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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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CAZymes clustered based on gene expression patterns (clusters A–I) show that some genes respond to multiple carbon sources while others are substrate-specific.mRNA expression changes (log2 expression ratios relative to glucose) for all 92 CAZyme genes differentially expressed on at least 1 polysaccharide relative to glucose were separated into nine clusters using K-means. Plot centers are expression on glucose and concentric rings show log2 up-regulation on the following carbon sources: cellobiose (Cb), filter paper cellulose (Cl), starch (Sa), xylose (Xo), xylan (Xy), arabinan (Ar), galacturonic acid (Ga), homogalacturonan (Hg), galactan (Gl), galactomannan (Gm), raw corn stover (Co). Gene membership of clusters is shown in Table S9.
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pgen-1004773-g003: CAZymes clustered based on gene expression patterns (clusters A–I) show that some genes respond to multiple carbon sources while others are substrate-specific.mRNA expression changes (log2 expression ratios relative to glucose) for all 92 CAZyme genes differentially expressed on at least 1 polysaccharide relative to glucose were separated into nine clusters using K-means. Plot centers are expression on glucose and concentric rings show log2 up-regulation on the following carbon sources: cellobiose (Cb), filter paper cellulose (Cl), starch (Sa), xylose (Xo), xylan (Xy), arabinan (Ar), galacturonic acid (Ga), homogalacturonan (Hg), galactan (Gl), galactomannan (Gm), raw corn stover (Co). Gene membership of clusters is shown in Table S9.

Mentions: The differentially expressed CAZymes are putatively classified by the CAZy database as 67 glycoside hydrolases, 6 carbohydrate esterases, 4 polysaccharide lyases, 14 glycosyl transferases, and 2 CBM proteins. We analyzed the specificity of the CAZyme transcriptional response by K-means clustering the expression profiles of these genes (Fig. 3, Table S9). Cluster A consists six genes that were highly up-regulated on multiple substrates: the GH26 cphy1071, the GH11 cphy2105, two GH18 chitinases cphy1799 and cphy1800[26], the GH9 cellulase cphy3367[27][28] and the GH48 cellulase cphy3368[29]. Clusters B–F respond to specific polysaccharides such as homogalacturonan (clusters B,C), starch (cluster D), xylan (cluster E), and cellulose/arabinan (cluster F). C. phytofermentans thus perceives signals from individual polysaccharides and responds by up-regulating specific transcriptional regulons that enable it to tailor its complement of CAZymes to the polysaccharide substrate.


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)

CAZymes clustered based on gene expression patterns (clusters A–I) show that some genes respond to multiple carbon sources while others are substrate-specific.mRNA expression changes (log2 expression ratios relative to glucose) for all 92 CAZyme genes differentially expressed on at least 1 polysaccharide relative to glucose were separated into nine clusters using K-means. Plot centers are expression on glucose and concentric rings show log2 up-regulation on the following carbon sources: cellobiose (Cb), filter paper cellulose (Cl), starch (Sa), xylose (Xo), xylan (Xy), arabinan (Ar), galacturonic acid (Ga), homogalacturonan (Hg), galactan (Gl), galactomannan (Gm), raw corn stover (Co). Gene membership of clusters is shown in Table S9.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004773-g003: CAZymes clustered based on gene expression patterns (clusters A–I) show that some genes respond to multiple carbon sources while others are substrate-specific.mRNA expression changes (log2 expression ratios relative to glucose) for all 92 CAZyme genes differentially expressed on at least 1 polysaccharide relative to glucose were separated into nine clusters using K-means. Plot centers are expression on glucose and concentric rings show log2 up-regulation on the following carbon sources: cellobiose (Cb), filter paper cellulose (Cl), starch (Sa), xylose (Xo), xylan (Xy), arabinan (Ar), galacturonic acid (Ga), homogalacturonan (Hg), galactan (Gl), galactomannan (Gm), raw corn stover (Co). Gene membership of clusters is shown in Table S9.
Mentions: The differentially expressed CAZymes are putatively classified by the CAZy database as 67 glycoside hydrolases, 6 carbohydrate esterases, 4 polysaccharide lyases, 14 glycosyl transferases, and 2 CBM proteins. We analyzed the specificity of the CAZyme transcriptional response by K-means clustering the expression profiles of these genes (Fig. 3, Table S9). Cluster A consists six genes that were highly up-regulated on multiple substrates: the GH26 cphy1071, the GH11 cphy2105, two GH18 chitinases cphy1799 and cphy1800[26], the GH9 cellulase cphy3367[27][28] and the GH48 cellulase cphy3368[29]. Clusters B–F respond to specific polysaccharides such as homogalacturonan (clusters B,C), starch (cluster D), xylan (cluster E), and cellulose/arabinan (cluster F). C. phytofermentans thus perceives signals from individual polysaccharides and responds by up-regulating specific transcriptional regulons that enable it to tailor its complement of CAZymes to the polysaccharide substrate.

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
Related in: MedlinePlus