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1.
J Oleo Sci ; 69(10): 1273-1280, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-32908102

RESUMO

The study involved the isolation and identification of a member of Streptomyces griseorubens and the identification of its secondary metabolite content. Two extract samples were prepared by using butanol and chloroform. In the analyses of the extracts TLC, FT-IR, and GC-MS were employed. Butanol extract appeared to be dominated by three different pyrrole compounds (43.59%), while two fatty acids, linoleic- and erucic acids, were the most abundant secondary metabolites in the chloroform extract, 27.57% and 12.34%, respectively. Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-compound was represented by a single and distinct band on the thin layer chromatography plate. In GC-MS spectra, it also constituted 13.50% of the butanol extract.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Ácido Linoleico/análise , Pirróis/análise , Streptomyces/química , Streptomyces/metabolismo , Butanóis , Cromatografia em Camada Delgada , Ácidos Erúcicos/análise , Cromatografia Gasosa-Espectrometria de Massas , Streptomyces/isolamento & purificação
2.
PLoS One ; 15(9): e0239005, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946485

RESUMO

The two-domain bacterial laccases oxidize substrates at alkaline pH. The role of natural phenolic compounds in the oxidation of substrates by the enzyme is poorly understood. We have studied the role of ferulic and caffeic acids in the transformation of low molecular weight substrates and of soil humic acid (HA) by two-domain laccase of Streptomyces puniceus (SpSL, previously undescribed). A gene encoding a two-domain laccase was cloned from S. puniceus and over-expressed in Escherichia coli. The recombinant protein was purified by affinity chromatography to an electrophoretically homogeneous state. The enzyme showed high thermal stability, alkaline pH optimum for the oxidation of phenolic substrates and an acidic pH optimum for the oxidation of K4[Fe(CN)6] (potassium ferrocyanide) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt). Phenolic compounds were oxidized with lower efficiency than K4[Fe(CN)6] and ABTS. The SpSL did not oxidize 3.4-dimethoxybenzoic alcohol and p-hydroxybenzoic acid neither in the absence of phenolic acids nor in their presence. The enzyme polymerized HA-the amount of its high molecular weight fraction (>80 kDa) increased at the expense of low MW fraction (10 kDa). The addition of phenolic acids as potential mediators did not cause the destruction of HA by SpSL. In the absence of the HA, the enzyme polymerized caffeic and ferulic acids to macromolecular fractions (>80 kDa and 10-12 kDa). The interaction of SpSL with HA in the presence of phenolic acids caused an increase in the amount of HA high MW fraction and a two-fold increase in the molecular weight of its low MW fraction (from 10 to 20 kDa), suggesting a cross-coupling reaction. Infrared and solution-state 1H-NMR spectroscopy revealed an increase in the aromaticity of HA after its interaction with phenolic acids. The results of the study expand our knowledge on the transformation of natural substrates by two-domain bacterial laccases and indicate a potentially important role of the enzyme in the formation of soil organic matter (SOM) at alkaline pH values.


Assuntos
Lacase/metabolismo , Solo/química , Streptomyces/metabolismo , Ácidos Cafeicos/metabolismo , Clonagem Molecular/métodos , Ácidos Cumáricos/metabolismo , Substâncias Húmicas , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Oxirredução , Proteínas Recombinantes/genética , Microbiologia do Solo , Streptomyces/genética , Especificidade por Substrato/genética
3.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908132

RESUMO

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Biocatálise , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/ultraestrutura , Ensaios Enzimáticos , Metilação , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Estreptovaricina/biossíntese , Estreptovaricina/química , Estreptovaricina/metabolismo
4.
Nat Commun ; 11(1): 4022, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782248

RESUMO

One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Policetídeo Sintases/genética , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Estrutura Molecular , Família Multigênica/genética , Policetídeo Sintases/metabolismo , Sirolimo/química , Sirolimo/metabolismo , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo
5.
Nucleic Acids Res ; 48(14): 8188-8202, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32672817

