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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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Cleavage A, binding B, and CAZy database classification C of purified enzymes.A Polysaccharide cleavage was quantified as nmol reducing sugar released per milligram enzyme per minute: >160 red, 80–160 orange, 40–80 yellow, 20–40 green, <20 gray. B Binding to insoluble polysaccharides was quantified as percentage enzyme bound to substrate: >30% red, 20–30% orange, 15–20% yellow, 10–15% green, <10% gray. C CAZy database classifications: glycoside hydrolases (GH), carbohydrate esterases (CE), polysaccharide lyases (PL), and carbohydrate binding domains (CBM). Among 56 purified CAZymes, only the 32 enzymes for which activities were found are shown.
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pgen-1004773-g004: Cleavage A, binding B, and CAZy database classification C of purified enzymes.A Polysaccharide cleavage was quantified as nmol reducing sugar released per milligram enzyme per minute: >160 red, 80–160 orange, 40–80 yellow, 20–40 green, <20 gray. B Binding to insoluble polysaccharides was quantified as percentage enzyme bound to substrate: >30% red, 20–30% orange, 15–20% yellow, 10–15% green, <10% gray. C CAZy database classifications: glycoside hydrolases (GH), carbohydrate esterases (CE), polysaccharide lyases (PL), and carbohydrate binding domains (CBM). Among 56 purified CAZymes, only the 32 enzymes for which activities were found are shown.

Mentions: A set of 56 CAZymes up-regulated on polysaccharides were His-tagged, overexpressed, purified, and their abilities to bind and cleave polysaccharides were quantified. The CAZy database classifies these enzymes putatively as 47 glycoside hydrolases, 4 polysaccharide lyases, and 4 carbohydrate esterases (Table S2); putative glycosyltransferases were not examined as they are not involved in polysaccharide catabolism [6]. Thirty-two enzymes have significant cleavage or binding activities (Fig. 4, Table S11). Some substrates such as β-1,4-galactan appear to be cut by a single, highly active enzyme, while multiple CAZymes from the same family degrade other substrates such as xylan (GH10), mannan (GH26), starch (GH13), and HG (PL9). CAZymes from multiple families together depolymerize substrates such as xyloglucan (GH2,5,12,31), glucomannan (GH5,GH9,GH26) and galactomannan (GH5,GH26).


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)

Cleavage A, binding B, and CAZy database classification C of purified enzymes.A Polysaccharide cleavage was quantified as nmol reducing sugar released per milligram enzyme per minute: >160 red, 80–160 orange, 40–80 yellow, 20–40 green, <20 gray. B Binding to insoluble polysaccharides was quantified as percentage enzyme bound to substrate: >30% red, 20–30% orange, 15–20% yellow, 10–15% green, <10% gray. C CAZy database classifications: glycoside hydrolases (GH), carbohydrate esterases (CE), polysaccharide lyases (PL), and carbohydrate binding domains (CBM). Among 56 purified CAZymes, only the 32 enzymes for which activities were found are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230839&req=5

pgen-1004773-g004: Cleavage A, binding B, and CAZy database classification C of purified enzymes.A Polysaccharide cleavage was quantified as nmol reducing sugar released per milligram enzyme per minute: >160 red, 80–160 orange, 40–80 yellow, 20–40 green, <20 gray. B Binding to insoluble polysaccharides was quantified as percentage enzyme bound to substrate: >30% red, 20–30% orange, 15–20% yellow, 10–15% green, <10% gray. C CAZy database classifications: glycoside hydrolases (GH), carbohydrate esterases (CE), polysaccharide lyases (PL), and carbohydrate binding domains (CBM). Among 56 purified CAZymes, only the 32 enzymes for which activities were found are shown.
Mentions: A set of 56 CAZymes up-regulated on polysaccharides were His-tagged, overexpressed, purified, and their abilities to bind and cleave polysaccharides were quantified. The CAZy database classifies these enzymes putatively as 47 glycoside hydrolases, 4 polysaccharide lyases, and 4 carbohydrate esterases (Table S2); putative glycosyltransferases were not examined as they are not involved in polysaccharide catabolism [6]. Thirty-two enzymes have significant cleavage or binding activities (Fig. 4, Table S11). Some substrates such as β-1,4-galactan appear to be cut by a single, highly active enzyme, while multiple CAZymes from the same family degrade other substrates such as xylan (GH10), mannan (GH26), starch (GH13), and HG (PL9). CAZymes from multiple families together depolymerize substrates such as xyloglucan (GH2,5,12,31), glucomannan (GH5,GH9,GH26) and galactomannan (GH5,GH26).

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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