Project description:Glycans, the major source of energy for the human gut microbiota (HGM), are metabolised by numerous members of the Bacteroides genus. Arabinogalactan proteins (AGPs) are a ubiquitous and highly heterogenous group of plant glycans in which a B1,3-galactan backbone and B1,6-galactan side chains are conserved features. Diversity is provided by the extensive and highly variable nature of the sugars that decorate both the backbone and side chain galactans. The mechanisms by which nutritionally relevant AGPs are degraded at a cellular and biochemical level are poorly understood, as is the impact of this process on the ecology of the HGM. Here we have explored how the HGM organism Bacteroides thetaiotaomicron metabolises simple and highly complex AGPs. We propose a sequential degradative model in which a repertoire of exo-acting family GH43 B1,3-galactanases releases backbone galactose units that are attached to the side chains. The oligosaccharide side chains are depolymerized by the synergistic action of exo-acting enzymes in which catalytic interactions are dependent on whether degradation is initiated by a lyase or glycoside hydrolase. We identified an a-L-arabinofuranosidase and B-D-glucuronidase that are the founders of two previously undiscovered glycoside hydrolase families. The crystal structures of the backbone exo-B1,3-galactanases identified a key specificity determinant and reveals significant departure from the canonical catalytic apparatus of other family GH43 enzymes. Growth studies of 20 HGM Bacteroidetes species on a complex AGP revealed three keystone organisms that facilitated utilisation of fragments of the glycan by the 17 other bacteria, which thus acted as recipients. The ability to function as a keystone organism was conferred by a surface endo-B1,3-galactanase, which, when engineered into a recipient enabled this bacterium to utilise complex AGPs and facilitate the growth of the other Bacteroidetes species. This study underpins future pre- and probiotic strategies to exploit AGPs to manipulate the function of the HGM.

Project description:Background: Saprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers [GSE33852]. Results: In this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study. Conclusions: By comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production. Six samples in total consisting of duplicate shake flask Aspergillus niger cultures from three conditions: glucose 48 h, willow 24 h, willow 24 h + glucose 5 h

Project description:Background: Saprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers [GSE33852]. Results: In this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study. Conclusions: By comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production.

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Expression profiling of Cellvibrio japonicus using either glucose or xyloglucan

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