RESUMO

Quorum-sensing (QS) mediated dynamic regulation has emerged as an effective strategy for optimizing product titers in microbes. However, these QS-based circuits are often created on heterologous systems and require careful tuning via a tedious testing/optimization process. This hampers their application in industrial microbes. Here, we design a novel QS circuit by directly integrating an endogenous QS system with CRISPRi (named EQCi) in the industrial rapamycin-producing strain Streptomyces rapamycinicus. EQCi combines the advantages of both the QS system and CRISPRi to enable tunable, autonomous, and dynamic regulation of multiple targets simultaneously. Using EQCi, we separately downregulate three key nodes in essential pathways to divert metabolic flux towards rapamycin biosynthesis and significantly increase its titers. Further application of EQCi to simultaneously regulate these three key nodes with fine-tuned repression strength boosts the rapamycin titer by ∼660%, achieving the highest reported titer (1836 ± 191 mg/l). Notably, compared to static engineering strategies, which result in growth arrest and suboptimal rapamycin titers, EQCi-based regulation substantially promotes rapamycin titers without affecting cell growth, indicating that it can achieve a trade-off between essential pathways and product synthesis. Collectively, this study provides a convenient and effective strategy for strain improvement and shows potential for application in other industrial microorganisms.


Assuntos
Sistemas CRISPR-Cas , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Percepção de Quorum , Streptomyces/genética , Sirolimo/metabolismo , Streptomyces/metabolismo
6.
J Med Microbiol ; 69(8): 1040-1048, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32692643

RESUMO

Given the increased reporting of multi-resistant bacteria and the shortage of newly approved medicines, researchers have been looking towards extreme and unusual environments as a new source of antibiotics. Streptomyces currently provides many of the world's clinical antibiotics, so it comes as no surprise that these bacteria have recently been isolated from traditional medicine. Given the wide array of traditional medicines, it is hoped that these discoveries can provide the much sought after core structure diversity that will be required of a new generation of antibiotics. This review discusses the contribution of Streptomyces to antibiotics and the potential of newly discovered species in traditional medicine. We also explore how knowledge of traditional medicines can aid current initiatives in sourcing new and chemically diverse antibiotics.


Assuntos
Antibacterianos/isolamento & purificação , Descoberta de Drogas/tendências , Microbiologia do Solo , Streptomyces/metabolismo , Animais , Antibacterianos/biossíntese , Cavernas/química , Invertebrados/química , Medicina Tradicional , Peptídeo Sintases/metabolismo , Plantas Medicinais/química , Policetídeo Sintases/metabolismo , Poríferos/química , Streptomyces/química , Streptomyces/enzimologia
7.
Arch Microbiol ; 202(9): 2481-2492, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32617605

RESUMO

In this study, we tested the ability of Streptomyces to use for their growth benzene, toluene, ethylbenzene, and o-, m-, p-xylenes as sole source of carbon and energy. These bacteria were isolated from agricultural soils and activated sludge samples from a wastewater treatment plant. The results show that Streptomyces are capable of degrading at least one of the BTEX compounds. Among them, 3 isolates from activated sludge called (U, F and V) and a single isolate (SA13) isolated from an agricultural soil, can use as the sole source of carbon and energy, all of these BTEX compounds at concentrations of 1500 mg/L. Based on the analysis of the 16S rRNA gene sequence, two active strains were identified as Streptomyces fimicarius, Streptomyces cavourensis, Streptomyces flavogriseus and Streptomyces pratensis. These strains can be excellent candidates for the bioremediation of the telluric and aquatic sites polluted by these xenobiotics.


Assuntos
Microbiologia do Solo , Poluentes do Solo/metabolismo , Solo , Streptomyces/metabolismo , Benzeno/metabolismo , Derivados de Benzeno/metabolismo , Biodegradação Ambiental , RNA Ribossômico 16S/genética , Esgotos/química , Esgotos/microbiologia , Solo/química , Streptomyces/genética , Streptomyces/isolamento & purificação , Tolueno/metabolismo , Xilenos/metabolismo
8.
Food Chem ; 330: 127225, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569931

RESUMO

The whole genome of Streptomyces violascens (=ATCC 27968) was sequenced and the cloning and expression of OUC-Lipase 6 were conducted in Bacillus subtilis WB800. The recombinant enzyme belongs to the lipolytic enzymes family V. OUC-Lipase 6 showed optimal activity at 30 °C and pH 9.0, and retained 90.2% of its activity in an alkaline buffer (pH 8.0, 30 °C and 96 h). OUC-Lipase 6 showed good stability under medium temperature conditions (residual activity of 68.8%, pH 8.0, 45 °C and 96 h). OUC-Lipase 6 could selectively hydrolyze fatty acids on the glyceride backbone, thus improving the contents of DHA and EPA in codfish oil. OUC-Lipase 6 also showed regioselectivity, resulting in a better enrichment efficiency for EPA than DHA. After hydrolyzing for 36 h via OUC-Lipase 6, the contents of EPA and DHA were improved to 3.24-fold and 1.98-fold, respectively.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Lipase/metabolismo , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Genoma Bacteriano , Glicerídeos/química , Hidrólise , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismo , Especificidade por Substrato
9.
Proc Natl Acad Sci U S A ; 117(19): 10265-10270, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32350138

