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.
Highlights
Plants annually produce 200 billion tons of lignocellulosic biomass [1], which is metabolized by specialized microbes in diverse environments
Elucidating how microbes depolymerize and metabolize plant biomass is important to understand carbon flow in the environment, to promote healthy human nutrition and prevent disease [6], and to develop industrial processes based on cellulosic bioconversion
We assimilated our results into a model of C. phytofermentans polysaccharide catabolism that shows degradation by active carbohydrate-active enzyme (CAZyme) and uses mRNA expression profiles to predict how these substrates are transported and metabolized (Fig. 6)
Summary
Plants annually produce 200 billion tons of lignocellulosic biomass [1], which is metabolized by specialized microbes in diverse environments. Most of plant biomass is in the cell wall, a macromolecular network of phenolic lignin and three types of polysaccharides (cellulose, hemicelluloses, and pectins) whose relative abundances vary widely among species and tissues (Table S1). We demonstrate a strategy for systematic analysis of the enzymatic machinery used by microbes to degrade and metabolize plant polysaccharides. Among these microbes, the plant-fermenting clostridia are of particular interest for being a dominant group in the human gut microbiome [6] and top candidates to transform cellulosic biomass into fuels and commodities [16], [17]. We focused on up-regulated CAZyme genes and determined how they are organized into regulons that respond to specific
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