Unveiling Xylanolytic Enzymes Production of Talaromyces wortmannii DR49 on Industrial Agro Wastes

Abstract Xylan degradation is an important step in different industries, such as in biorefinery for biomass hydrolysis. Talaromyces wortmannii is a known fungus due to second metabolite production but only few works showed the xylanolytic potential of this fungus. In this way, the aim of this study was to evaluate the production of xylanolytic enzymes from T. wortmannii DR49 on industrial agro wastes. Cultivation in shake flask showed highest xylanase titration (10.3 U/mL; 9.5 U/mL) for wheat bran (WB) and hydrothermal pretreated sugar cane bagasse (HB); in β-xylosidase production WB and xylose were the best carbon sources (0.57 U/mL; 0.34 U/mL) respectively. STR cultivation revealed that 29°C and pH 6.0 were the best conditions for xylanase (14.5 U/mL) and β-xylosidase (1.7 U/mL) production. T. wortmannii DR49 showed to be a potential candidate for xylanolytic enzymes production using agro wastes in bioreactors, which has never been previously reported in this fungus.

Endophytic Actinomycetes as Potential Producers of Hemicellulases and Related Enzymes for Plant Biomass Degradation

Abstract Tailor made enzymatic preparation must be design to hydrolyze efficiently plant biomass, once that each plant biomass possesses a distinct cell wall composition. Most of actinomycetes used for plant cell wall degradation are focused on the cellulases and xylanases production. However, a wide range of enzymes must be produced for an efficient degradation of lignocellulose materials. During the last decade several unusual environments were studied to obtain strains that produce glycohydrolases with innovator characteristics. In this context, the present work concerned the selection of endophytic actinomycetes as producers of hemicellulases and related enzymes with different enzymatic profiles, for use in the deconstruction of lignocellulosic biomass. A total of 45 Brazilian actinomycetes previously isolated from plants (endophytics) and soil were prospected for hemicellulases and β-glucosidase production. Four strains highlighted for hemicellulase production (DR61, DR63, DR69 and DR66) and were selected for cultivation under other inductors substrates (xylan and pectin). All strains belong to Streptomyces genera and have their extracts tested for degradation of several hemicellulolytic substrates. The strains presented different glicohydrolyse enzymes profiles mainly for xylans and glucans that can be used for specific formulations of enzymes applied on the biomass deconstruction, principally on sugar cane bagasse.

Screening of polyketide genes from Brazilian Atlantic Forest soil / Evaluación de genes biosintéticos de policétidos provenientes de suelo de la selva atlántica brasileñae / Avaliação de genes biossintéticos de policetídeos provenientes do solo da Mata Atlântica Brasileira

Abstract Soil is a large source of microorganisms with potential to produce bioactive compounds. Since most of them cannot be cultured, metagenomics has become a useful tool in order to evaluate this potential. The aim of this study was to screen biosynthetic polyketide genes (PKS) present in a metagenomic library constructed from a soil sample isolated from the Brazilian Atlantic Forest. The library comprises 5000 clones with DNA inserts between 40 and 50 Kb. The characterization of the biosynthetic gene clusters of these molecules is a promising alternative to elucidate the biotechnological potential of bioactive compounds in microbial communities. The PKS genes were screened using degenerated primers. The positive clones for PKS systems were isolated, and their nucleotide sequences analysed with bioinformatics tools. The screening yielded two positive clones for PKS II genes. Furthermore, variations in the sequences of the PKS II genes from the metagenomic library were observed when compared with sequences of ketosynthases' databases. With these findings we gain insight into the possible relation between new biosynthetic genes and the production of new secondary metabolites.

Resumen El suelo es una fuente importante de microrganismos con potencial para producir compuestos bioactivos. Dado que la gran mayoría de estos microorganismos no puede cultivarse, la metagenómica se ha convertido en una herramienta útil para evaluar dicho potencial. El objetivo del presente estudio fue evaluar los genes biosintéticos de policétidos (PKS) presentes en una biblioteca metagenómica construida a partir de una muestra de suelo aislada de la selva atlántica brasileña. La biblioteca comprende 5000 clones con insertos de DNA entre 40 y 50 Kb. La caracterización de clústeres de genes biosintéticos de estas moléculas es una alternativa promisoria para elucidar el potencial biotecnológico de los compuestos bioactivos en comunidades microbianas. Los genes biosintéticos de PKS se evaluaron usando cebadores degenerados. Se aislaron los clones positivos para sistemas PKS y sus secuencias de nucleótidos se analizaron con herramientas bioinformáticas. La evaluación arrojó dos clones positivos para genes de PKS II. Además, se observaron variaciones en las secuencias de genes de PKS II de la biblioteca metagenómica cuando se compararon con las secuencias de las bases de datos de cetosintasas. Estos hallazgos proporcionan nueva información sobre la posible relación entre nuevos genes biosintéticos y la producción de nuevos metabolitos secundarios.

