Complete genome of a new Firmicutes species belonging to the dominant human colonic microbiota (‘Ruminococcus bicirculans’) reveals two chromosomes and a selective capacity to utilize plant glucans.
Wegmann, U., Louis, P., Goesmann, A., Henrissat, B., Duncan, S. H. & Flint, H. J. (2014). Environmental Microbiology, 16 (9), 2879–2890.
The recently isolated bacterial strain 80/3 represents one of the most abundant 16S rRNA phylotypes detected in the healthy human large intestine and belongs to the Ruminococcaceae family of Firmicutes. The completed genome sequence reported here is the first for a member of this important family of bacteria from the human colon. The genome comprises two large chromosomes of 2.24 and 0.73 Mbp, leading us to propose the name Ruminococcus bicirculans for this new species. Analysis of the carbohydrate active enzyme complement suggests an ability to utilize certain hemicelluloses, especially β-glucans and xyloglucan, for growth that was confirmed experimentally. The enzymatic machinery enabling the degradation of cellulose and xylan by related cellulolytic ruminococci is however lacking in this species. While the genome indicated the capacity to synthesize purines, pyrimidines and all 20 amino acids, only genes for the synthesis of nicotinate, NAD+, NADP+ and coenzyme A were detected among the essential vitamins and co-factors, resulting in multiple growth requirements. In vivo, these growth factors must be supplied from the diet, host or other gut microorganisms. Other features of ecological interest include two type IV pilins, multiple extracytoplasmic function-sigma factors, a urease and a bile salt hydrolase.
Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis.
Larsbrink, J., Tuveng, T. R., Pope, P. B., Bulone, V., Eijsink, V. G., Brumer, H. & McKee, L. S. (2017). Journal of Proteomics, 156, 63-74.
Together with fungi, saprophytic bacteria are central to the decomposition and recycling of biomass in forest environments. The Bacteroidetes phylum is abundant in diverse habitats, and several species have been shown to be able to deconstruct a wide variety of complex carbohydrates. The genus Chitinophaga is often enriched in hotspots of plant and microbial biomass degradation. We present a proteomic assessment of the ability of Chitinophaga pinensis to grow on and degrade mannan polysaccharides, using an agarose plate-based method of protein collection to minimise contamination with exopolysaccharides and proteins from lysed cells, and to reflect the realistic setting of growth on a solid surface. We show that select Polysaccharide Utilisation Loci (PULs) are expressed in different growth conditions, and identify enzymes that may be involved in mannan degradation. By comparing proteomic and enzymatic profiles, we show evidence for the induced expression of enzymes and PULs in cells grown on mannan polysaccharides compared with cells grown on glucose. In addition, we show that the secretion of putative biomass-degrading enzymes during growth on glucose comprises a system for nutrient scavenging, which employs constitutively produced enzymes. Significance of this study: Chitinophaga pinensis belongs to a bacterial genus which is prominent in microbial communities in agricultural and forest environments, where plant and fungal biomass is intensively degraded. Such degradation is hugely significant in the recycling of carbon in the natural environment, and the enzymes responsible are of biotechnological relevance in emerging technologies involving the deconstruction of plant cell wall material. The bacterium has a comparatively large genome, which includes many uncharacterised carbohydrate-active enzymes. We present the first proteomic assessment of the biomass-degrading machinery of this species, focusing on mannan, an abundant plant cell wall hemicellulose. Our findings include the identification of several novel enzymes, which are promising targets for future biochemical characterisation. In addition, the data indicate the expression of specific Polysaccharide Utilisation Loci, induced in the presence of different growth substrates. We also highlight how a constitutive secretion of enzymes which deconstruct microbial biomass likely forms part of a nutrient scavenging process.
The transcription factor PDR-1 is a multi-functional regulator and key component of pectin deconstruction and catabolism in Neurospora crassa.
Thieme, N., Wu, V. W., Dietschmann, A., Salamov, A. A., Wang, M., Johnson, J., Singan, V. R., Grigoriev, I. V., Glass, N. L., Somerville, C. R., & Benz, J. P. (2017). Biotechnology for Biofuels, 10(1), 149.
Background: Pectin is an abundant component in many fruit and vegetable wastes and could therefore be an excellent resource for biorefinery, but is currently underutilized. Fungal pectinases already play a crucial role for industrial purposes, such as for foodstuff processing. However, the regulation of pectinase gene expression is still poorly understood. For an optimal utilization of plant biomass for biorefinery and biofuel production, a detailed analysis of the underlying regulatory mechanisms is warranted. In this study, we applied the genetic resources of the filamentous ascomycete species Neurospora crassa to screen for transcription factors that play a major role in pectinase induction. Results: The pectin degradation regulator-1 (PDR-1) was identified through a transcription factor mutant screen in N. crassa. The Δpdr-1 mutant exhibited a severe growth defect on pectin and all tested pectin-related poly- and monosaccharides. Biochemical as well as transcriptional analyses of WT and the Δpdr-1 mutant revealed that while PDR-1-mediated gene induction was dependent on the presence of L-rhamnose, it also strongly affected the degradation of the homogalacturonan backbone. The expression of the endo-polygalacturonase gh28-1 was greatly reduced in the Δpdr-1 mutant, while the expression levels of all pectate lyase genes increased. Moreover, a pdr-1 overexpression strain displayed substantially increased pectinase production. Promoter analysis of the PDR-1 regulon allowed refinement of the putative PDR-1 DNA-binding motif. Conclusions: PDR-1 is highly conserved in filamentous ascomycete fungi and is present in many pathogenic and industrially important fungi. Our data demonstrate that the function of PDR-1 in N. crassa combines features of two recently described transcription factors in Aspergillus niger (RhaR) and Botrytis cinerea (GaaR). The results presented in this study contribute to a broader understanding of how pectin degradation is orchestrated in filamentous fungi and how it could be manipulated for optimized pectinase production.
