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      Temporal Expression Dynamics of Plant Biomass-Degrading Enzymes by a Synthetic Bacterial Consortium Growing on Sugarcane Bagasse

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          Abstract

          Plant biomass (PB) is an important source of sugars useful for biofuel production, whose degradation efficiency depends on synergistic and dynamic interactions of different enzymes. Here, using a metatranscriptomics-based approach, we explored the expression of PB-degrading enzymes in a five-species synthetic bacterial consortium during cultivation on sugarcane bagasse as a unique carbon source. By analyzing the temporal expression dynamics of a selection of enzymes we revealed the functional role of each consortium member and disentangled the potential interactions between them. Based on normalized expression values and the taxonomic affiliation of all the transcripts within thirty carbohydrate-active enzyme (CAZy) families, we observed a successional profile. For instance, endo-glucanases/-xylanases (e.g., GH8, GH10, and GH16) were significantly expressed at 12 h, whereas exo-glucanases (e.g., GH6 and GH48) and α-arabinosidases/β-xylosidases (e.g., GH43) were highly expressed at 48 h. Indeed, a significant peak of extracellular β-xylosidase activity was observed at this stage. Moreover, we observed a higher expression of several CAZy families at 12–48 h, suggesting easy access to the main plant polysaccharides. Based on this evidence, we predicted that the highest level of collaboration between strains takes place at the initial stages of growth. Here, Paenibacillus, Brevundimonas, and Chryseobacterium were the most important contributors, whereas Stenotrophomonas was highly active at the end of the culture (96–192 h) without contributing to a large extent to the expression of lignocellulolytic enzymes. Our results contribute to the understanding of enzymatic and ecological mechanisms within PB-degrading microbial consortia, yielding new perspectives to improve the PB saccharification processes.

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          Novel enzymes for the degradation of cellulose

          The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first “extracting” these chains from their crystalline matrix.
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            Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation.

            The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.
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              Plant-polysaccharide-degrading enzymes from Basidiomycetes.

              Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                26 February 2018
                2018
                : 9
                : 299
                Affiliations
                [1] 1Microbial Ecology Cluster, Groningen Institute for Evolutionary Life Sciences, University of Groningen , Groningen, Netherlands
                [2] 2Department of Biological Sciences, Universidad de los Andes , Bogotá, Colombia
                Author notes

                Edited by: Suhelen Egan, University of New South Wales, Australia

                Reviewed by: Johan Larsbrink, Chalmers University of Technology, Sweden; François Thomas, UMR8227 Laboratoire de Biologie Intégrative des Modèles Marins, France; Guillermina Hernandez-Raquet, Institut National de la Recherche Agronomique (INRA), France; Fabio M. Squina, Universidade de Sorocaba, Brazil

                *Correspondence: Diego Javier Jiménez, djimenez1909@ 123456gmail.com

                This article was submitted to Microbial Symbioses, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2018.00299
                5834485
                29535687
                8083c04a-3ba8-40b4-954d-9ef916119457
                Copyright © 2018 Jiménez, Chaib De Mares and Salles.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 21 September 2017
                : 08 February 2018
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 61, Pages: 13, Words: 0
                Funding
                Funded by: Fundação de Amparo à Pesquisa do Estado de São Paulo 10.13039/501100001807
                Award ID: 729004006
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                bacterial consortium,expression dynamics,lignocellulolytic enzymes,metatranscriptomics,sugarcane bagasse,synergism

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