RESUMO

Coformycin and pentostatin are structurally related N-nucleoside inhibitors of adenosine deaminase characterized by an unusual 1,3-diazepine nucleobase. Herein, the cof gene cluster responsible for coformycin biosynthesis is identified. Reconstitution of the coformycin biosynthetic pathway in vitro demonstrates that it overlaps significantly with the early stages of l-histidine biosynthesis. Committed entry into the coformycin pathway takes place via conversion of a shared branch point intermediate to 8-ketocoformycin-[Formula: see text]-monophosphate catalyzed by CofB, which is a homolog of succinylaminoimidazolecarboxamide ribotide (SAICAR) synthetase. This reaction appears to proceed via a Dieckmann cyclization and a retro-aldol elimination, releasing ammonia and D-erythronate-4-phosphate as coproducts. Completion of coformycin biosynthesis involves reduction and dephosphorylation of the CofB product, with the former reaction being catalyzed by the NADPH-dependent dehydrogenase CofA. CofB also shows activation by adenosine triphosphate (ATP) despite the reaction requiring neither a phosphorylated nor an adenylated intermediate. This may serve to help regulate metabolic partitioning between the l-histidine and coformycin pathways.


Assuntos
Adenosina Desaminase/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Coformicina/biossíntese , Família Multigênica , Streptomyces/genética , Adenosina Desaminase/metabolismo , Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Fosforilação , Streptomyces/metabolismo
10.
PLoS One ; 15(5): e0232927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396566

RESUMO

Tetraene macrolides remain one of the most reliable fungicidal agents as resistance of fungal pathogens to these antibiotics is relatively rare. The modes of action and biosynthesis of polyene macrolides had been the focus of research over the past few years. However, few studies have been carried out on the overproduction of polyene macrolides. In the present study, cumulative drug-resistance mutation was used to obtain a quintuple mutant G5-59 with huge tetraene macrolide overproduction from the starting strain Streptomyces diastatochromogenes 1628. Through DNA sequence analysis, the mutation points in the genes of rsmG, rpsL and rpoB were identified. Additionally, the growth characteristic and expression level of tetrRI gene (belonging to the large ATP binding regulator of LuxR family) involved in the biosynthesis of tetraene macrolides were analyzed. As examined with 5L fermentor, the quintuple mutant G5-59 grew very well and the maximum productivity of tetramycin A, tetramycin P and tetrin B was as high as 1735, 2811 and 1500 mg/L, which was 8.7-, 16- and 25-fold higher than that of the wild-type strain 1628, respectively. The quintuple mutant G5-59 could be useful for further improvement of tetraene macrolides production at industrial level.


Assuntos
Proteínas de Bactérias/genética , Reatores Biológicos/microbiologia , Macrolídeos/metabolismo , Mutação , Streptomyces/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Fermentação , Engenharia Metabólica , Metiltransferases/genética , Proteínas Ribossômicas/genética , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/metabolismo
11.
J Ind Microbiol Biotechnol ; 47(4-5): 413-423, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32367443

RESUMO

CRISPR-Cas9 has proven as a very powerful gene editing tool for Actinomyces, allowing scarless and precise genome editing in selected strains of these biotechnologically relevant microorganisms. However, its general application in actinomycetes has been limited due to its inefficacy when applying the system in an untested strain. Here, we provide evidence of how Cas9 levels are toxic for the model actinomycetes Streptomyces coelicolor M145 and Streptomyces lividans TK24, which show delayed or absence of growth. We overcame this toxicity by lowering Cas9 levels and have generated a set of plasmids in which Cas9 expression is either controlled by theophylline-inducible or constitutive promoters. We validated the targeting of these CRISPR-Cas9 system using the glycerol uptake operon and the actinorhodin biosynthesis gene cluster. Our results highlight the importance of adjusting Cas9 expression levels specifically in strains to gain optimum and efficient gene editing in Actinomyces.


