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Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

Boutard M, Cerisy T, Nogue PY, Alberti A, Weissenbach J, Salanoubat M, Tolonen AC - PLoS Genet. (2014)

Bottom Line: These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates.CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms.We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

View Article: PubMed Central - PubMed

Affiliation: Genoscope, CEA, DSV, IG, Évry, France; CNRS-UMR8030, Évry, France; Department of Biology, Université d'Évry Val d'Essonne, Évry, France.

ABSTRACT
Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

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Members of the same CAZy family vary in polysaccharide cleavage activities and CAZymes can by potentiated by other enzymes.A Variation in cleavage activities of GH10 enzymes on xylan. B GH5 and C GH26 family members differ in their activities and substrate specificities on amorphous cellulose (red), glucomannan (green), xyloglucan (violet), galactomannan (yellow), mannan (gray). Enzyme activities in A–C are nmol reducing sugar released per milligram enzyme per minute. D–G CAZyme mixtures have higher activities than the individual enzymes. D Cphy1163 and Cphy3367 alone and together on amorphous cellulose. E Cphy2105, Cphy3009, and Cphy3207 alone and the latter two enzymes plus Cphy2105 on xylan. F Cphy1719 and Cphy1071 alone and together on glucomannan. G Cphy1687, Cphy2567, and Cphy3310 alone and the latter two enzymes plus Cphy1687 on homogalacturonan. In D–G, enzyme activities are shown as reducing sugar (nmol) produced by individual and combined enzymes. The fraction of the reducing sugar produced by the mixed enzymes that exceeds the sum of the individual enzymes is shown in green.
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pgen-1004773-g005: Members of the same CAZy family vary in polysaccharide cleavage activities and CAZymes can by potentiated by other enzymes.A Variation in cleavage activities of GH10 enzymes on xylan. B GH5 and C GH26 family members differ in their activities and substrate specificities on amorphous cellulose (red), glucomannan (green), xyloglucan (violet), galactomannan (yellow), mannan (gray). Enzyme activities in A–C are nmol reducing sugar released per milligram enzyme per minute. D–G CAZyme mixtures have higher activities than the individual enzymes. D Cphy1163 and Cphy3367 alone and together on amorphous cellulose. E Cphy2105, Cphy3009, and Cphy3207 alone and the latter two enzymes plus Cphy2105 on xylan. F Cphy1719 and Cphy1071 alone and together on glucomannan. G Cphy1687, Cphy2567, and Cphy3310 alone and the latter two enzymes plus Cphy1687 on homogalacturonan. In D–G, enzyme activities are shown as reducing sugar (nmol) produced by individual and combined enzymes. The fraction of the reducing sugar produced by the mixed enzymes that exceeds the sum of the individual enzymes is shown in green.

Mentions: Thirty-two CAZy families have multiple members, which often have divergent cleavage activities and cellular localizations. Cphy1510 has the highest activity among the four GH10 active on xylan (Fig. 5A). Cphy3010, the GH10 with lowest activity, is the only one lacking a secretion signal, supporting it acts intracellularly on xylo-oligosaccharides while the other GH10 are extracellular. Members of the GH5 family act on a wide range of polysaccharides [32]. C. phytofermentans encodes 3 GH5 enzymes, among which one is active on galactomannan and two on xyloglucan (Fig. 5B). The GH5 Cphy1163 has no activity on either of these substrates, but is the most active on cellulose and glucomannan. The 3 GH26 also vary in substrate specificities (Fig. 5C); all the GH26 are similarly active on β-mannan, but only Cphy1071 has cellulase activity and it has lower activity on gluco- and galactomannan. Sequenced-based families are thus useful to make general substrate predictions for CAZymes, but experiments are needed to determine substrate range and catalytic efficiency.


Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

Boutard M, Cerisy T, Nogue PY, Alberti A, Weissenbach J, Salanoubat M, Tolonen AC - PLoS Genet. (2014)

Members of the same CAZy family vary in polysaccharide cleavage activities and CAZymes can by potentiated by other enzymes.A Variation in cleavage activities of GH10 enzymes on xylan. B GH5 and C GH26 family members differ in their activities and substrate specificities on amorphous cellulose (red), glucomannan (green), xyloglucan (violet), galactomannan (yellow), mannan (gray). Enzyme activities in A–C are nmol reducing sugar released per milligram enzyme per minute. D–G CAZyme mixtures have higher activities than the individual enzymes. D Cphy1163 and Cphy3367 alone and together on amorphous cellulose. E Cphy2105, Cphy3009, and Cphy3207 alone and the latter two enzymes plus Cphy2105 on xylan. F Cphy1719 and Cphy1071 alone and together on glucomannan. G Cphy1687, Cphy2567, and Cphy3310 alone and the latter two enzymes plus Cphy1687 on homogalacturonan. In D–G, enzyme activities are shown as reducing sugar (nmol) produced by individual and combined enzymes. The fraction of the reducing sugar produced by the mixed enzymes that exceeds the sum of the individual enzymes is shown in green.
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Related In: Results  -  Collection

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pgen-1004773-g005: Members of the same CAZy family vary in polysaccharide cleavage activities and CAZymes can by potentiated by other enzymes.A Variation in cleavage activities of GH10 enzymes on xylan. B GH5 and C GH26 family members differ in their activities and substrate specificities on amorphous cellulose (red), glucomannan (green), xyloglucan (violet), galactomannan (yellow), mannan (gray). Enzyme activities in A–C are nmol reducing sugar released per milligram enzyme per minute. D–G CAZyme mixtures have higher activities than the individual enzymes. D Cphy1163 and Cphy3367 alone and together on amorphous cellulose. E Cphy2105, Cphy3009, and Cphy3207 alone and the latter two enzymes plus Cphy2105 on xylan. F Cphy1719 and Cphy1071 alone and together on glucomannan. G Cphy1687, Cphy2567, and Cphy3310 alone and the latter two enzymes plus Cphy1687 on homogalacturonan. In D–G, enzyme activities are shown as reducing sugar (nmol) produced by individual and combined enzymes. The fraction of the reducing sugar produced by the mixed enzymes that exceeds the sum of the individual enzymes is shown in green.
Mentions: Thirty-two CAZy families have multiple members, which often have divergent cleavage activities and cellular localizations. Cphy1510 has the highest activity among the four GH10 active on xylan (Fig. 5A). Cphy3010, the GH10 with lowest activity, is the only one lacking a secretion signal, supporting it acts intracellularly on xylo-oligosaccharides while the other GH10 are extracellular. Members of the GH5 family act on a wide range of polysaccharides [32]. C. phytofermentans encodes 3 GH5 enzymes, among which one is active on galactomannan and two on xyloglucan (Fig. 5B). The GH5 Cphy1163 has no activity on either of these substrates, but is the most active on cellulose and glucomannan. The 3 GH26 also vary in substrate specificities (Fig. 5C); all the GH26 are similarly active on β-mannan, but only Cphy1071 has cellulase activity and it has lower activity on gluco- and galactomannan. Sequenced-based families are thus useful to make general substrate predictions for CAZymes, but experiments are needed to determine substrate range and catalytic efficiency.

Bottom Line: These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates.CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms.We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

View Article: PubMed Central - PubMed

Affiliation: Genoscope, CEA, DSV, IG, Évry, France; CNRS-UMR8030, Évry, France; Department of Biology, Université d'Évry Val d'Essonne, Évry, France.

ABSTRACT
Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

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