Resumo O solo é uma fonte de importante de microrganismos com potencial para produzir compostos bioativos. Considerando que a maioria destes microrganismos não se pode cultivar, a metagenômica tem se convertido em uma ferramenta útil para avaliar este potencial. O objetivo deste estudo foi avaliar os genes biossintéticos de policetídeos (PKS) presentes em uma biblioteca metagenômica construída a partir de uma amostra de solo isolada da Mata Atlântica brasileira. A biblioteca compreende 5000 clones com insertos de DNA entre 40 e 50 Kb. A caracterização de clusters de genes biossintéticos destas moléculas é uma alternativa promissora para elucidar o potencial biotecnológico de compostos bioativos em comunidades microbianas. Os genes PKS foram avaliados usando primers degenerados. Os clones positivos para sistemas PKS foram isolados e suas sequências de nucleotídeos foram analisadas com ferramentas de bioinformática. A avaliação forneceu dois clones positivos para genes PKS II. Além disso, variações nas sequências dos genes PKS II da biblioteca metagenômica foram observadas quando comparadas com sequências da base de dados de cetosintases. Com estas descobertas obtivemos uma visão sobre uma possível relação entre novos genes biossintéticos e a produção de novos metabólitos secundários.

Evaluación de la presencia del gen mer A implicado en la detoxificación de mercurio a partir de actinomicetos nativos del humedal de La Conejera / Evaluation of the presence of mer A gen implied in the mercury detoxification from native actinomycetes of La Conejera wetland

Se aislaron diez cepas de actinomicetos nativos del humedal La Conejera a partir de muestras de sedimento y agua, con el fin de determinar la capacidad de resistencia y detoxificación de mercurio, evaluando la presencia del gen mer A involucrado en el proceso. La prueba de resistencia fue realizada mediante un test de sensibilidad tomando concentraciones desde 3,68x10-3 mM hasta 10 mM de HgCl2. Se realizaron curvas de crecimiento de 192 h para las cepas resistentes al mercurio, en un medio suplementado con 0,01 mM y 0,05 mM de HgCl2, realizando una fermentación discontinua y midiendo el crecimiento por la técnica de peso seco. A partir de estos resultados se determinó un tiempo de adaptación de 24 h y una producción máxima de biomasa de 2,72 g·L-1 a la hora 72. Paralelamente, se realizó la secuenciación de los genes de resistencia al mercurio de Streptomyces lividans 1326, por medio del diseño de los oligonucleótidos capaces de amplificar el extremos 3’ del gen mer A, codificador de la enzima mercurio reductasa. Se optimizaron las condiciones de amplificación por PCR para obtener un producto amplificado de 686 pb aproximadamente. Finalmente, se realizó una electroforesis en campo pulsado comprobando la presencia de plásmidos en la cepa KH7 con tamaños aproximados de 50, 90 y 300 Kb, no integrados al cromosoma y posiblemente asociados a la resistencia presentada frente al metal.

Ten native actinomycete strains were isolated from water and sediment samples collected in La Conejera wet-lands to assess mercury resistance and detoxification ability for evaluating the presence of the mer A gene involved in that process. Sensitivity assays were performed using 3.68 x 10-3 mM to 10 mM HgCl2 concentrations. Growth curves were generated for each resistant strain after 192 h in discontinuous fermentation in medium supplemented with 0.01 mM and 0.05 mM HgCl2. Biomass growth was then measured by dry weight, maximum value (2.37 g·L-1) being obtained after 72 h. Primer pairs were designed based on the sequence encoding the mercury-reductase enzyme in Streptomyces lividans 1326. PCR conditions for amplifying that region were standardised. The mer A gene was identified in the KH7 strain, resulting in an amplified product of around 686 bp. The KH7 strain electrophoresis profile obtained by pulsed field gel electrophoresis showed 50, 90 and 300 Kb plasmid DNA which could be related to metal resistance in this strain.