Assuntos
Sistemas CRISPR-Cas , Recombinação Genética , Streptomyces/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Família Multigênica , Plasmídeos/genética , Streptomyces/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
12.
Enzyme Microb Technol ; 136: 109530, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32331723

RESUMO

L-glutamate oxidase (LGOX) catalyzes the oxidative deamination of l-glutamate to α-ketoglutarate (α-KG) with the formation of ammonia and hydrogen peroxide. Consequently, identifying a novel LGOX with high enzymatic activity is a prime target for industrial biotechnology. In this study, error-prone PCR mutagenesis of Streptomyces mobaraensis LGOX followed by high-throughput screening was performed to yield four single point mutants with improved enzymatic activity, termed F94L, S280T, I282M and H533R. Moreover, site-saturation mutagenesis at these four residues was employed, yielding two additionally improved mutants, termed I282L and H533L. Subsequently, we employed combinatorial mutagenesis of two, three and four point mutants, and the best mutant S280TH533L showed 90 % higher enzymatic activity than the wild-type control. The data also showed that the presence of these point mutations greatly enhanced enzymatic activity, but did not alter its optimum temperature and pH. Furthermore, the S280TH533L mutant had the maximal velocity (Vmax) of 231.3 µmol/mg/min and the Michaelis-Menten constant (KM) of 2.7 mM, which were the highest Vmax and lowest KM values of LGOX reported so far. Finally, we developed a whole-cell biocatalyst for α-KG production by co-expression of both S280TH533L mutant and KatE catalase. Randomized ribosome binding site (RBS) sequences were introduced to generate vectors with varying expression levels of S280TH533L and KatE, and two optimized co-expression strains were obtained after screening. The α-KG production reached a maximum titer of 181.9 g/L after 12 h conversation using the optimized whole-cell biocatalyst, with a molar conversion rate of substrate higher than 86.3 % in the absence of exogenous catalase, while the molar conversion rate of substrate using the wild-type biocatalyst was less than 30 %. Taken together, these data suggest that the engineering of LGOX has great potentials to enhance the industrial production of α-KG.


Assuntos
Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Ácidos Cetoglutáricos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Ácido Glutâmico/metabolismo , Microbiologia Industrial , Mutagênese Sítio-Dirigida , Mutação Puntual , Especificidade por Substrato
13.
Nat Commun ; 11(1): 1614, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32235841

RESUMO

The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.


Assuntos
Bactérias/metabolismo , Óxido Nítrico/metabolismo , Triazóis/metabolismo , Azaguanina/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos , Vias Biossintéticas/genética , Genes Bacterianos/genética , Óxido Nítrico Sintase/metabolismo , Nitrogênio , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo , Biologia Sintética
14.
Proc Natl Acad Sci U S A ; 117(13): 7392-7400, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32188788

RESUMO

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Streptomyces/metabolismo , Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Hidrólise , Mycobacterium tuberculosis/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais/fisiologia , Streptococcus pneumoniae/metabolismo
15.
J Appl Microbiol ; 129(3): 652-664, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32196866

RESUMO

AIMS: This study aimed to apply the volatile organic compounds from Streptomyces philanthi RL-1-178 (VOCs RL-1-178) as a fumigant to protect soybean seeds against the two aflatoxin-producing fungi in stored soybean seeds. METHODS AND RESULTS: The antifungal bioassay tests on potato dextrose agar (PDA) dishes showed that 30 g l-1 wheat seed inoculum of S. philanthi RL-1-178 exhibited total (100%) inhibition on Aspergillus parasiticus TISTR 3276 and Aspergillus flavus PSRDC-4. Identification of the VOCs RL-1-178 using GC-MS revealed 39 compounds with the most abundant substances being geosmin (13·75%) followed by l-linalool (13·55%), 2-mercaptoethanol (9·71%) and heneicosane (5·96%). Comparison on the efficacy of the VOCs RL-1-178 (at 30 g l-1 wheat seed culture) and their four major components (100 µl l-1 each) on the suppression of the two aflatoxin-producing fungi on PDA plates revealed that the VOCs RL-1-178 as well as geosmin, l-linalool and 2-mercaptoethanol completely inhibited (100%) mycelial growth while heneicosane showed only 70·7% inhibition. Use of the VOCs RL-1-178 (30 g l-1 ) as a biofumigant on stored soybean seeds resulted in complete protection (100%) against the infection as well as complete inhibition on production of aflatoxin (B1 , B2 and G2 ) (analysed by HPLC) by the two aflatoxin-producing fungi. CONCLUSIONS: The VOCs RL-1-178 displayed strong inhibitory effects on A. parasiticus TISTR 3276 and A. flavus PSRDC-4 as well as inhibited aflatoxin (B1 , B2 and G2 ) production. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the VOCs RL-1-178 can be applied as a biofumigant to control the two aflatoxin-producing fungi on stored seeds products.


