Biotechnology of polyketides: new breath of life for the novel antibiotic genetic pathways discovery through metagenomics

The discovery of secondary metabolites produced by microorganisms (e.g., penicillin in 1928) and the beginning of their industrial application (1940) opened new doors to what has been the main medication source for the treatment of infectious diseases and tumors. In fact, approximately 80 years after the discovery of the first antibiotic compound, and despite all of the warnings about the failure of the "goose that laid the golden egg," the potential of this wealth is still inexorable: simply adjust the focus from "micro" to "nano", that means changing the look from microorganisms to nanograms of DNA. Then, the search for new drugs, driven by genetic engineering combined with metagenomic strategies, shows us a way to bypass the barriers imposed by methodologies limited to isolation and culturing. However, we are far from solving the problem of supplying new molecules that are effective against the plasticity of multi- or pan-drug-resistant pathogens. Although the first advances in genetic engineering date back to 1990, there is still a lack of high-throughput methods to speed up the screening of new genes and design new molecules by recombination of pathways. In addition, it is necessary an increase in the variety of heterologous hosts and improvements throughout the full drug discovery pipeline. Among numerous studies focused on this subject, those on polyketide antibiotics stand out for the large technical-scientific efforts that established novel solutions for the transfer/engineering of major metabolic pathways using transposons and other episomes, overcoming one of the main methodological constraints for the heterologous expression of major pathways. In silico prediction analysis of three-dimensional enzymatic structures and advances in sequencing technologies have expanded access to the metabolic potential of microorganisms.

Crescimento in vitro e aclimatização de Cattleya loddigesii Lindley (Orchidaceae) com carvão ativado sob dois espectros luminosos

The aim of this study was to evaluate the effect of different concentrations of activated charcoal in ½ MS (half concentration of macronutrients) culture medium under two light spectra on the in vitro growth of Cattleya loddigesii seedlings. Plantlets with approximately 90 days were subcultured under eight treatments, consisting of different active charcoal concentrations (0; 0.5; 1.0 and 2.0g L-1) in ½ MS medium combined with white and red light spectra. After 180 days of germination, biometric data, fresh weight, and the level of photosynthetic pigments were evaluated. Plantlets acclimatized in a greenhouse were evaluated for survival after 120 days. Total chlorophyll, chlorophyll a, and carotenoid concentrations were higher in treatments under white light, while red light promoted greater chlorophyll b, plantlets with fewer and shorter roots, and high ex vitro mortality. The addition of 2.0g L-1 of active charcoal to the culture medium and the use of white light provided greater development efficiency both on in vitro culture and ex vitro plant survival.

O objetivo deste estudo foi avaliar a adição de concentrações de carvão ativado em meio de cultura ½ MS (com metade da concentração dos macronutrientes) sob dois espectros luminosos para a obtenção de plântulas in vitro de Cattleya loddigesii. Plântulas com aproximadamente 90 dias foram subcultivadas em oito tratamentos, nos quais foi testada a adição ao meio de cultura ½ MS com carvão ativado (0; 0,5; 1,0 e 2,0g L-1) e combinados sob espectro de luz branca e luz vermelha. Após 180 dias da germinação, foram mensurados dados biométricos (raiz e parte aérea), massa fresca e teores de pigmentos fotossintéticos. Em plântulas aclimatizadas em casa de vegetação, foram avaliadas a taxa de sobrevivência após 120 dias. As concentrações de clorofila total, clorofila a e carotenoides foram maiores nos tratamentos sob luz branca, enquanto a luz vermelha influenciou significativamente maior clorofila b, plântulas com menos raízes e de menor comprimento e elevada mortalidade ex vitro. A adição de 2,0g L-1 de carvão ativado ao meio de cultura e o uso de luz branca proporcionaram maior eficiência de desenvolvimento tanto para as culturas in vitro quanto para a sobrevivência ex vitro das plantas.

Crescimento in vitro e aclimatização de Cattleya loddigesii Lindley (Orchidaceae) com carvão ativado sob dois espectros luminosos

The aim of this study was to evaluate the effect of different concentrations of activated charcoal in ½ MS (half concentration of macronutrients) culture medium under two light spectra on the in vitro growth of Cattleya loddigesii seedlings. Plantlets with approximately 90 days were subcultured under eight treatments, consisting of different active charcoal concentrations (0; 0.5; 1.0 and 2.0g L-1) in ½ MS medium combined with white and red light spectra. After 180 days of germination, biometric data, fresh weight, and the level of photosynthetic pigments were evaluated. Plantlets acclimatized in a greenhouse were evaluated for survival after 120 days. Total chlorophyll, chlorophyll a, and carotenoid concentrations were higher in treatments under white light, while red light promoted greater chlorophyll b, plantlets with fewer and shorter roots, and high ex vitro mortality. The addition of 2.0g L-1 of active charcoal to the culture medium and the use of white light provided greater development efficiency both on in vitro culture and ex vitro plant survival.

