Lysohexaenetides A and B, linear lipopeptides from Lysobacter sp. DSM 3655 identified by heterologous expression in Streptomyces / 中国天然药物

Lysobacter harbors a plethora of cryptic biosynthetic gene clusters (BGCs), albeit only a limited number have been analyzed to date. In this study, we described the activation of a cryptic polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) gene cluster (lsh) in Lysobacter sp. DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp. S001. As a result of this methodology, we were able to isolate two novel linear lipopeptides, lysohexaenetides A (1) and B (2), from the recombinant strain S001-lsh. Furthermore, we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs. This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes, particularly in the absence of genetic manipulation tools.

Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Engineering the precursor supply pathway in Streptomyces gilvosporeus for overproduction of natamycin / 生物工程学报

Natamycin is a safe and efficient antimycotics which is widely used in food and medicine industry. The polyene macrolide compound, produced by several bacterial species of the genus Streptomyces, is synthesized by type Ⅰ polyketide synthases using acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA as substrates. In this study, four pathways potentially responsible for the supply of the three precursors were evaluated to identify the effective precursor supply pathway which can support the overproduction of natamycin in Streptomyces gilvosporeus, a natamycin-producing wild-type strain. The results showed that over-expressing acetyl-CoA synthetase and methylmalonyl-CoA mutase increased the yield of natamycin by 44.19% and 20.51%, respectively, compared with the wild type strain under shake flask fermentation. Moreover, the yield of natamycin was increased by 66.29% compared with the wild-type strain by co-overexpression of acetyl-CoA synthetase and methylmalonyl-CoA mutase. The above findings will facilitate natamycin strain improvement as well as development of strains for producing other polyketide compounds.

Molluscicidal activity of the secondary metabolites from Streptomyces nigrogriseolus XD 2-7 against Oncomelania hupensis and its preliminary mechanisms of molluscicidal actions / 中国血吸虫病防治杂志

OBJECTIVE@#To evaluate the storage stability of metabolites from actinomycetes Streptomyces nigrogriseolus XD 2-7 and the mollcuscicidal activity against Oncomelania hupensis in the laboratory, and to preliminarily explore the mechanisms of the molluscicidal activity.@*METHODS@#The fermentation supernatant of S. nigrogriseolus XD 2-7 was prepared and stored at -20, 4 °C and 28 °C without light for 10 d; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation supernatant was boiled in a 100 °C water bath for 30 min and recovered to room temperature, and then the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The pH values of the fermentation supernatant were adjusted to 4.0, 6.0 and 9.0 with concentrated hydrochloric acid and sodium hydroxide, and the fermentation supernatant was stilled at room temperature for 12 h, with its pH adjusted to 7.0; then, the molluscicidal effect was tested against O. hupensis following immersion for 72 h. The fermentation product of S. nigrogriseolus XD 2-7was isolated and purified four times with macroporous resin, silica gel and octadecylsilane bonded silica gel. The final products were prepared into solutions at concentrations of 10.00, 5.00, 2.50, 1.25 mg/L and 0.63 mg/L, and the molluscicidal effect of the final productswas tested against O. hupensis following immersion for 72 h, while dechlorination water served as blank controls, and 0.10 mg/L niclosamide served as positive control. The adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were measured in in O. hupensis soft tissues using high performance liquid chromatography (HPLC) following exposure to the final purified fermentation products of S. nigrogriseolus XD 2-7.@*RESULTS@#After the fermentation supernatant of S. nigrogriseolus XD 2-7 was placed at -20, 4 °C and 28 °C without light for 10 d, immersion in the stock solution and solutions at 10- and 50-fold dilutions for 72 h resulted in a 100% (30/30) O. hupensis mortality. Following boiling at 100 °C for 30 min, immersion in the stock solution and solutions at 10- and 50-fold dilutions for 72 h resulted in a 100.00% (30/30) O. hupensis mortality. Following storage at pH values of 4.0 and 6.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2-7 for 72 h resulted in a 100.00% (30/30) O. hupensis mortality, and following storage at a pH value of 9.0 for 12 h, immersion in the fermentation supernatant of S. nigrogriseolus XD 2-7 for 72 h resulted in a 33.33% (10/30) O. hupensis mortality (χ2 = 30.000, P < 0.05). The minimum concentration of the final purified fermentation products of S. nigrogriseolus XD 2-7 was 1.25 mg/L for achieving a 100% (30/30) O. hupensis mortality. The ATP level was significantly lower in O. hupensis soft tissues exposed to 0.10 mg/L and 1.00 mg/L of the final purified fermentation products of S. nigrogriseolus XD 2-7 than in controls (F = 7.274, P < 0.05), while no significant difference was detected in the ADP level between the treatment group and controls (F = 2.485, P > 0.05).@*CONCLUSIONS@#The active mollcuscicidal ingredients of the S. nigrogriseolus XD 2-7 metabolites are maintained stably at -20, 4 °C and 28 °C for 10 d, and are heat and acid resistant but not alkali resistant. The metabolites from S. nigrogriseolus XD 2-7 may cause energy metabolism disorders in O. hupensis, leading to O. hupensis death.

