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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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C. phytofermentans growth on pectic A–E, hemicellulosic F–J, and glucan K–L. Polysaccharides: homogalacturonan A, rhamnogalacturonan I B, galactan C, arabinan D, arabinogalactan II E, xylan F, arabinoxylan G, glucomannan H, galactomannan I, xyloglucan J, carboxymethylcellulose K, starch L. Growth was measured as OD600 every 15 minutes. Each point is the mean of six cultures; red lines show one standard deviation.
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pgen-1004773-g001: C. phytofermentans growth on pectic A–E, hemicellulosic F–J, and glucan K–L. Polysaccharides: homogalacturonan A, rhamnogalacturonan I B, galactan C, arabinan D, arabinogalactan II E, xylan F, arabinoxylan G, glucomannan H, galactomannan I, xyloglucan J, carboxymethylcellulose K, starch L. Growth was measured as OD600 every 15 minutes. Each point is the mean of six cultures; red lines show one standard deviation.

Mentions: We developed a high resolution, microtiter anaerobic growth assay that shows C. phytofermentans ferments diverse plant polysaccharides (Fig. 1) and their constituent monosaccharides (Fig. S1), but with widely varying cell yields and growth rates (Table S4). It also forms colonies on solid medium containing each polysaccharide except arabinogalactan II (AGII) (Fig. S2). Growth was fastest on HG (Fig. 1A, generation time 0.70h), similar to rumen microbes that digest pectin more rapidly than cellulose and hemicellulose [19]. Although C. phytofermentans ferments both galacturonic acid (Fig. S1F) and rhamnose (Fig. S1H), cell yield was low on RGI (Fig. 1B). C. phytofermentans grows well on galactan (Fig. 1C), xylans (Fig. 1F–G), mannans (Fig. 1H–I), xyloglucan (Fig. 1J), and starch (Fig. 1L). Limited growth on AGII (Fig. 1E) relative to galactan supports that C. phytofermentans cleaves β-1,4 galactan, but not the β-1,3 and β-1,6-galactose bonds in AGII. Poor growth on arabinan (Fig. 1D) is similar to arabinose (Fig. S1G), suggesting this sugar is transported or metabolized inefficiently. C. phytofermentans grows well on cellulose plates (Fig. S2) and solubilizes cellulosic substrates such as filter paper and raw corn stover (Fig. S3), but weak growth on carboxymethylcellulose (CMC) might result from either lack of a suitable endoglucanase or carboxymethyl side groups inhibiting its metabolism.


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)

C. phytofermentans growth on pectic A–E, hemicellulosic F–J, and glucan K–L. Polysaccharides: homogalacturonan A, rhamnogalacturonan I B, galactan C, arabinan D, arabinogalactan II E, xylan F, arabinoxylan G, glucomannan H, galactomannan I, xyloglucan J, carboxymethylcellulose K, starch L. Growth was measured as OD600 every 15 minutes. Each point is the mean of six cultures; red lines show one standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004773-g001: C. phytofermentans growth on pectic A–E, hemicellulosic F–J, and glucan K–L. Polysaccharides: homogalacturonan A, rhamnogalacturonan I B, galactan C, arabinan D, arabinogalactan II E, xylan F, arabinoxylan G, glucomannan H, galactomannan I, xyloglucan J, carboxymethylcellulose K, starch L. Growth was measured as OD600 every 15 minutes. Each point is the mean of six cultures; red lines show one standard deviation.
Mentions: We developed a high resolution, microtiter anaerobic growth assay that shows C. phytofermentans ferments diverse plant polysaccharides (Fig. 1) and their constituent monosaccharides (Fig. S1), but with widely varying cell yields and growth rates (Table S4). It also forms colonies on solid medium containing each polysaccharide except arabinogalactan II (AGII) (Fig. S2). Growth was fastest on HG (Fig. 1A, generation time 0.70h), similar to rumen microbes that digest pectin more rapidly than cellulose and hemicellulose [19]. Although C. phytofermentans ferments both galacturonic acid (Fig. S1F) and rhamnose (Fig. S1H), cell yield was low on RGI (Fig. 1B). C. phytofermentans grows well on galactan (Fig. 1C), xylans (Fig. 1F–G), mannans (Fig. 1H–I), xyloglucan (Fig. 1J), and starch (Fig. 1L). Limited growth on AGII (Fig. 1E) relative to galactan supports that C. phytofermentans cleaves β-1,4 galactan, but not the β-1,3 and β-1,6-galactose bonds in AGII. Poor growth on arabinan (Fig. 1D) is similar to arabinose (Fig. S1G), suggesting this sugar is transported or metabolized inefficiently. C. phytofermentans grows well on cellulose plates (Fig. S2) and solubilizes cellulosic substrates such as filter paper and raw corn stover (Fig. S3), but weak growth on carboxymethylcellulose (CMC) might result from either lack of a suitable endoglucanase or carboxymethyl side groups inhibiting its metabolism.

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