Laccases from actinobacteria: characterization, designing of new primers and heterologous expression in the yeast Kluyveromyces marxianus UFV-3

Dentre as enzimas multicobre oxidases, as lacases, por sua grande variedade de substratos, tem atraído atenção tanto para pesquisa básica quanto aplicada a atividades industriais e ambientais. Com relação às lacases de origem microbiana, pouco se conhece sobre aquelas produzidas por bactérias. Neste trabalho, destaca-se o filo Actinobacteria, ordem Actinomycetales, devido à sua reconhecida importância biotecnológica e sua habilidade para degradação de compostos recalcitrantes, sendo muitos deles substratos de lacases. Tendo em vista a demanda por grande volume de enzima por parte das diversas indústrias, e a dificuldade de manipulação dos produtores nativos, é sugerida aqui a expressão heteróloga de lacases de actinobactérias na levedura Kluyveromyces marxianus, em virtude das propriedades industriais que a mesma apresenta, tais como alta velocidade do crescimento, termotolerância e status Generally Regarded as Safe (GRAS). O objetivo deste trabalho foi obter uma linhagem de Kluyveromyces marxianus portando gene de lacase de actinobactéria, capaz de expressá-lo e secretar a enzima ativa; buscando- se, para isso, isolar actinobactérias produtoras de lacase a partir de solo, caracterizar a enzima, isolar um gene de lacase, clonar em vetor de expressão e, por fim, transformar K. marxianus UFV-3. A partir de dados da literatura, realizou-se uma revisão crítica relacionada ao tema, e as sequências gênicas que codificam proteínas caracterizadas foram identificadas e utilizadas na síntese de oligonucleotídeos iniciadores que se revelaram adequados para detecção e isolamento do gene neste grupo, havendo distinção de genes correspondentes a proteínas com duas estruturas distintas. Havendo o isolamento de um dos genes, o mesmo foi clonado em cassete de expressão para integração no genoma da K. marxinus UFV-3; os dados sugerem que houve sucesso na integração.The great diversity in substrate range oxidized by laccase enzymes has attracted attention in both areas of research: basic, and applied to industrial and environmental activities. Among microbial laccases, little is known about the ones produced by bacteria. In this work, it is highlighted the phylum Actinobacteria, order Actinomycetales, due to its recognized biotechnological importance and capacity of degrading recalcitrant compounds. Owing to the large amounts of enzyme required for industrial application, summed to the difficulty in manipulation of native producers, heterologous expression is here suggested using the yeast Kluyveromyces marxianus as host, which presents the industrial-relevant properties such as fast growth, thermotolerance and status generally regarded as safe (GRAS). The objective of this work was to obtain a strain of Kluyveromyces marxianus expressing and secreting a laccase from actinobacteria; and, in order to accomplish the main objective, we searched for isolating laccase-producing actinobacteria from soil, characterizing the enzyme, isolating a laccase gene, cloning into expression vector and, finally, transforming K. marxianus UFV-3. In this work, an isolation from soil samples provided laccase-producer colonies, having one of the selected been characterized. From literature data, a critical review was produced, and available gene sequences that codify characterized proteins were employed for designing oligonucleotide primers. The new primers proved to be effective for detection of laccase genes in actinobacteria, and, furthermore, allowed distinction of genes that corresponded to two structurally different laccases. After selection of a complete laccase gene, it was cloned into an expression cassette for integration into K. marxianus UFV-3 genome, and data suggest successful integration

Internalization of Kluyveromyces lactis lactose permease in response to carbon sources

A permease de lactose de Kluyveromyces lactis, Lac12, media o transporte de lactose e o de galactose de baixa afinidade. Aqui é apresentado o estudo do efeito de fontes de carbono na internalização de Lac12 através do uso de linhagens contendo o gene quimérico LAC12-GFP. Quando células de K. lactis pré-cultivadas em galactose ou lactose foram transferidas para um novo meio, Lac12-GFP foi removida da membrana plasmática e localizada intracelularmente. Surpreendentemente, mesmo a presença de galactose ou lactose no novo meio de transferência causou essa internalização, e a resposta celular foi diferente para esse dois açúcares. Os resultados obtidos revelam que o processo de internalização é dependente do tipo de açúcar presente e de sua concentração. A internalização de Lac12-GFP causou redução nas taxas de captação de lactose[C14] e também foi observada em uma linhagem mutante Klsnf1; portanto, esse evento independe da atividade de KlSnf1. Evidências indicam que glicose-6-fosfato é o sinal intracelular, uma vez que a internalização foi induzida por 2-deoxiglicose, e a inibição da atividade da enzima fosfoglimutase por lítio impediu a internalização por galactose, mas não por lactose ou glicose. A internalização não ocorreu em 6-deoxiglicose, e, em ausência de síntese protéica, o evento foi irreversível.Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we have investigated the effects of carbon sources on internalization of Lac12 by using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and localized intracellularly. Surprisingly, even galactose or lactose in the new media caused the internalization, and cells responded differently to theses two sugars. Our results reveal that this process is dependent of sugar species and also sugar concentration. Lac12-GFP internalization causes reduction on [C14]lactose uptake rates and also occurs in a Klsnf1 mutant strain; thereby, it is independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, since internalization was induced by 2-deoxyglucose and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in absence of protein synthesis.Fundação de Amparo à Pesquisa do Estado de Minas Gerai

The role of mycorrhization helper bacteria in the establishment and action of ectomycorrhizae associations

More than 95 % short roots of most terrestrial plants are colonized by mycorrhizal fungi as soon as they emerge in the upper soil profiles. The establishment of mycorrhizal association involves profound morphological and physiological changes in root and fungus. It is affected by other rhizospheric microorganisms, specifically by the bacteria. Bacteria may have developed mechanisms of selective interaction with surrounding microorganisms, with neutral or positive effects on mycorrhizal associations, but negative effect on root pathogens in general. Because of the beneficial effect of bacteria on mycorrhizae, the concept of Mycorrhization Helper Bacteria (MHB) was created. Five main actions of MHB on mycorrhizae were proposed: in the receptivity of root to the mycobiont, in root-fungus recognition, in fungal growth, in the modification of rhizospheric soil and in the germination of fungal propagules. MHB appear to develop a gradation of specificity for the mycobiont, but little or no specificity for the host plant in symbiosis. One of the main groups of MHB is the fluorescent Pseudomonas, well represented in diversity and cell density studies of mycorrhizal associations. This review covers the activity of MHB in the establishment of ectomycorrhizae, taking as model the effects of Pseudomonas sp. described in scientific literature

Characterization of a thermotolerant laccase produced by Streptomyces sp. SB086

Laccases have become desirable enzymes for application in many industrial processes. Nowadays, most of these enzymes are obtained from fungi. Among prospective studies for bacterial laccase genes, some have included actinomycetes, but only a few studies have characterized the enzyme produced. Thus, we have isolated a laccase-producing actinomycete from forest soil under restoration process and further aimed to characterize its produced enzyme. The isolate SB086 was assigned to the Streptomyces genus by a combination of phenotypical, chemical and phylogenetic properties. Our data indicate that the bacterium produces a thermotolerant laccase. The maximum activity was obtained in the pH range 4.0–5.0 and at 50 °C in reaction mixture containing 5 mM CuSO4; thermal stability was noted at 60 °C and 70 °C—a well-desired characteristic for industry. The active enzyme presented a high molecular mass (over 100 kDa) and was less sensitive to inhibition by metal ions than generally described for bacterial laccases. Our findings support in silico data of bacterial laccase secretion, and reinforce the view that actinomycetes may be a rich source of laccase for industrial application