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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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Model of polysaccharide degradation and metabolism by C. phytofermentans. CAZymes (shown as the number of enzymes in CAZy families) are based on purified activities and are intra- or extracellular based on putative secretion signals.Metabolic enzymes are shown as NCBI numbers and are proposed based on mRNA expression. Rhamnose transport and assimilation is based on pathway from [55]. Abbreviations are D-galacturonic acid (GA), L-rhamnose (R), D-mannose (M), D-glucose (Gc), D-galactose (G), D-xylose (X), L-arabinose (A), fructose (F), phosphate (P), pentose phosphate pathway (PPP), dihydroxyacetone-phosphate (DHAP), glyceraldehyde-3-phosphate (G3P). For each substrate, the number of significantly up-regulated extracellular solute binding proteins (ESB) and ABC transporters (ABC) are shown. Shaded regions show metabolism of glucose (green), mannose (blue), xylose and arabinose (yellow), rhamnose (orange), and galacturonic acid (red).
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pgen-1004773-g006: Model of polysaccharide degradation and metabolism by C. phytofermentans. CAZymes (shown as the number of enzymes in CAZy families) are based on purified activities and are intra- or extracellular based on putative secretion signals.Metabolic enzymes are shown as NCBI numbers and are proposed based on mRNA expression. Rhamnose transport and assimilation is based on pathway from [55]. Abbreviations are D-galacturonic acid (GA), L-rhamnose (R), D-mannose (M), D-glucose (Gc), D-galactose (G), D-xylose (X), L-arabinose (A), fructose (F), phosphate (P), pentose phosphate pathway (PPP), dihydroxyacetone-phosphate (DHAP), glyceraldehyde-3-phosphate (G3P). For each substrate, the number of significantly up-regulated extracellular solute binding proteins (ESB) and ABC transporters (ABC) are shown. Shaded regions show metabolism of glucose (green), mannose (blue), xylose and arabinose (yellow), rhamnose (orange), and galacturonic acid (red).

Mentions: We assimilated our results into a model of C. phytofermentans polysaccharide catabolism that shows degradation by active CAZymes and uses mRNA expression profiles to predict how these substrates are transported and metabolized (Fig. 6). Unlike other clostridia that transport sugars with numerous phosphotransferase systems (PTS) [35][36], C. phytofermentans encodes a single, lowly expressed PTS and also lacks the symporters to transport xylose and arabinose [37]. Instead, C. phytofermentans responds to carbon sources by up-regulating between two (galacturonic acid) and twenty-two (arabinan) ABC transporters (Fig. 6). Expression changes support that oligosaccharides and monosaccharides are uptaken by distinct transporters. For example, different ABC transporters are up-regulated on xylose and xylan. Similarly, different transporters respond to glucose, cellobiose, and cellulose. Intracellular cellodextrins are cleaved by at least one cellodextin phosphorylase (GH94); hexoses are phosphorylated, likely by a ROK hexokinase (Cphy0329) and a putative galactokinase (Cphy2237), and fed into glycolysis. While hexokinases may have wide substrate activity [38], poor growth on mannose could be due to inefficient mannose phosphorylation. The pentoses xylose and arabinose are isomerized and metabolized by the pentose phosphate pathway (PPP). Weak growth on arabinose could be due to inefficient transport or the lack of the phosphoketolase in the PPP enabling rapid L-arabinose metabolism by C. acetobutylicum[39].


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)

Model of polysaccharide degradation and metabolism by C. phytofermentans. CAZymes (shown as the number of enzymes in CAZy families) are based on purified activities and are intra- or extracellular based on putative secretion signals.Metabolic enzymes are shown as NCBI numbers and are proposed based on mRNA expression. Rhamnose transport and assimilation is based on pathway from [55]. Abbreviations are D-galacturonic acid (GA), L-rhamnose (R), D-mannose (M), D-glucose (Gc), D-galactose (G), D-xylose (X), L-arabinose (A), fructose (F), phosphate (P), pentose phosphate pathway (PPP), dihydroxyacetone-phosphate (DHAP), glyceraldehyde-3-phosphate (G3P). For each substrate, the number of significantly up-regulated extracellular solute binding proteins (ESB) and ABC transporters (ABC) are shown. Shaded regions show metabolism of glucose (green), mannose (blue), xylose and arabinose (yellow), rhamnose (orange), and galacturonic acid (red).
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Related In: Results  -  Collection

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pgen-1004773-g006: Model of polysaccharide degradation and metabolism by C. phytofermentans. CAZymes (shown as the number of enzymes in CAZy families) are based on purified activities and are intra- or extracellular based on putative secretion signals.Metabolic enzymes are shown as NCBI numbers and are proposed based on mRNA expression. Rhamnose transport and assimilation is based on pathway from [55]. Abbreviations are D-galacturonic acid (GA), L-rhamnose (R), D-mannose (M), D-glucose (Gc), D-galactose (G), D-xylose (X), L-arabinose (A), fructose (F), phosphate (P), pentose phosphate pathway (PPP), dihydroxyacetone-phosphate (DHAP), glyceraldehyde-3-phosphate (G3P). For each substrate, the number of significantly up-regulated extracellular solute binding proteins (ESB) and ABC transporters (ABC) are shown. Shaded regions show metabolism of glucose (green), mannose (blue), xylose and arabinose (yellow), rhamnose (orange), and galacturonic acid (red).
Mentions: We assimilated our results into a model of C. phytofermentans polysaccharide catabolism that shows degradation by active CAZymes and uses mRNA expression profiles to predict how these substrates are transported and metabolized (Fig. 6). Unlike other clostridia that transport sugars with numerous phosphotransferase systems (PTS) [35][36], C. phytofermentans encodes a single, lowly expressed PTS and also lacks the symporters to transport xylose and arabinose [37]. Instead, C. phytofermentans responds to carbon sources by up-regulating between two (galacturonic acid) and twenty-two (arabinan) ABC transporters (Fig. 6). Expression changes support that oligosaccharides and monosaccharides are uptaken by distinct transporters. For example, different ABC transporters are up-regulated on xylose and xylan. Similarly, different transporters respond to glucose, cellobiose, and cellulose. Intracellular cellodextrins are cleaved by at least one cellodextin phosphorylase (GH94); hexoses are phosphorylated, likely by a ROK hexokinase (Cphy0329) and a putative galactokinase (Cphy2237), and fed into glycolysis. While hexokinases may have wide substrate activity [38], poor growth on mannose could be due to inefficient mannose phosphorylation. The pentoses xylose and arabinose are isomerized and metabolized by the pentose phosphate pathway (PPP). Weak growth on arabinose could be due to inefficient transport or the lack of the phosphoketolase in the PPP enabling rapid L-arabinose metabolism by C. acetobutylicum[39].

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.

Show MeSH