Felipe S. Chambergo

Core Microbial Functional Activities in Ocean Environments Revealed by Global Metagenomic Profiling Analyses

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the worlds oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

Background: Molecular features underlying enzyme function in extreme environments are poorly understood. Results: Identification of the basis for thermostability, halophilicity, and detoxification activity in a mercuric reductase from hot deep-sea brine. Conclusion: A small number of structural modifications accounts for the enzymes robustness. Significance: This work defines novel adaptations that enable enzymes to cope with multiple abiotic stressors simultaneously. A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+ in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Fungal biodiversity to biotechnology

Fungal habitats include soil, water, and extreme environments. With around 100,000 fungus species already described, it is estimated that 5.1 million fungus species exist on our planet, making fungi one of the largest and most diverse kingdoms of eukaryotes. Fungi show remarkable metabolic features due to a sophisticated genomic network and are important for the production of biotechnological compounds that greatly impact our society in many ways. In this review, we present the current state of knowledge on fungal biodiversity, with special emphasis on filamentous fungi and the most recent discoveries in the field of identification and production of biotechnological compounds. More than 250 fungus species have been studied to produce these biotechnological compounds. This review focuses on three of the branches generally accepted in biotechnological applications, which have been identified by a color code: red, green, and white for pharmaceutical, agricultural, and industrial biotechnology, respectively. We also discuss future prospects for the use of filamentous fungi in biotechnology application.

Solution structure of the C-terminal domain of multiprotein bridging factor 1 (MBF1) of Trichoderma reesei.

Solution structure of the C-terminal domain of multiprotein bridging factor 1 (MBF1) of Trichoderma reesei Roberto K. Salinas,* Cesar M. Camilo, Simona Tomaselli, Estela Y. Valencia, Chuck S. Farah, Hamza El-Dorry, and Felipe S. Chambergo* 1 Institute of Chemistry, University of São Paulo, São Paulo SP 05508-900, Brazil 2 Laboratorio NMR, ISMAC, CNR, Milano 20133, Italy 3 Department of Biology and the Science and Research Technology Center, The American University in Cairo, Cairo, Egypt 4 School of Arts, Sciences and Humanities, University of São Paulo, São Paulo SP 03828-000, Brazil

Mini-review: Brazilian fungi diversity for biomass degradation.

Brazil houses over 10% of the total number of known species on Earth, with a great diversity of plants and fungi. The collection, isolation, identification and conservation of filamentous fungi with relevance to agriculture, pharmaceutical, food and biotechnological industries in Biological Resource Centers (CRBs) is very important to the development of a nations scientific and technological infrastructure. In Brazil, 36 fungal collections are registered in the database of International Collections. Several federal and state programs have encouraged the formation of a researchers network in order to study natural resources and the nations biodiversity. In this context, Brazilian researchers have been on the frontiers of knowledge, investigating the enzymatic systems from native filamentous fungi with potential for biomass degradation and biotechnological application. In this review, we address recent progress in Brazilian fungal research, focusing on the identification and study of fungi and enzymes with potential for biomass degradation and application in bioenergy.

A new species of Backusella (Mucorales) from a Cerrado reserve in Southeast Brazil

Cerrado (Brazilian savanna) areas present a high number of endemic and unknown species and consequently have been included among the world’s biodiversity hotspots. After a survey of zygomycetes from Cerrado areas of the “Reserva Biológica de Mogi Guaçu” (RBMG) in São Paulo State, we have found a Mucor-like fungus that produces very large sporangiospores and clusters of giant cells visible to the naked-eye. This isolate was characterized morphologically, based on partial LSU (28S) and complete ITS rDNA sequences. Maximum likelihood phylogenetic analyses support its inclusion within the recently emended genus Backusella.

Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool.

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90°C for 1h at pH from 8 to 11.5, while maintaining its biological activity.

Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity

The data presented in this article are related to the research article entitled “Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization” [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay.

Purification and characterization of a lectin with refolding ability from Genipa americana bark

Genipa americana L., commonly known as genipap, is a plant with economical and medicinal importance, and a promising source of bioactive compounds. Lectins are carbohydrate-binding proteins with several biotechnological applications. This study reports the isolation and characterization of a G. americana bark lectin (GaBL). A single chromatographic procedure on Sephacryl S-100 resulted in isolation of GaBL, a protein with native molecular weight of over 200 kDa and pI 4.02, whose hemagglutinating activity was inhibited by lactose and fetuin, not affected by ions (Ca2+ and Mg2+), and stable upon heating (303-393 K) as well as over the pH range 5-10. The highest activity was found at a temperature lower than 333 K and pH 5. The secondary structure was analyzed by circular dichroism and showed a prevalence of beta structures and unordered forms. GaBL was able to partially refold in acidic pH conditions when dissolved in PBS buffer at pH 7.4. In conclusion, GaBL was purified in milligram quantities with high stability against different conditions, and is a new biomaterial with potential biotechnological applications.