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1.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 932-941, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567276

RESUMO

Endo-ß-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-ß-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×106 U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.


Assuntos
Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Streptomyces , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/genética , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética
2.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101840

RESUMO

The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The Vmax and Km values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g-1 min-1 and 5.7 10-4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Doenças das Plantas/microbiologia , Polímeros/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/isolamento & purificação , Ácidos Graxos/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solanum tuberosum/microbiologia , Streptomyces/genética
3.
J Agric Food Chem ; 68(10): 3184-3194, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32105462

RESUMO

Enzymatic hydrolysis of xylan represents a promising way to produce xylooligosaccharide (XOS), which is a novel ingredient in functional food. However, the recalcitrance of xylan in natural lignocellulosic biomass entails effective and robust xylanases. In the present study, we reported the isolation of a thermophilic Streptomyces sp. B6 from mushroom compost producing high xylanase activity. Two xylanases of Streptomyces sp. B6 belonging to GH10 (XynST10) and GH11 (XynST11) families were thus identified and biochemically characterized to be robust enzymes with high alkaline- and thermostability. Direct hydrolysis of neutralized viscose fiber production waste using XynST10 and XynST11 showed that while XynST10 produced 23.22 g/L XOS with a degree of polymerization (DP) of 2-4 and 9.27 g/L xylose, XynST11 produced much less xylose (1.19 g/L) and a higher amounts of XOS with a DP = 2-4 (28.29 g/L). Thus, XynST11 holds great potential for the production of XOS from agricultural and industrial waste.


Assuntos
Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Glucuronatos/química , Oligossacarídeos/química , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Glucuronatos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Resíduos Industriais/análise , Oligossacarídeos/metabolismo , Streptomyces/química , Streptomyces/genética , Xilose/química , Xilose/metabolismo
4.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-31932540

RESUMO

Chitin amendment is an agricultural management strategy for controlling soil-borne plant disease. We previously reported an exponential decrease in chitin added to incubated upland soil. We herein investigated the transition of the bacterial community structure in chitin-degrading soil samples over time and the characteristics of chitinolytic bacteria in order to elucidate changes in the chitinolytic bacterial community structure during chitin degradation. The addition of chitin to soil immediately increased the population of bacteria in the genus Streptomyces, which is the main decomposer of chitin in soil environments. Lysobacter, Pseudoxanthomonas, Cellulosimicrobium, Streptosporangium, and Nonomuraea populations increased over time with decreases in that of Streptomyces. We isolated 104 strains of chitinolytic bacteria, among which six strains were classified as Lysobacter, from chitin-treated soils. These results suggested the involvement of Lysobacter as well as Streptomyces as chitin decomposers in the degradation of chitin added to soil. Lysobacter isolates required yeast extract or casamino acid for significant growth on minimal agar medium supplemented with glucose. Further nutritional analyses demonstrated that the six chitinolytic Lysobacter isolates required methionine (Met) to grow, but not cysteine or homocysteine, indicating Met auxotrophy. Met auxotrophy was also observed in two of the five type strains of Lysobacter spp. tested, and these Met auxotrophs used d-Met as well as l-Met. The addition of Met to incubated upland soil increased the population of Lysobacter. Met may be a factor increasing the population of Lysobacter in chitin-treated upland soil.


Assuntos
Bactérias/isolamento & purificação , Bactérias/metabolismo , Quitina/farmacologia , Metionina/metabolismo , Microbiota/efeitos dos fármacos , Microbiologia do Solo , Bactérias/classificação , Bactérias/genética , Quitina/análise , Quitina/metabolismo , Lysobacter/classificação , Lysobacter/genética , Lysobacter/isolamento & purificação , Lysobacter/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Solo/química , Streptomyces/classificação , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo
5.
Nat Commun ; 11(1): 80, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900404

RESUMO

To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Policetídeo Sintases/química , Policetídeo Sintases/genética , Streptomyces/enzimologia , Proteínas de Bactérias/metabolismo , Macrolídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química , Engenharia de Proteínas , Streptomyces/química , Streptomyces/genética , Especificidade por Substrato
6.
Science ; 367(6476): 458-463, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31896661

