Deconstruction of Lignin: From Enzymes to Microorganisms.

Lignocellulosic residues are low-cost abundant feedstocks that can be used for industrial applications. However, their recalcitrance currently makes lignocellulose use limited. In natural environments, microbial communities can completely deconstruct lignocellulose by synergistic action of a set of enzymes and proteins. Microbial degradation of lignin by fungi, important lignin degraders in nature, has been intensively studied. More recently, bacteria have also been described as able to break down lignin, and to have a central role in recycling this plant polymer. Nevertheless, bacterial deconstruction of lignin has not been fully elucidated yet. Direct analysis of environmental samples using metagenomics, metatranscriptomics, and metaproteomics approaches is a powerful strategy to describe/discover enzymes, metabolic pathways, and microorganisms involved in lignin breakdown. Indeed, the use of these complementary techniques leads to a better understanding of the composition, function, and dynamics of microbial communities involved in lignin deconstruction. We focus on omics approaches and their contribution to the discovery of new enzymes and reactions that impact the development of lignin-based bioprocesses.

Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Seasonal effects in a lake sediment archaeal community of the Brazilian Savanna.

The Cerrado is a biome that corresponds to 24% of Brazil's territory. Only recently microbial communities of this biome have been investigated. Here we describe for the first time the diversity of archaeal communities from freshwater lake sediments of the Cerrado in the dry season and in the transition period between the dry and rainy seasons, when the first rains occur. Gene libraries were constructed, using Archaea-specific primers for the 16S rRNA and amoA genes. Analysis revealed marked differences between the archaeal communities found in the two seasons. I.1a and I.1c Thaumarchaeota were found in greater numbers in the transition period, while MCG Archaea was dominant on the dry season. Methanogens were only found in the dry season. Analysis of 16S rRNA sequences revealed lower diversity on the transition period. We detected archaeal amoA sequences in both seasons, but there were more OTUs during the dry season. These sequences were within the same cluster as Nitrosotalea devanaterra's amoA gene. The principal coordinate analysis (PCoA) test revealed significant differences between samples from different seasons. These results provide information on archaeal diversity in freshwater lake sediments of the Cerrado and indicates that rain is likely a factor that impacts these communities.

Characterization of soil bacterial assemblies in Brazilian savanna-like vegetation reveals acidobacteria dominance.

The Brazilian Cerrado is the second largest biome in Brazil and is considered a biodiversity hotspot. In this work, we compared the bacterial communities in Cerrado soil associated with four types of native vegetation (Cerrado Denso, Cerrado sensu stricto, Campo Sujo, and Mata de Galeria) by ribosomal RNA intergenic spacer analysis, terminal fragment restriction length polymorphism and pyrosequencing. The fingerprinting results were very similar. The bacterial communities of Cerrado Denso and Cerrado sensu stricto grouped together and were distinct from those in Campo Sujo and Mata de Galeria. Pyrosequencing generated approximately 40,000 16S rRNA gene sequences per sample and allowed the identification of 17 phyla in soil samples under Cerrado vegetation. Acidobacteria were dominant in all areas studied with a relative frequency of 40-47 %, followed closely by Proteobacteria accounting for 34-40 % of the sequences. Results from all molecular techniques used suggested that the bacterial communities of Cerrado sensu stricto and Cerrado Denso are very similar to each other, while Campo Sujo forms a separate group, and Mata de Galeria is the most distinct with higher species richness. This is the first extensive study of native Cerrado soil microbiota, an important but endangered biome.

Bacteria and Archaea community structure in the rumen microbiome of goats (Capra hircus) from the semiarid region of Brazil.