An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins.
Munoz-Munoz, J., Cartmell, A., Terrapon, N., Baslé, A., Henrissat, B. & Gilbert, H. J. (2017). Journal of Biological Chemistry, jbc-M117.
The human gut microbiota utilizes complex carbohydrates as major nutrients. The requirement for efficient glycan degrading systems exerts a major selective selection pressure on this microbial community. Thus, we propose that this microbial ecosystem represents a substantial resource for discovering novel carbohydrate active enzymes. To test this hypothesis we screened the potential enzymatic functions of hypothetical proteins encoded by genes of Bacteroides thetaiotaomicron that were upregulated by arabinogalactan arabinogalactan proteins or AGPs. Although AGPs are ubiquitous in plants, there is a paucity of information on their detailed structure, the function of these glycans in planta and the mechanisms by which they are depolymerized in microbial ecosystems. Here we have discovered a new polysaccharide lyase family that is specific for the L-rhamnose-alpha1,4-D-glucuronic acid linkage that caps the side chains of complex AGPs. The reaction product generated by the lyase, delta4,5-unsaturated uronic acid, is removed from AGP by a glycoside hydrolase located in family GH105, producing the final product 4-deoxy-β-L-threo-hex-4-enepyranosyl-uronic acid. The crystal structure of a member of the novel lyase family revealed a catalytic domain that displays an (alpha/alpha6)6 barrel fold. In the centre of the barrel is a deep pocket, which, based on mutagenesis data and amino acid conservation, comprises the active site of the lyase. A tyrosine is the proposed catalytic base in the beta-elimination reaction. This study illustrates how highly complex glycans can be used as a scaffold to discover new enzyme families within microbial ecosystems where carbohydrate metabolism is a major evolutionary driver.
Improved starch recovery from potatoes by enzymes and reduced water holding of the residual fibres.
Ramasamy, U. R., Lips, S., Bakker, R., Gruppen, H. & Kabel, M. A. (2014). Carbohydrate Polymers, 113, 256-263.
During the industrial extraction of starch from potatoes (Seresta), some starch remains within undisrupted potato cells in the fibrous side-stream. The aim of this study was to investigate if enzymatic degradation of cell wall polysaccharides (CWPs) can enhance starch recovery and lower the water holding capacity (WHC) of the “fibre” fraction. The use of a pectinase-rich preparation recovered 58% of the starch present in the “fibre” fraction. Also, the “fibre” fraction retained only 40% of the water present in the non-enzyme treated “fibre”. This was caused by the degradation of pectins, in particular arabinogalactan side chains calculated as the sum of galactosyl and arabinosyl residues.
Comparison of structure and antioxidant activity of polysaccharides extracted from the leaves of Plantago major L., P. media L. and P. lanceolate. L.
Lukova, P. K., Karcheva-Bahchevanska, D. P.,Nikolova, M. M.,Iliev , I. N. & Mladenov, R. D. (2017). Bulgarian Chemical Communications, 49, 282-288.
In the current study for the first time were investigated the chemical composition and antioxidant activity of polysaccharides isolated from three indigenous for Bulgaria species of Plantago genus - Plantago major L., Plantago lanceolata L. and Plantago media L. Crude polysaccharides were extracted from fresh leaves with water and dilute acid and their yield was between 0.64% and 2.79%. The chemical composition of water-extractable polysaccharides (WEPs) and total acid-extractable polysaccharides (TAEPs) of Plantago leaves was evaluated by HPLC analysis. The phytochemical data revealed the presence of branched heteropolysaccharides with different neutral/acidic monosaccharide ratio. The predominant monosaccharide unit of WEPs was galacturonic acid (62.64% - 70.58%). Additionally, there were registered small amounts of arabinose and rhamnose. In TAEPs among with galacturonic acid (36.93% - 41.46%), significant amounts of neutral monosaccharides as galactose (22.80% - 46.11%) and rhamnose (16.96% - 35.74%) were determined. Two types of analyses were used to evaluate the antioxidant activity of Plantago isolated polysaccharides: DPPH and FRAP assay. Based on DPPH method, WEPs exhibited stronger radical scavenging ability (29.39% - 40.08%) compared to TAEPs (19.44% - 24.15%). In parallel, WEPs showed greater rate of ferric reducing power (103.71 - 137.83 µM TE/5 mg Ps) compared to TAEPs (34.63 - 117.66 µM TE/5 mg Ps). Although lower than synthetic BHT, Plantago polysaccharides revealed antioxidant potential and could be further explored as promising natural antioxidants for the nutraceutical and pharmaceutical industries.
An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.
Sabbadin , F., Hemsworth, G. R., Ciano, L., Henrissat, B., Dupree, P., Tryfona, T., Marques, R. D. S., Sweeney, S. T., Besser, K., Elias, L., Pesante, G., Li, Y., Dowle, A. A., Bates, R., Gomez, L. D., Simister, R., Davies, G. J., Walton, P. H., Bruce, N. C., McQueen-Mason, S. J. (2018). Nature Communications, In Press.
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over twenty members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin and was recruited by early arthropods with possible roles in remodelling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes towards cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.