RESUMO

Molecular shape defines function in both biological and material settings, and chemists have developed an ever-increasing vernacular to describe these shapes. Noncanonical atropisomers-shape-defined molecules that are formally topologically trivial but are interconvertible only by complex, nonphysical multibond torsions-form a unique subset of atropisomers that differ from both canonical atropisomers (e.g., binaphthyls) and topoisomers (i.e., molecules that have identical connectivity but nonidentical molecular graphs). Small molecules, in contrast to biomacromolecules, are not expected to exhibit such ambiguous shapes. Using total synthesis, we found that the peptidic alkaloid tryptorubin A can be one of two noncanonical atropisomers. We then devised a synthetic strategy that drives the atropospecific synthesis of a noncanonical atrop-defined small molecule.


Assuntos
Produtos Biológicos/metabolismo , Peptídeos Cíclicos/biossíntese , Sequência de Aminoácidos , Produtos Biológicos/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismo , Xanthomonas/genética , Xanthomonas/metabolismo
7.
World J Microbiol Biotechnol ; 36(1): 13, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31897764

RESUMO

Streptomyces is famous for its capability to produce the most abundant antibiotics in all kingdoms. All Streptomyces antibiotics are natural products, whose biosynthesis from the so-called gene clusters are elaborately regulated by pyramidal transcriptional regulatory cascades. In the past decades, scientists have striven to unveil the regulatory mechanisms involved in antibiotic production in Streptomyces. Here we mainly focus on three aspects of the regulation on antibiotic production. 1. The onset of antibiotic production triggered by hormones and their coupled receptors as regulators; 2. The cascades of global and pathway-specific regulators governing antibiotic production; 3. The feedback regulation of antibiotics and/or intermediates on the gene cluster expression for their coordinated production. This review will summarize how the antibiotic production is stringently regulated in Streptomyces based on the signaling, and lay a theoretical foundation for improvement of antibiotic production and potentially drug discovery.


Assuntos
Antibacterianos/metabolismo , Redes Reguladoras de Genes , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Hormônios/metabolismo , Família Multigênica , Streptomyces/genética
8.
Nucleic Acids Res ; 48(5): 2401-2411, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31970401

RESUMO

Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC-MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Genes Essenciais , Holoenzimas/metabolismo , Lisina/metabolismo , Fator sigma/metabolismo , Streptomyces/metabolismo , Acetilação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Regiões Promotoras Genéticas , Ligação Proteica , Fator sigma/química , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento
9.
Microb Cell Fact ; 19(1): 3, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906943

RESUMO

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.


Assuntos
Antibacterianos/biossíntese , Antifúngicos/química , Vias Biossintéticas/genética , Engenharia Metabólica/métodos , Streptomyces/genética , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Edição de Genes/métodos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Polienos/química , Streptomyces/metabolismo
10.
Nucleic Acids Res ; 48(2): 709-718, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31713613

RESUMO

DNA interstrand crosslinks (ICLs) induced by the highly genotoxic agent azinomycin B (AZB) can cause severe perturbation of DNA structure and even cell death. However, Streptomyces sahachiroi, the strain that produces AZB, seems almost impervious to this danger because of its diverse and distinctive self-protection machineries. Here, we report the identification of a novel endonuclease-like gene aziN that contributes to drug self-protection in S. sahachiroi. AziN expression conferred AZB resistance on native and heterologous host strains. The specific binding reaction between AziN and AZB was also verified in accordance with its homology to drug binding proteins, but no drug sequestering and deactivating effects could be detected. Intriguingly, due to the high affinity with the drug, AziN was discovered to exhibit specific recognition and binding capacity with AZB-mediated ICL structures, further inducing DNA strand breakage. Subsequent in vitro assays demonstrated the structure-specific endonuclease activity of AziN, which cuts both damaged strands at specific sites around AZB-ICLs. Unravelling the nuclease activity of AziN provides a good entrance point to illuminate the complex mechanisms of AZB-ICL repair.


