Microbial enrichment and meta-omics analysis identify CAZymes from mangrove sediments with unique properties.

Although lignocellulose is the most abundant and renewable natural resource for biofuel production, its use remains under exploration because of its highly recalcitrant structure. Its deconstruction into sugar monomers is mainly driven by carbohydrate-active enzymes (CAZymes). To develop highly efficient and fast strategies to discover biomass-degrading enzymes for biorefinery applications, an enrichment process combined with integrative omics approaches was used to identify new CAZymes. The lignocellulolytic-enriched mangrove microbial community (LignoManG) established on sugarcane bagasse (SB) was enriched with lignocellulolytic bacteria and fungi such as Proteobacteria, Bacteroidetes, Basidiomycota, and Ascomycota. These microbial communities were able to degrade up to 55 % of the total SB, indicating the production of lignocellulolytic enzymes. Metagenomic analysis revealed that the LignoManG harbors 18.042 CAZyme sequences such as of cellulases, hemicellulases, carbohydrate esterases, and lytic polysaccharide monooxygenase. Similarly, our metaproteomic analysis depicted several enzymes from distinct families of different CAZy families. Based on the LignoManG data, a xylanase (coldXynZ) was selected, amplified, cloned, expressed, and biochemically characterized. The enzyme displayed psicrofilic properties, with the highest activity at 15 °C, retaining 77 % of its activity when incubated at 0 °C. Moreover, molecular modeling in silico indicated that coldXynZ is composed of a TIM barrel, which is a typical folding found in the GH10 family, and displayed similar structural features related to cold-adapted enzymes. Collectively, the data generated in this study represent a valuable resource for lignocellulolytic enzymes with potential biotechnological applications.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Penicillium echinulatum secretome analysis reveals the fungi potential for degradation of lignocellulosic biomass.

BACKGROUND: The enzymatic degradation of lignocellulosic materials by fungal enzyme systems has been extensively studied due to its effectiveness in the liberation of fermentable sugars for bioethanol production. Recently, variants of the fungus Penicillium echinulatum have been described as a great producer of cellulases and considered a promising strain for the bioethanol industry. RESULTS: Penicillium echinulatum, wild-type 2HH and its mutant strain S1M29, were grown on four different carbon sources: cellulose, sugar cane bagasse pretreated by steam explosion (SCB), glucose, and glycerol for 120 h. Samples collected at 24, 96, and 120 h were used for enzymatic measurement, and the 96-h one was also used for secretome analysis by 1D-PAGE LC-MS/MS. A total of 165 proteins were identified, and more than one-third of these proteins belong to CAZy families. Glycosyl hydrolases (GH) are the most abundant group, being represented in larger quantities by GH3, 5, 17, 43, and 72. Cellobiohydrolases, endoglucanases, ß-glycosidases, xylanases, ß-xylosidases, and mannanases were found, and in minor quantities, pectinases, ligninases, and amylases were also found. Swollenin and esterases were also identified. CONCLUSIONS: Our study revealed differences in the two strains of P. echinulatum in several aspects in which the mutation improved the production of enzymes related to lignocellulosic biomass deconstruction. Considering the spectral counting analysis, the mutant strain S1M29 was more efficient in the production of enzymes involved in cellulose and hemicellulose degradation, despite having a nearly identical CAZy enzymatic repertoire. Moreover, S1M29 secretes more quantities of protein on SCB than on cellulose, relevant information when considering the production of cellulases using raw materials at low cost. Glucose, and especially glycerol, were used mainly for the production of amylases and ligninases.

Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant ß-glucosidase from the termite Nasutitermes takasagoensis.

Termites are well-known cellulose decomposers and can give researchers insights into how to utilize lignocellulosic biomass in the actual scenario of energy consumption. In this work, an endogenous ß-glucosidase from the midgut of the higher termite Nasutitermes takasagoensis was purified to homogeneity by Ni(2+) affinity chromatography and its properties were characterized. This ß-glucosidase (G1mgNtBG1), which belongs to glycoside hydrolase family 1, is a homotrimer in its native form, with a molecular mass of 169.5 kDa, as demonstrated by gel filtration chromatography. The enzyme displayed maximum activity at pH 5.5 and had broad substrate specificities toward several saccharides, including cellobiose. G1mgNtBG1 showed a relatively high temperature optimum of 65°C and one of the highest levels of glucose tolerance among several ß-glucosidases already characterized, with a K(i) of 600 mM glucose. To examine the applicability of G1mgNtBG1 in biomass conversion, we compared the thermostability and glucose tolerance of G1mgNtBG1 with those of Novozym 188. We found that G1mgNtBG1 was more thermostable after 5 h of incubation at 60°C and more resistant to glucose inhibition than Novozym 188. Furthermore, our result suggests that G1mgNtBG1 acts synergistically with Celluclast 1.5 L in releasing reducing sugars from Avicel. Thus, G1mgNtBG1 seems to be a potential candidate for use as a supplement in the hydrolysis of biomass.

Expression and one-step purification of recombinant proteins using an alternative episomal vector for the expression of N-tagged heterologous proteins in Pichia pastoris.

Here we report the construction of an alternative episomal vector, pBGP3, which allows the expression of heterologous proteins with N-terminal hexahistidine and myc-epitope tags in Pichia pastoris. To test the usefulness of pBGP3, four cellulases from termites were expressed. Production was confirmed by activity assays and Western blot using anti-c-Myc antibody. Purification was performed by single-step Ni(2+)-affinity chromatography, which confirmed the efficiency of pBGP3.

Heterologous expression and characterization of a glucose-stimulated ß-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae.

Neotermes koshunensis is a lower termite that secretes endogenous ß-glucosidase in the salivary glands. This ß-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-ß-D-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn(2+) and glycerol. The K(m) and V(max) values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-ß-D-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.