Microorganismos xerófilos cultivables de la zona semiárida de la Tatacoa (Colombia) / Culture xerophile microorganisms from the Tatacoa semiarid zone (Colombia)

Resumen Introducción. Los microorganismos xerófilos han adquirido una mayor relevancia para la realización de investigaciones relacionadas con sus mecanismos adaptativos frente al estrés hídrico, así como la caracterización e identificación de sus hábitats. En Colombia, las zonas semiáridas y desérticas como el desierto de la Tatacoa han sido poco estudiadas a nivel microbiano. Objetivo. Aislar y caracterizar microorganismos xerófilos provenientes del suelo de la zona semiárida de la Tatacoa del departamento del Huila (Colombia). Materiales y métodos. Se colectaron muestras en los sectores denominados localmente como Cuzco y La Victoria, las cuales fueron procesadas para el aislamiento de microorganismos xerófilos en medio selectivo M40Y para posterior caracterización macro y microscópica, así como evaluación mediante pruebas bioquímicas para la utilización de sustratos. Resultados. Fueron aislados 29 morfotipos entre los cuales se pudieron diferenciar: Bacilos y cocos Gram positivos presentes exclusivamente en el sector del Cuzco y bacterias filamentosas ramificadas (actinobacterias) únicamente en el sector de La Victoria. Se estableció la presencia de los géneros Streptomyces, Micrococcus y Corynebacterium. Conclusiones. La presencia de microorganismos relacionados con los géneros anteriormente mencionados permitirá comprender las posibles interacciones que se presentan en este ecosistema, lo que aportará al desarrollo de este lugar como un posible análogo para estudios de búsqueda de vida en otros planetas como Marte. Además, incentivar estudios más detallados donde se puedan recuperar microorganismos que sean de utilidad para diferentes procesos biotecnológicos.

Abstract Introduction. Xerophilic microorganisms have become more relevant for conducting research related to their adaptive mechanisms against water stress, as well as the characterization and identification of their habitats. In Colombia, semi-arid and desert areas such as the Tatacoa desert have been little studied at the microbial level. Objective. Isolation and characterization of cultivable xerophilic microorganisms from the soil of the semi-arid zone of Tatacoa, in the department of Huila (Colombia). Material and methods. For this, samples were collected in the sectors locally called Cuzco and La Victoria, which were processed for the isolation of xerophilic microorganisms in selective M40Y medium for subsequent macro and microscopic characterization, as well as evaluation by biochemical tests for the use of substrates. Results. 29 morphotypes were isolated among which it was possible to differentiate: Gram-positive bacilli and cocci present exclusively in the Cuzco sector and branched filamentous bacteria (Actinobacteria) only in the La Victoria sector. The presence of the genera Streptomyces, Micrococcus and Corynebacterium was established. Conclusions. The presence of microorganisms related to the mentioned genera will allow us to understand the possible interactions that occur in this ecosystem, which will contribute to the development of this place as a possible analogue of studies for the search for life on other planets such as Mars. In addition, promote more detailed studies where microorganisms that are useful for different biotechnological processes can be recovered.

