycin phosphate. We also performed a spectrophotometric analysis to determine the production of ADP coupled to NADH oxidation. Here are the kinetic parameters of the streptomycin phosphotransferase APH(3″)-Id: Km 80.4 µM, Vmax 6.45 µmol/min/mg and kcat 1.73 s-1. We demonstrated for the first time the ability of the aminoglycoside phototransferase (APH(3″)-Id) to undergo autophosphorylation in vitro. The 3D structures of APH(3″)-Id in its unliganded state and in ternary complex with streptomycin and ADP were obtained. The structure of the ternary complex is the first example of this class of enzymes with bound streptomycin. Comparison of the obtained structures with those of other aminoglycoside phosphotransferases revealed peculiar structure of the substrate-binding pocket reflecting its specificity to a particular antibiotic.


Assuntos
Proteínas de Bactérias/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Streptomyces rimosus/enzimologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biologia Computacional , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Filogenia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Estreptomicina/farmacologia
20.
J Ind Microbiol Biotechnol ; 46(5): 697-708, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30697650

RESUMO

Rimocidin is a polyene macrolide that exhibits a strong inhibitory activity against a broad range of plant-pathogenic fungi. In this study, fermentation optimization and ribosome engineering technology were employed to enhance rimocidin production in Streptomyces rimosus M527. After the optimization of fermentation, rimocidin production in S. rimosus M527 increased from 0.11 ± 0.01 to 0.23 ± 0.02 g/L during shake-flask experiments and reached 0.41 ± 0.05 g/L using 5-L fermentor. Fermentation optimization was followed by the generation of mutants of S. rimosus M527 through treatment of the strain with different concentrations of gentamycin (Gen) or rifamycin. One Genr mutant named S. rimosus M527-G37 and one Rifr mutant named S. rimosus M527-R5 showed increased rimocidin production. Double-resistant (Genr and Rifr) mutants were selected using S. rimosus M527-G37 and S. rimosus M527-R5, and subsequently tested. One mutant, S. rimosus M527-GR7, which was derived from M527-G37, achieved the greatest cumulative improvement in rimocidin production. In the 5-L fermentor, the maximum rimocidin production achieved by S. rimosus M527-GR7 was 25.36% and 62.89% greater than those achieved by S. rimosus M527-G37 and the wild-type strain S. rimosus M527, respectively. Moreover, in the mutants S. rimosus M527-G37 and S. rimosus M527-GR7 the transcriptional levels of ten genes (rimAsr to rimKsr) located in the gene cluster involved in rimocidin biosynthesis were all higher than those in the parental strain M527 to varying degrees. In addition, after expression of the single rimocidin biosynthetic genes in S. rimosus M527 a few recombinants showed an increase in rimocidin production. Expression of rimE led to the highest production.


Assuntos
Farmacorresistência Bacteriana , Microbiologia Industrial/métodos , Família Multigênica , Mutação , Streptomyces rimosus/metabolismo , Antibacterianos/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica , Engenharia Genética , Macrolídeos/metabolismo , Polienos/metabolismo , Ribossomos
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