Assuntos
Aspergillus/efeitos dos fármacos , Fumigação/métodos , Controle Biológico de Vetores/métodos , Soja/microbiologia , Streptomyces/metabolismo , Aflatoxinas/biossíntese , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Sementes/microbiologia
16.
J Appl Microbiol ; 129(3): 565-574, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32145135

RESUMO

AIM: The aim of the present work was to investigate the overexpression of the wysR gene in Streptomyces albulus var. wuyiensis strain CK-15 based on the ΔwysR3 mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. METHODS AND RESULTS: We developed a method to create a wysR overexpression strain based on the ΔwysR3 mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into ΔwysR3 mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression of wysR gene in ΔwysR3 mutant. CONCLUSION: We can conclude that overexpression of wysR gene in ΔwysR3 mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed that wysR regulated wuyiencin biosynthesis by modulating other putative regulatory genes and bld, whi, chp, rdl and ram family genes are crucial for the morphological development. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of wysR gene in the ΔwysR3 mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by which wysR overexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Fermentação , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mutação , Plasmídeos , Streptomyces/genética
17.
Microb Cell Fact ; 19(1): 71, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32192516

RESUMO

Notonesomycin A is a 32-membered bioactive glycosylated macrolactone known to be produced by Streptomyces aminophilus subsp. notonesogenes 647-AV1 and S. aminophilus DSM 40186. In a high throughput antifungal screening campaign, we identified an alternative notonesomycin A producing strain, Streptomyces sp. A793, and its biosynthetic gene cluster. From this strain, we further characterized a new more potent antifungal non-sulfated analogue, named notonesomycin B. Through CRISPR-Cas9 engineering of the biosynthetic gene cluster, we were able to increase the production yield of notonesomycin B by up to 18-fold as well as generate a strain that exclusively produces this analogue.


Assuntos
Antifúngicos/isolamento & purificação , Macrolídeos/isolamento & purificação , Streptomyces/genética , Antifúngicos/metabolismo , Clonagem Molecular , Macrolídeos/metabolismo , Família Multigênica , Streptomyces/metabolismo
19.
Sci Rep ; 10(1): 2003, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029878

RESUMO

Streptomyces bacteria are known for their prolific production of secondary metabolites, many of which have been widely used in human medicine, agriculture and animal health. To guide the effective prioritization of specific biosynthetic gene clusters (BGCs) for drug development and targeting the most prolific producer strains, knowledge about phylogenetic relationships of Streptomyces species, genome-wide diversity and distribution patterns of BGCs is critical. We used genomic and phylogenetic methods to elucidate the diversity of major classes of BGCs in 1,110 publicly available Streptomyces genomes. Genome mining of Streptomyces reveals high diversity of BGCs and variable distribution patterns in the Streptomyces phylogeny, even among very closely related strains. The most common BGCs are non-ribosomal peptide synthetases, type 1 polyketide synthases, terpenes, and lantipeptides. We also found that numerous Streptomyces species harbor BGCs known to encode antitumor compounds. We observed that strains that are considered the same species can vary tremendously in the BGCs they carry, suggesting that strain-level genome sequencing can uncover high levels of BGC diversity and potentially useful derivatives of any one compound. These findings suggest that a strain-level strategy for exploring secondary metabolites for clinical use provides an alternative or complementary approach to discovering novel pharmaceutical compounds from microbes.


Assuntos
Proteínas de Bactérias/genética , Produtos Biológicos/metabolismo , Família Multigênica , Metabolismo Secundário/genética , Streptomyces/metabolismo , Animais , Antibacterianos/metabolismo , Antineoplásicos/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Mineração de Dados , Desenvolvimento de Medicamentos/métodos , Genoma Bacteriano , Genômica , Filogenia , Streptomyces/genética
20.
Sci Rep ; 10(1): 1756, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019976

RESUMO

Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline-L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purification yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including different types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confirmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products.


Assuntos
Antineoplásicos/metabolismo , Produtos Biológicos/metabolismo , Furanos/metabolismo , Streptomyces/metabolismo , Células A549 , Antibacterianos/metabolismo , Bioensaio/métodos , Linhagem Celular Tumoral , Genoma Bacteriano/genética , Células HCT116 , Humanos , Família Multigênica/genética , Filogenia , Streptomyces/genética
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