High-level soluble expression of phospholipase D from Streptomyces chromofuscus in Escherichia coli by combinatorial optimization

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

Novel angucycline/angucyclinone family of natural products discovered between 2010 and 2020 / 生物工程学报

Angucyclines/angucyclinones are a large group of polycyclic aromatic polyketides and their producers are widely distributed in nature. This family of natural products attracts great attention because of their diverse biological activities and unique chemical structures. With the development of synthetic biology and the exploitation of the actinomycetes from previously unexplored environments, angucyclines/angucyclinones-like natural products with new skeletons were continuously discovered, thus enriching the structural diversity of this family. In this review we summarize the new angucyclines/angucyclinones analogues discovered in the last decade (2010-2020) by using different strategies, such as changing cultivation conditions, genetic modification, genome mining, bioactivity-guided compound isolation, and fermentation of actinomycetes from underexplored environments. We also discuss the role of synthetic biology in the discovery and development of new compounds of the angucycline/angucyclinone family.

Improvement of natural product production in Streptomyces by manipulating pathway-specific regulators / 生物工程学报

Streptomyces are major sources of bioactive natural products. Genome sequencing reveals that Streptomyces have great biosynthetic potential, with an average of 20-40 biosynthetic gene clusters each strain. However, most natural products from Streptomyces are produced in low yields under regular laboratory cultivation conditions, which hamper their further study and drug development. The production of natural products in Streptomyces is controlled by the intricate regulation mechanisms. Manipulation of the regulatory systems that govern secondary metabolite production will strongly facilitate the discovery and development of natural products of Streptomyces origin. In this review, we summarize progresses in pathway-specific regulators from Streptomyces in the last five years and highlight their role in improving the yields of corresponding natural products.

Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques / 生物工程学报

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.

Cytotoxic activity of methanolic extract of Streptomyces sp. strain KSF 83 on growth of human breast and colon cancer cells

Aims@#The attention for new and effective anticancer drugs but less toxic is increasing over time. Streptomyces is the most important and well-known source of their bioactive compound production with useful bioactivities. This work aimed for evaluation of the anticancer potential of methanolic extract of Streptomyces sp. strain KSF 83 against non-cancerous cell lines (CCD-841-CoN), breast (MCF-7, MDA-MB-231) and colon cancer cell lines (HT-29, HCT-116).@*Methodology and results@#The characteristic of the strain KSF 83 was identified by morphology and 16S rRNA sequencing and results confirmed that the strain belonged to the genus of Streptomyces. The crude substance was produced via submerged fermentation from the strain and methanol solvent was used to extract the culture filtrate. Methanolic extract possessed low toxicity against CCD-841-CoN with only 18% of inhibition activity at the 400 µg/mL. Among all tested cancer cells, the methanolic extract was able to inhibit the growth of all cancer cells tested with MCF-7 was the highest anticancer activity recorded. The methanolic extract also exhibited cytotoxicity in a range of EC50 of 65.79 μg/mL to 262.40 μg/mL. This study revealed the anticancer potential of Streptomyces sp. strain KSF 83, which could be sources of prospective anticancer drugs against breast and colon cancer.@*Conclusion, significance and impact of study@# The extract of KSF 83 was non-toxic toward normal cell lines and able to inhibit the growth of breast and cancer cell lines, thus it can be a potential source of the anticancer drug against breast and colon cancer.

Advances in the biosynthesis of natamycin and its regulatory mechanisms / 生物工程学报

Natamycin is a polyene macrolide antibiotics with strong and broad spectrum antifungal activity. It not only effectively inhibits the growth and reproduction of fungi, but also prevents the formation of some mycotoxins. Consequently, it has been approved for use as an antifungal food preservative in most countries, and is also widely used in agriculture and healthcare. Streptomyces natalensis and Streptomyces chatanoogensis are the main producers of natamycin. This review summarizes the biosynthesis and regulatory mechanism of natamycin, as well as the strategies for improving natamycin production. Moreover, the future perspectives on natamycin research are discussed.

Effect of pH soil and irrigation regimes on management of potato scab

Potato scab caused by different species of phytopathogenic Streptomyces is considered one of the main bacterial diseases of economic crop importance worldwide. Several studies are being carried out in order to control the disease, but until now, there is no efficient way to do this. Some management strategies have been investigated including application of chemical and biological products and utilization of resistant cultivars of potato but there are few reports about the impact of pH and irrigation regimes on the disease. The present study aimed to evaluate the effects of these last two factors on the incidence and severity of potato scab caused by S. scabiei, S. acidiscabies, Streptomyces sp., S. caviscabies and S. europaeiscabiei in assays at pH 4.0, 4.5, 5.0, 5.5, 6.5 and 7.5; and irrigation regimes of once a week, alternate days and daily in greenhouse conditions. The experimental design for the pH tests was randomized blocks arranged in a 5x2 factorial scheme, with 5 replications and 3x2 for the irrigation regimes with 5 replications. The pH tests showed significant differences between the treatments and pH 4,0 - 4,5 presented lower incidence and severity of the disease for the most species tested but no significant differences were observed between the irrigation regimes. The soil acidification is considered a classic strategy for management of the disease and the results obtained herein corroborated this hypothesis.

Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

Response surface methodology-artificial neural network based optimization and strain improvement of cellulase production by Streptomyces sp / Metodologia de superfície de resposta - otimização baseada em redes neurais artificiais e melhoria de estirpes na produção de celulases por Streptomyces sp

Thirty-seven different colonies were isolated from decomposing logs of textile industries. From among these, a thermotolerant, gram-positive, filamentous soil bacteria Streptomyces durhamensis vs15 was selected and screened for cellulase production. The strain showed clear zone formation on the CMC agar plate after Gram's iodine staining. Streptomyces durhamensis vs15 was further confirmed for cellulase production by estimating the reducing sugars through the dinitrosalicylic acid (DNS) method. The activity was enhanced by sequential mutagenesis using three mutagens of ultraviolet irradiation (UV), N methyl-N'-nitro-N-nitrosoguanidine (NTG), and Ethyl methanesulfonate (EMS). After mutagenesis, the cellulase activity of GC23 (mutant) was improved to 1.86-fold compared to the wild strain (vs15). Optimal conditions for the production of cellulase by the GC 23 strain were evaluated using Response Surface Methodology (RSM) and Artificial Neural Network (ANN). The effects of pH, temperature, duration of incubation, and substrate concentration on cellulase production were evaluated. Optimal conditions for the production of cellulase enzyme using Carboxymethyl cellulose as a substrate are 55 ºC of temperature, pH of 5.0, and incubation for 40 h. The cellulase activity of the mutant Streptomyces durhamensis GC23 was further optimized to 2-fold of the activity of the wild type by RSM and ANN

Trinta e sete colônias diferentes foram isoladas de toras em decomposição das indústrias têxteis. Dentre estes, uma bactéria do solo filamentosa termotolerante, Gram-positiva, Streptomyces durhamensis vs15, foi selecionada e rastreada quanto à produção de celulase. A cepa mostrou uma formação de zona clara na placa de ágar CMC após a coloração com iodo Gram. Streptomyces durhamensis vs15 foi ainda confirmado para a produção de celulase, estimando os açúcares redutores pelo método do ácido dinitrosalicílico (DNS). A atividade foi aprimorada por mutagênese sequencial usando três mutagênicos de irradiação ultravioleta (UV), N metil-N'-nitro-N-nitrosoguanidina (NTG) e metanossulfonato de etil (EMS). Após a mutagênese, a atividade celulase do GC23 (mutante) foi melhorada para 1,86 vezes em comparação com a cepa selvagem (vs15). As condições ideais para a produção de celulase pela cepa GC 23 foram avaliadas usando a Metodologia de Superfície de Resposta (RSM) e a Rede Neural Artificial (RNA). Os efeitos do pH, temperatura, duração da incubação e concentração de substrato na produção de celulase foram avaliados. As condições ideais para a produção da enzima celulase usando Carboximetilcelulose como substrato são 55 ° C de temperatura, pH de 5,0 e incubação por 40 h. A atividade da celulase do mutante Streptomyces durhamensis GC23 foi ainda otimizada para 2 vezes a atividade do tipo selvagem por RSM e RNA.