Assuntos
Proteínas de Transporte/química , DNA/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas de Transporte/genética , Reagentes para Ligações Cruzadas/química , DNA/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Endonucleases/genética , Humanos , Naftalenos/química , Streptomyces/genética
11.
J Ind Microbiol Biotechnol ; 47(1): 73-81, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31705217

RESUMO

Streptomyces and other closely-related actinobacteria are important sources of bioactive molecules. Streptomyces synthetic biology and genetics empower therapeutic and agrichemical development through strain improvement and biosynthetic understanding. Such efforts rely on the availability of developed molecular toolsets. Among these tools, vectors that enable combinatorial chromosomal manipulations are particularly desirable. Towards developing tools for facile multiplex engineering, we herein describe the development of new integrating vectors derived from BD1 subgroup actinophage OzzyJ (ϕOZJ). By demonstrating the transformation of several Streptomyces spp. using ϕOZJ-derived vectors, we reveal their potential for strain engineering. We further report the development of new ϕC31 and ϕBT1-based vectors having orthogonal resistance, replication and integration features for concomitant transformation with our ϕOZJ-derived vectors. Importantly, the resulting compatible vector panel enabled us to demonstrate the transfer of up to three plasmids each into Streptomyces venezuelae, Streptomyces roseosporus and Streptomyces pristinaespiralis during a single conjugation experiment. To our knowledge this is the first documentation of conjugation-mediated multiplex plasmid transformation, a useful approach for rapid combinatorial strain development.


Assuntos
Actinobacteria/genética , Bacteriófagos/genética , Vetores Genéticos , Streptomyces/genética , Plasmídeos/genética , Biologia Sintética
12.
Mol Cell ; 77(3): 586-599.e6, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31810759

RESUMO

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.


Assuntos
GMP Cíclico/análogos & derivados , Fator sigma/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , GMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Domínios Proteicos , RNA Bacteriano/metabolismo , Esporos Bacterianos/metabolismo , Streptomyces/genética
13.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 91-96, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31833535

RESUMO

Distamycin (DST) is a well-characterized DNA minor groove binder with antivirus activity and antitumor potency. Two separate gene clusters (a 28-kb cluster and a 7-kb cluster) have recently been identified to coordinately encode the biosynthetic machinery of DST in Streptomyces netropsis. Here we report a gene cassette, which is linked to the aforementioned smaller dst gene cluster and plays an important role in the self-resistance to DST in S. netropsis. This cassette consists of three uncharacterized genes that might be implicated in DNA replication/repair. Knockout of the cassette led to the decrease in the production of DST, while heterologous expression of part of the cassette in S. lividans made it become resistant to both DST and mitomycin C, another DNA-binding agent. More interestingly, homologs of these three genes were found in genomes of other actinomyces that produce DNA-binding antibiotics, suggesting that a novel common mechanism in addition to pumping may enable these strains to resist the cytotoxic metabolites they produced.


Assuntos
Antibacterianos/farmacologia , Reparo do DNA/genética , Replicação do DNA/genética , Distamicinas/farmacologia , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Streptomyces/genética , Antibacterianos/biossíntese , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/farmacologia , Distamicinas/biossíntese , Escherichia coli/genética , Técnicas de Inativação de Genes , Mitomicina/farmacologia , Família Multigênica/genética , Streptomyces/efeitos dos fármacos , Streptomyces lividans/efeitos dos fármacos
14.
Enzyme Microb Technol ; 132: 109391, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731956

RESUMO

Styrene monooxygenases (SMOs) are two-component enzymes known to catalyze the epoxidation of styrene to (S)-styrene oxide. In this work, we identified a new oxygenase component, named StStyA, from the genome of Streptomyces sp. NRRL S-31. StStyA displayed complementary stereoselectivity to all of the known SMOs when coupled with a known reductase component (PsStyB), which made it the first natural SMO that produces (R)-styrene oxide. Accordingly, a plasmid co-expressing StStyA and PsStyB was constructed, which led to an artificial two-component SMO, named StStyA/B. When applied in the bio-epoxidation of nine aromatic alkenes, the enzyme showed activity toward five alkenes, and consistently displayed (R)-selectivity. Excellent stereoselectivity was achieved for all five substrates with enantiomeric excesses ranging from 91% to >99%ee.