The function of lexical motifs in the organization of the Actinomycetes 5S rRNAs

This work shows results obtained by employing the linguistic method to identify biologically meaningful sites in Actinomycetes 5S rRNAs. The approach adopted identifies triplet-words, along the base sequence of 5S rRNA, located mainly at the alpha and beta domains of the 5S secondary structure. There are triplet-words representing universal protein binding sites that include important prokaryote signatures, and sites strategically located in critical regions related to the formation of the 5S ribonucleoproteins (RNP) complex. In those sites, where the GC pressure promoted substitutions, the analysis demonstrates that alterations did not affect their biological significance. Sites formed by GGY (or more rarely GGR), continued to play an important role as ribosomal proteins rpL18 and rpL5 protein receptors. The data suggest that instead of increasing the molecular variability, expected for the diversity in species and habitats occupied for the group, GC pressure functioned as a reducer mechanism for the inter-specific diversity.

Neste trabalho são apresentados resultados obtidos empleando o método linguístico para identificar sítios no 5S rRNAs de actinomicetes com significado biológico. A abordagem identificou palavras-tripletes, junto com a sequência de bases do 5S rRNAs, localizados principalmente nos domínios alfa e beta da estrutura secundária. Entre eles, existem palavras-tripletes que representam sítios de ligação de proteínas universais, que incluem importantes assinaturas procarióticas, além de sítios estrategicamente colocados em regiões críticas relacionados com a formação do complexo 5S ribonucleoproteína (RNP). Nestes sítios, onde a pressão GC promove substituições, as alterações não afetaram seu significado biológico. Sítios formados por GGY (ou mais raramente GER), jogam um papel importante como receptores de proteínas ribossomicas rpL18 e rpL5. Os dados também sugerem que ao contrário de aumentar a variabilidade molecular, esperada pela diversidade em espécies e habitats ocupados pelo grupo, GC funciona como um mecanismo para diversidade inter-específica.

Carbon catabolite repression of retamycin production by Streptomyces olindensis ICB20

Retamycin is an anthracyclinic antitumoral complex produced by Streptomyces olindensis ICB20. In this work the influence of different glucose concentrations in the feed medium on the production of retamycin was studied. Chemostat cultures employing glucose concentration varying between 10 g/L and 25 g/L showed that use of high glucose concentration resulted in catabolite repression of the biosynthesis of the antitumoral. The highest specific retamycin production rate, qRTM = 7.8 mg/g.h, was obtained when glucose concentration was 10 g/L. The lowest value of qRTM, 2.5 mg/g.h, was observed when glucose concentration was 20 and 25 g/L. The residual glucose concentration varied from 0 to 13 g/L, as the glucose concentration in the feed was increased from 10 to 25 g/L.

A retamicina é um complexo antitumoral antraciclínico produzido por Streptomyces olindensis ICB20. Neste trabalho estudou-se a influência de diferentes concentrações de glicose no meio de alimentação sobre a produção de retamicina. Os resultados de cultivos contínuos mostraram que o uso de elevadas concentrações de glicose resultou em repressão catabólica da biossíntese do antitumoral. A maior velocidade específica de produção de retamicina, qRTM = 7,8 mg/g.h, foi obtida quando a concentração de glicose foi de 10 g/L. O menor valor de qRTM, 2,5 mg/g.h, foi observado quando a concentração de glicose foi de 20 e 25 g/L. A concentração de glicose residual aumentou de 0 a 13 g/L conforme a concentração de glicose na alimentação foi incrementada de 10 a 25 g/L.

Phenotypic stability and plasmid detection in Actinobacillus actinomycetemcomitans

Neste estudo foram avaliadas a estabalidade da atividade hemolítica, a resistência a antibióticos e a detecção de plasmídios em cepas orais de A. actinomycetemcomitans. As características foram estßveis durante os diferentes repiques sucessivos realizados nas condições de nosso experimento. Todos os isolados de A. actinomycetemcomitans testados, perderam ou mudaram algums características fenotípicas tal como morfologia colonial e o crescimento em meio líquido.