Actividad antimicrobiana de Streptomyces sp. 6E3 aislado de concentrado de mineral* / Streptomyces sp. 6E3 antimicrobial activity isolated from mineral concentrate

RESUMEN El objetivo de este estudio fue determinar la actividad antimicrobiana de un cultivo de Streptomyces sp. 6E3 aislado de minerales frente a diferentes cepas patógenas, producir un extracto y estimar la concen tración mínima inhibitoria (CMI) de las fracciones contra Staphylococcus aureus resistente a meticilina (SARM). La cepa Streptomyces sp. 6E3 mostró actividad antimicrobiana principalmente contra Staphy lococcus aureus (S. aureus). Cinco de las seis fracciones presentaron actividad antimicrobiana y la más efectiva dio una CMI de 0,88 ug/mL frente a S. aureus ATCC 33862, 0,44 ug/mL frente a S. aureus ATCC 43300 y 1,76 ug/mL frente a S. aureus cepa SARM. Streptomyces sp. 6E3 tiene un potencial antimicrobiano frente a cepas de S. aureus resistentes a meticilina y no resistentes, siendo de interés la realización de más estudios sobre sus metabolitos activos.

ABSTRACT The objectives of this study were to determine the antimicrobial activity of a culture of Streptomyces sp. 6E3 isolated from minerals against different pathogenic strains, to produce an extract and to estimate the minimum inhibitory concentration (MIC) of the fractions against methicillin-resistant Staphylococ cus aureus (MRSA). Streptomyces sp. 6E3 showed antimicrobial activity primarily against Staphylococcus aureus (S. aureus). Five of the six fractions presented antimicrobial activity and the most effective gave a MIC of 0.88 ug / mL against S. aureus ATCC 33862, 0.44 ug / mL against S. aureus ATCC 43300 and 1.76 ug / mL vs. a S. aureus MRSA strain. Streptomyces sp. 6E3 has an antimicrobial potential against S. aureus strains resistant to methicillin and non-resistant, being of interest carrying out of more studies on its active metabolites.

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Cloning, expression and characterization of a new endo-β-N-acetylglucosaminidase from Streptomyces alfalfae / 生物工程学报

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-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.

Effects of combination of phenazine-1-carboxylic acid from Streptomyces kebangsaanensis with amphotericin B on clinical Fusarium solani species

Aim@#A novel endophyte, Streptomyces kebangsaanensis was isolated from the stem of a Malaysian ethnomedicinal plant, Portulaca oleracea in 2013. Studies on S. kebangsaanensis crude extract showed that it had antifungal activities and further work led to isolation of a novel compound, phenazine-1-carboxylic acid (PCA). This study investigated the combinatorial effect of PCA isolated from S. kebangsaanensis with amphotericin B on the growth of four clinical Fusarium solani isolates. @*Methodology and results@#Disk diffusion assay showed that the crude extract of S. kebangsaaneesis inhibited growth of all four F. solani isolates. Whereas, the compound PCA from this extract inhibited two of the tested F. solani isolates, UZ541/12, and UZ667/13 at minimum inhibitory concentration of 18.00 µg/mL Combinations of this compound with amphotericin B, reduced the minimum inhibitory concentration of amphotericin B for these two isolates from 8 to 0.13 µg/mL and 4 to 0.03 µg/mL respectively. Analysis of fractional inhibitory concentration index showed that a borderline synergism is present between the compound and amphotericin B. @*Conclusion, significance and impact of the study@#These results indicate PCA may be useful in improving actions of available drugs against antimicrobial resistant microorganisms.

Shunt products of aminoansamycins from aas1 overexpressed mutant strain of Streptomyces sp. S35 / 中国天然药物

Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.

A Thermotolerant Xylan-Degrading Enzyme Is Produced by Streptomyces malaysiensis AMT-3 Using by-Products From the Food Industry

Abstract This study evaluated the production of endoxylanases by Streptomyces malaysiensis AMT-3 in submerged fermentation using by-products of the food industry at 28ºC. In shake-flasks experiments, the highest endoxylanase activity of 45.8 U.mL-1 was observed within 6 days in a medium containing (w/v) 2.5% wheat bran and 1.2% corn steep liquor. The same culture conditions were used to evaluate the enzyme production in a 2 L stirred tank reactor under different agitation (300, 450 and 600 rev.min-1) and aeration (30 and 60 L.h-1) conditions. The use of 450 rev.min-1 coupled to an aeration of 90 L.h-1 resulted on 81.3 U.mL-1 endoxylanase activity within 5 days. The effect of temperature and pH on endoxylanase activity and stability showed the highest activity at 60 ºC and pH 6.0. Zymography showed the presence of three xylanolytic bands with molecular masses of 690, 180 and 142 kDa. The results showed that the thermotolerant actinobacterial endoxylanase can be produced in high titers using by-product of the food industry.