Assuntos
Proteínas de Bactérias/metabolismo , Oxigenases/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Biocatálise , Compostos de Epóxi/metabolismo , Cinética , Oxigenases/genética , Streptomyces/genética
15.
Talanta ; 208: 120439, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816710

RESUMO

Marine Streptomyces is a potential source of novel bioactive natural products in medicine and agriculture. The current discrimination and screening method of Streptomyces isolates is not accurate and time-consuming, and a novel method is necessary. In this study, a protein profiling method based on an ultrahigh resolution 15 T Matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) was established and applied for differentiation and bioactivity screening of marine Streptomyces isolates. To obtain robust protein profiling, the effects of the protein extraction method, the matrix-solvent, the sample deposition mode, and the culture time of isolates on protein profiling were thoroughly studied, the optimal conditions were obtained. To evaluate the performance of the developed MALDI-FTICR MS method, MALDI-time of flight (TOF) MS and 16S rRNA were applied in parallel to analyze 25 marine Streptomyces isolates. We found that the clustering result of MALDI-FTICR MS was more similar to that of 16S rRNA than MALDI-TOF MS. And MALDI-FTICR MS could effectively indicate the antibacterial activity of Streptomyces isolates against three plant pathogenic bacteria including Xanthomonas campestris, Xanthomonas oryzae and Erwinia carotovora. Furthermore, a differential protein/peptide was defined and successfully applied to predict antibacterial activity of blind samples. This study demonstrated that MALDI-FTICR MS has great potential to discriminate and screen complex microorganisms, especially those closely related strains.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Proteômica/métodos , Streptomyces/classificação , Streptomyces/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Análise de Fourier , Pectobacterium/efeitos dos fármacos , Pectobacterium/crescimento & desenvolvimento , RNA Bacteriano , RNA Ribossômico 16S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Streptomyces/genética , Xanthomonas/efeitos dos fármacos , Xanthomonas/crescimento & desenvolvimento
16.
BMC Genomics ; 20(1): 994, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856709

RESUMO

BACKGROUND: Bacteria within the genus Streptomyces remain a major source of new natural product discovery and as soil inoculants in agriculture where they promote plant growth and protect from disease. Recently, Streptomyces spp. have been implicated as important members of naturally disease-suppressive soils. To shine more light on the ecology and evolution of disease-suppressive microbial communities, we have sequenced the genome of three Streptomyces strains isolated from disease-suppressive soils and compared them to previously sequenced isolates. Strains selected for sequencing had previously showed strong phenotypes in competition or signaling assays. RESULTS: Here we present the de novo sequencing of three strains of the genus Streptomyces isolated from disease-suppressive soils to produce high-quality complete genomes. Streptomyces sp. GS93-23, Streptomyces sp. 3211-3, and Streptomyces sp. S3-4 were found to have linear chromosomes of 8.24 Mb, 8.23 Mb, and greater than 7.5 Mb, respectively. In addition, two of the strains were found to have large, linear plasmids. Each strain harbors between 26 and 38 natural product biosynthetic gene clusters, on par with previously sequenced Streptomyces spp. We compared these newly sequenced genomes with those of previously sequenced organisms. We see substantial natural product biosynthetic diversity between closely related strains, with the gain/loss of episomal DNA elements being a primary driver of genome evolution. CONCLUSIONS: Long read sequencing data facilitates large contig assembly for high-GC Streptomyces genomes. While the sample number is too small for a definitive conclusion, we do not see evidence that disease suppressive soil isolates are particularly privileged in terms of numbers of biosynthetic gene clusters. The strong sequence similarity between GS93-23 and previously isolated Streptomyces lydicus suggests that species recruitment may contribute to the evolution of disease-suppressive microbial communities.


Assuntos
Genoma Bacteriano , Microbiologia do Solo , Streptomyces/genética , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Fenótipo , Análise de Sequência de DNA , Streptomyces/isolamento & purificação , Streptomyces/metabolismo
17.
Appl Microbiol Biotechnol ; 103(23-24): 9373-9378, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31728585

RESUMO

Bacteria modulate their physiological behavior by responding to various signal molecules. The signals are received by cognate receptors, which usually mediate transcriptional regulation. Streptomyces employ γ-butyrolactones (GBLs) and cognate GBL receptors (GblRs) to regulate secondary metabolism and morphological development. However, there are additional transcriptional regulators called pseudo GblR regulators, which cannot bind GBLs and are not directly associated with GBL synthase. The pseudo GblR regulators may act as transcriptional repressors and respond to antibiotic signals. They play regulatory roles in coordination of antibiotic biosynthesis by connecting the hormone feed-forward loops and the antibiotic feedback loops. As the TetR family members, they might also have evolutionary roles between the transcriptional regulators of quorum sensing and antibiotic resistance. Understanding the regulatory and evolutionary roles of the pseudo GblR family would be helpful for fine-tuning regulation of antibiotic biosynthesis and resistance.


