The Carbohydrate-Active Enzymes (CAZymes) required for cellulose deconstruction in Cellvibrio japonicus are part of an efficient cellodextrin uptake system
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PROVIDER: PRJNA1211626 | ENA |
REPOSITORIES: ENA
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Project description:Microbial deconstruction of plant polysaccharides is important for environmental nutrient cycling, and bacteria proficient at this process have extensive suites of polysaccharide-specific enzymes. In the Gram-negative saprophyte Cellvibrio japonicus, genome annotation suggests that 17 genes are predicted to encode Carbohydrate-Active enZymes (CAZymes) with roles in cellulose degradation, however previous work suggested that only a subset of these genes is essential. Building upon that work, here we identify the required and minimally sufficient set of enzymes for complete degradation of cellulose using a combination of transcriptomics, gene deletion analysis, heterologous expression studies, and metabolite analysis. We identified six CAZyme-encoding required for cellulose deconstruction in C. japonicus, which are cel3B, cel5B, cel6A, lpmo10B, cbp2D, and cbp2E. These genes encode for a β-glucosidase, an endoglucanase, a cellbiohydrolase, a lytic polysaccharide mono-oxygenase, and two carbohydrate-binding proteins, respectively. These CAZyme-encoding genes are essential for growth using insoluble cellulose by C. japonicus, and sufficient using soluble cellulose when heterologously expressed in Escherichia coli. Moreover, during C. japonicus grow using insoluble cellulose we detected no cellodextrins in the medium, which suggests that cello-oligosaccharide uptake is highly efficient. RNAseq analysis corroborates these results, as we observed several genes significantly up-regulated during growth on cellulose that encode TonB-dependent and ABC transporters. Our revised model of cellulose utilization by C. japonicus suggests a greater importance for the Cbp2D and Cbp2E proteins than previously thought and that rapid cellodextrin update by C. japonicus is a mechanism to maximize the energetic return on investment for the production and secretion of CAZymes.
2025-01-21 | GSE287208 | GEO
Project description:Plant mannans are a component of lignocellulose that may possess diverse compositions changing both in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides, specifically mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has ten predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential components of the mannan degradation apparatus in this bacterium. Transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode Carbohydrate Active Enzymes (CAZymes) when mannan substrates were actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme genes in the context of mannan utilization. Growth analysis of the mutant strains indicated that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α- galactosidase, and mannosidase, respectively, were influential to the deconstruction of galactomannan. Furthermore, our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the efficient and complete degradation of this hemicellulose by bacteria.
2022-06-28 | GSE206864 | GEO
Project description:Degradation of polysaccharides forms an essential arc in the carbon cycle, provides a percentage of our daily caloric intake, and is a major driver in the renewable chemical industry. Microorganisms proficient at degrading insoluble polysaccharides possess large numbers of carbohydrate active enzymes, many of which have been categorized as functionally redundant. Here we present data that suggests that carbohydrate active enzymes that have overlapping enzymatic activities can have unique, non-overlapping biological functions in the cell. Our comprehensive study to understand cellodextrin utilization in the soil saprophyte Cellvibrio japonicus found that only one of four predicted b-glucosidases is required in a physiological context. Gene deletion analysis indicated that only the cel3B gene product is essential for efficient cellodextrin utilization in C. japonicus and is constitutively expressed at high levels. Interestingly, expression of individual b-glucosidases in Escherichia coli K-12 enabled this non-cellulolytic bacterium to be fully capable of using cellobiose as a sole carbon source. Furthermore, enzyme kinetic studies indicated that the Cel3A enzyme is significantly more active than the Cel3B enzyme on the oligosaccharides but not disaccharides. Our approach for parsing related carbohydrate active enzymes to determine actual physiological roles in the cell can be applied to other polysaccharide-degradation systems.
2017-01-01 | GSE90955 | GEO
Project description:The search for new enzymes and microbial strains to degrade plant biomass is one of the most important strategies for improving the conversion processes in the production of environment-friendly chemicals and biofuels. In this study, we report a new Paenibacillus isolate, O199, which showed the highest efficiency for cellulose deconstruction in a screen of environmental isolates. Here, we provide a detailed description of the complex multi-component O199 enzymatic system involved in the degradation of lignocellulose. We examined the genome and the proteome of O199 grown on complex lignocellulose (wheat straw) and on microcrystalline cellulose. The genome contained 476 genes with domains assigned to carbohydrate-active enzyme (CAZyme) families, including 100 genes coding for glycosyl hydrolases (GHs) putatively involved in cellulose and hemicellulose degradation. Moreover, 31% of these CAZymes were expressed on cellulose and 29% on wheat straw. Proteomic analyses also revealed a complex and complete set of enzymes for deconstruction of cellulose (at least 22 proteins, including 4 endocellulases, 2 exocellulases, 2 cellobiohydrolases and 2 -glucosidases) and hemicellulose (at least 28 proteins, including 5 endoxylanases, 1 -xylosidase, 2 xyloglucanases, 2 endomannanases, 2 licheninases and 1 endo--1,3(4)-glucanase). Most of these proteins were secreted extracellularly and had numerous carbohydrate-binding domains (CBMs). In addition, O199 also secreted a high number of substrate-binding proteins (SBPs), including at least 42 proteins binding carbohydrates. Interestingly, both plant lignocellulose and crystalline cellulose triggered the production of a wide array of hydrolytic proteins, including cellulases, hemicellulases and other GHs. Our data provide an in-depth analysis of the complex and complete set of enzymes and accessory non-catalytic proteins—GHs, CBMs, transporters, and SBPs—implicated in the high cellulolytic capacity shown by this bacterial strain. The large diversity of hydrolytic enzymes and the extracellular secretion of most of them supports the use of Paenibacillus O199 as a candidate for second-generation technologies using paper or lignocellulosic agricultural wastes.
2016-04-26 | PXD003970 | Pride
Project description:Investigation of whole genome gene expression level changes in Cellvibrio japonicus wild-type, comparing glucose vs cellulose. Study was purposed with determining changes in polysaccharide degradation pathways during utilization of insoluble cellulose.
2014-10-07 | GSE62106 | GEO
Project description:Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 286 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks the seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3'-tag digital gene expression (DGE) reveals that 246 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryon strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryon strains. Using the 3'-tag digital gene expression (DGE), we compared the gene expression profiles among 2 homokaryotic V. volvacea strains PYd15 and PYd21, and one heterokaryotic strain H1521, which is a hybrid strain of PYd15 and PYd21.
2014-12-19 | E-GEOD-43019 | biostudies-arrayexpress
Project description:Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 286 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks the seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3'-tag digital gene expression (DGE) reveals that 246 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryon strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryon strains.
2014-12-19 | GSE43019 | GEO
Project description:Study of the secretome of Cellvibrio japonicus after growth on different chitins. Utilization of a novel plate method to enrich truly secreted proteins.
2016-05-18 | PXD002843 | Pride