Most studies present in the literature about the rumen microbiome have focused on cattle and sheep. This is the first report of the characterization of the bacterial and archaeal communities present in the liquid and solid-associated fractions of the rumen from free ranging Moxotó breed goats using 16S rRNA gene libraries. PCR was used to amplify the 16S rRNA gene with bacterial and archaeal universal primers and sequences from each library constructed were obtained. Sequences of Bacteria from the phyla Bacteroidetes and Firmicutes were predominant. The overall dominant classes in the rumen were Clostridia and Bacteroidia, which are known to play a role in plant fiber degradation in other ruminants. Unclassified Bacteria accounted for 4.7% of the liquid fraction sequences and 16.4% of the solid fraction sequences. From the archaeal libraries only sequences from the phylum Euryarcheota were identified and were assigned to the class Methanobacteria of the genera Methanobrevibacter and Methanosphaera. A group of Archaea not previously known to be associated with the rumen was identified: uncultured methanogens belonging to the "uncultured marine bacteria" groups II and III. The local water contained high salt concentrations and this may explain the presence of these groups in the Moxotó goat rumen.

Molecular phylogenetic diversity of bacteria associated with soil of the savanna-like Cerrado vegetation.

The Brazilian savanna-like vegetation of Cerrado is rapidly being converted to pasture and agricultural fields. A 16S rDNA-based approach was taken to study the bacterial community associated with the soil of a native cerrado area (sensu stricto) and an area that has been converted to pasture. The bacterial group most abundantly identified in cerrado sensu stricto soil was the alpha-Proteobacteria while in cerrado converted to pasture the Actinobacteria were the most abundant. Rarefaction curves indicate that the species richness of cerrado sensu stricto is greater than that of cerrado converted to pasture. Furthermore, lineage-through-time plots show that the expected richness of species present in cerrado sensu stricto soil is approximately 10 times greater than that of cerrado converted to pasture.

Plant cyclotides: an unusual class of defense compounds.

Plant cyclotides are unusual peptides with low molecular masses and a three-dimensional structure characterized by the presence of a cyclic fold. Synthetic peptides can adopt this circular conformation, but it is not a common feature for most members of other peptide groups. Cyclotides present a wide range of functions, such as the ability to induce stronger contractions during childbirth and anti-tumor activity. Additionally, some cyclotides present anti-viral, insecticidal or proteinase inhibitory activity. In this paper, we describe the structural and functional characteristics of plant cyclotides, their most conserved features and the development of these peptides for human health and biotechnological applications.

Molecular identification of four different alpha-amylase inhibitors from baru (Dipteryx alata) seeds with activity toward insect enzymes.

The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of alpha-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, alpha-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis alpha-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus alpha-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed alpha-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several alpha-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.

Identification of an alpha-amylase inhibitor from Pterodon pubescens with ability to inhibit cowpea weevil digestive enzymes.

Cowpea seeds (Vigna ungiculata) are widely cultivated by poor farmers in Latin America and Africa and are often severely damaged by the cowpea weevil Callosobruchus maculatus. A proteinaceous inhibitor of cowpea weevil digestive enzymes, PpAI, was purified from white sucupira seeds (Pterodon pubescens) and biochemically characterized in this study. Proteins were extracted from seeds and precipitated with ammonium sulfate at 100% saturation. This fraction was applied onto a Red-sepharose CL-6B column, and the retained peak showed 70% inhibitory activity toward larval C. maculatus digestive alpha-amylases. The retained peak was then purified using an analytical reversed-phase HPLC column. Purified PpAI showed 65% inhibitory activity against larval C. maculatus enzymes. Enzymatic assays also showed that the purified P. pubescens inhibitor was unable to reduce the activity of mammalian alpha-amylases, suggesting specificity toward insect enzymes. Moreover, artificial seeds containing PpAI were able to reduce larval weight by 36% and cause 55% mortality. Mass spectrometry and SDS-PAGE analyses indicated that PpAI showed a molecular mass of approximately 5.0 kDa. This alpha-amylase inhibitor, coming from a native Cerrado plant, could be used to construct a genetically engineered cowpea with enhanced resistance against weevil pests.

Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation.

The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.