Assuntos
Antibacterianos/biossíntese , Farmacorresistência Bacteriana/genética , Regulação Bacteriana da Expressão Gênica , Receptores de GABA-A/genética , Streptomyces/genética , 4-Butirolactona/metabolismo , Regiões Promotoras Genéticas , Percepção de Quorum , Proteínas Repressoras/genética , Metabolismo Secundário , Transdução de Sinais , Streptomyces/metabolismo , Fatores de Transcrição/genética
18.
Appl Microbiol Biotechnol ; 103(23-24): 9593-9606, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713669

RESUMO

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.


Assuntos
Engenharia Metabólica/métodos , Streptomyces/genética , Streptomyces/metabolismo , Tacrolimo/análogos & derivados , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Acil Coenzima A/metabolismo , Acil-CoA Desidrogenases/genética , Acil-CoA Desidrogenases/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Crotonatos/metabolismo , Expressão Gênica , Imunossupressores/metabolismo , Mutagênese , Tacrolimo/metabolismo , Raios Ultravioleta
19.
J Agric Food Chem ; 67(47): 13119-13126, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31686506

RESUMO

Phospholipids have been widely used in food, medicine, cosmetics, and other fields because of their unique chemical structure and healthcare functions. Phospholipase D (PLD) is a key biocatalyst for the biotransformation of phospholipids. Here, an autodisplay expression system was constructed for rapid screening of mutants, and PLD variants were recombined using DNA shuffling technology and three beneficial mutations were obtained. The results of enzymatic performance and sequence information comparison indicated that C-terminal amino acids exerted a greater impact on the correct folding of PLDs, and N-terminal amino acids are more important for catalytic reaction. The best-performing recombinant enzyme in transphosphatidylation reactions was Recom-34, with a phosphatidylserine content accounting for 80.3% of total phospholipids and a 3.24-fold increased conversion rate compared to the parent enzyme. This study demonstrates great significance for screening ideal biocatalysts, facilitating soluble expression of inclusion body proteins, and identifying key amino acids.


Assuntos
Proteínas de Bactérias/genética , Fosfatidilserinas/biossíntese , Fosfolipase D/genética , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Embaralhamento de DNA , Evolução Molecular Direcionada , Fosfolipase D/química , Fosfolipase D/metabolismo , Dobramento de Proteína , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismo
20.
J Microbiol Biotechnol ; 29(12): 1931-1937, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31693835

RESUMO

The heterologous expression of the Streptomyces natural product (NP) biosynthetic gene cluster (BGC) has become an attractive strategy for the activation, titer improvement, and refactoring of valuable and cryptic NP BGCs. Previously, a Streptomyces artificial chromosomal vector system, pSBAC, was applied successfully to the precise cloning of large-sized polyketide BGCs, including immunosuppressant tautomycetin and antibiotic pikromycin, which led to stable and comparable production in several heterologous hosts. To further validate the pSBAC system as a generally applicable heterologous expression system, the daptomycin BGC of S. roseosporus was cloned and expressed heterologously in a model Streptomyces cell factory. A 65-kb daptomycin BGC, which belongs to a non-ribosomal polypeptide synthetase (NRPS) family, was cloned precisely into the pSBAC which resulted in 28.9 mg/l of daptomycin and its derivatives in S. coelicolor M511(a daptomycin non-producing heterologous host). These results suggest that a pSBAC-driven heterologous expression strategy is an ideal approach for producing low and inconsistent Streptomyces NRPS-family NPs, such as daptomycin, which are produced low and inconsistent in native host.


Assuntos
Cromossomos Artificiais , Daptomicina/biossíntese , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , Daptomicina/farmacologia , Furanos/metabolismo , Genes Bacterianos , Vetores Genéticos , Lipídeos , Macrolídeos/metabolismo , Peptídeo Sintases , Policetídeos/metabolismo , Staphylococcus aureus/efeitos dos fármacos
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