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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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Related in: MedlinePlus

mRNA expression of all 171 CAZymes during growth on pectins A–C, hemicelluloses D–E, glucans F–H, and raw corn stover I relative to expression on glucose.Expression was quantified as log2(RPKM) with significantly differentially expressed genes on a given polysaccharide shown as triangles and unchanged genes as circles. The 56 purified CAZymes are red and others are blue.
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pgen-1004773-g002: mRNA expression of all 171 CAZymes during growth on pectins A–C, hemicelluloses D–E, glucans F–H, and raw corn stover I relative to expression on glucose.Expression was quantified as log2(RPKM) with significantly differentially expressed genes on a given polysaccharide shown as triangles and unchanged genes as circles. The 56 purified CAZymes are red and others are blue.

Mentions: We quantified mRNA expression by strand-specific RNA sequencing during log-phase growth on 8 polysaccharides, 3 monosaccharides, and raw corn stover as a complex biomass substrate. An average of 17.3 million mRNA reads were mapped per sample (Table S5), yielding expression (RPKM) values (Table S6) that were highly correlated (r2 = 0.96–0.99) between duplicate cultures for all conditions (Fig. S7). The reads were also highly strand-specific (Fig. S8), which will facilitate their future use for de novo transcriptome assembly, gene annotation and detection of antisense transcription. The fraction of reads mapping to CAZymes during growth on glucose was 2.0%, but this increased greatly on polysaccharides, especially cellulose (11.9%) and stover (31.0%). We assessed which genes were significantly differentially expressed on each polysaccharide relative to glucose using DESeq [25] (Table S7). Expression of CAZyme genes on polysaccharides relative to glucose (Fig. 2) shows that between 15 (cellobiose) and 40 (stover) CAZymes were significantly up-regulated per treatment (Table S8) with a total of 92 CAZymes up-regulated on at least one polysaccharide.


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)

mRNA expression of all 171 CAZymes during growth on pectins A–C, hemicelluloses D–E, glucans F–H, and raw corn stover I relative to expression on glucose.Expression was quantified as log2(RPKM) with significantly differentially expressed genes on a given polysaccharide shown as triangles and unchanged genes as circles. The 56 purified CAZymes are red and others are blue.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004773-g002: mRNA expression of all 171 CAZymes during growth on pectins A–C, hemicelluloses D–E, glucans F–H, and raw corn stover I relative to expression on glucose.Expression was quantified as log2(RPKM) with significantly differentially expressed genes on a given polysaccharide shown as triangles and unchanged genes as circles. The 56 purified CAZymes are red and others are blue.
Mentions: We quantified mRNA expression by strand-specific RNA sequencing during log-phase growth on 8 polysaccharides, 3 monosaccharides, and raw corn stover as a complex biomass substrate. An average of 17.3 million mRNA reads were mapped per sample (Table S5), yielding expression (RPKM) values (Table S6) that were highly correlated (r2 = 0.96–0.99) between duplicate cultures for all conditions (Fig. S7). The reads were also highly strand-specific (Fig. S8), which will facilitate their future use for de novo transcriptome assembly, gene annotation and detection of antisense transcription. The fraction of reads mapping to CAZymes during growth on glucose was 2.0%, but this increased greatly on polysaccharides, especially cellulose (11.9%) and stover (31.0%). We assessed which genes were significantly differentially expressed on each polysaccharide relative to glucose using DESeq [25] (Table S7). Expression of CAZyme genes on polysaccharides relative to glucose (Fig. 2) shows that between 15 (cellobiose) and 40 (stover) CAZymes were significantly up-regulated per treatment (Table S8) with a total of 92 CAZymes up-regulated on at least one polysaccharide.

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