O objetivo deste estudo foi avaliar a adição de concentrações de carvão ativado em meio de cultura ½ MS (com metade da concentração dos macronutrientes) sob dois espectros luminosos para a obtenção de plântulas in vitro de Cattleya loddigesii. Plântulas com aproximadamente 90 dias foram subcultivadas em oito tratamentos, nos quais foi testada a adição ao meio de cultura ½ MS com carvão ativado (0; 0,5; 1,0 e 2,0g L-1) e combinados sob espectro de luz branca e luz vermelha. Após 180 dias da germinação, foram mensurados dados biométricos (raiz e parte aérea), massa fresca e teores de pigmentos fotossintéticos. Em plântulas aclimatizadas em casa de vegetação, foram avaliadas a taxa de sobrevivência após 120 dias. As concentrações de clorofila total, clorofila a e carotenoides foram maiores nos tratamentos sob luz branca, enquanto a luz vermelha influenciou significativamente maior clorofila b, plântulas com menos raízes e de menor comprimento e elevada mortalidade ex vitro. A adição de 2,0g L-1 de carvão ativado ao meio de cultura e o uso de luz branca proporcionaram maior eficiência de desenvolvimento tanto para as culturas in vitro quanto para a sobrevivência ex vitro das plantas.

Crescimento in vitro e aclimatização de Cattleya loddigesii Lindley (Orchidaceae) com carvão ativado sob dois espectros luminosos

The aim of this study was to evaluate the effect of different concentrations of activated charcoal in ½ MS (half concentration of macronutrients) culture medium under two light spectra on the in vitro growth of Cattleya loddigesii seedlings. Plantlets with approximately 90 days were subcultured under eight treatments, consisting of different active charcoal concentrations (0; 0.5; 1.0 and 2.0g L-1) in ½ MS medium combined with white and red light spectra. After 180 days of germination, biometric data, fresh weight, and the level of photosynthetic pigments were evaluated. Plantlets acclimatized in a greenhouse were evaluated for survival after 120 days. Total chlorophyll, chlorophyll a, and carotenoid concentrations were higher in treatments under white light, while red light promoted greater chlorophyll b, plantlets with fewer and shorter roots, and high ex vitro mortality. The addition of 2.0g L-1 of active charcoal to the culture medium and the use of white light provided greater development efficiency both on in vitro culture and ex vitro plant survival.

O objetivo deste estudo foi avaliar a adição de concentrações de carvão ativado em meio de cultura ½ MS (com metade da concentração dos macronutrientes) sob dois espectros luminosos para a obtenção de plântulas in vitro de Cattleya loddigesii. Plântulas com aproximadamente 90 dias foram subcultivadas em oito tratamentos, nos quais foi testada a adição ao meio de cultura ½ MS com carvão ativado (0; 0,5; 1,0 e 2,0g L-1) e combinados sob espectro de luz branca e luz vermelha. Após 180 dias da germinação, foram mensurados dados biométricos (raiz e parte aérea), massa fresca e teores de pigmentos fotossintéticos. Em plântulas aclimatizadas em casa de vegetação, foram avaliadas a taxa de sobrevivência após 120 dias. As concentrações de clorofila total, clorofila a e carotenoides foram maiores nos tratamentos sob luz branca, enquanto a luz vermelha influenciou significativamente maior clorofila b, plântulas com menos raízes e de menor comprimento e elevada mortalidade ex vitro. A adição de 2,0g L-1 de carvão ativado ao meio de cultura e o uso de luz branca proporcionaram maior eficiência de desenvolvimento tanto para as culturas in vitro quanto para a sobrevivência ex vitro das plantas.

Molecular characterization of bacterial populations of different soils

Until recently, few studies were carried out in Brazil about diversity of bacterial soil communities. Aiming to characterize the bacterial population in the soil through 16S rRNA analysis, two types of soil have been analyzed: one of them characterized by intensive use where tomato, beans and corn were cultivated (CS); the other analyzed soil was under forest (FS), unchanged by man; both located in Guaíra, São Paulo State, Brazil. Using specific primers, 16S rRNA genes from metagenomic DNA in both soils were amplified by PCR, amplicons were cloned and 139 clones from two libraries were partially sequenced. The use of 16S rRNA analysis allowed identification of several bacterial populations in the soil belonging to the following phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria Verrucomicrobia in addition to the others that were not classified, beyond Archaea domain. Differences between FS and CS libraries were observed in size phyla. A larger number of phyla and, consequently, a greater bacterial diversity were found in the under-forest soil. These data were confirmed by the analyses of genetic diversity that have been carried out. The characterization of bacterial communities of soil has made its contribution by providing facts for further studies on the dynamics of bacterial populations in different soil conditions in Brazil.

Até o momento poucos estudos foram realizados no Brasil a respeito da diversidade de comunidades bacterianas no solo. Com o objetivo de caracterizar as populações bacterianas presentes no solo através da análise do gene 16S rRNA, foram analisados dois solos: um caracterizado pelo uso intensivo, principalmente para a produção de tomate, feijão e milho (CS); e outro sob floresta (FS), não modificado pelo homem, ambos do município de Guaíra, no estado de São Paulo, Brasil. Usando oligonucleotídeos específicos, de genes 16S rRNA do DNA metagenomico de ambos os solos foram amplificados por PCR, amplicons foram clonados e 139 clones de duas bibliotecas foram seqüenciados. O uso da técnica de 16S rRNA, gerou a identificação de diferentes populações de bactérias de solo pertencentes aos filos Acidobacteria Actinobacteria Bacteroidetes Firmicutes Proteobacteria Verrucomicrobia, Archaea, além das não classificadas. Diferenças entre as bibliotecas FS e CS foram observadas no tamanho dos filos. Um grande número de filos e, consequentemente, uma grande diversidade bacteriana foi observada no solo sob floresta. Estes dados foram confirmados pela análise de diversidade genética realizada. A caracterização de comunidades do solo apresentada neste trabalho contribuiu fornecendo dados para estudos posteriores sobre a dinâmica das populações bacterianas em solos de diferentes condições no Brasil.