Abstract
Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.
Highlights
The utilization of lignocellulosic biomass for production of liquid fuels is an important and promising alternative energy production
Through analysis of the draft genome sequence of R. albus 8, we previously uncovered many genes predicted to be involved in cellulose depolymerization, including 17 putative endoglucanases, three putative cellobiohydrolases, and two putative cellobiose phosphorylases
The structure of the R. albus 8 enzymes are similar to those found in other Firmicutes, including the Cel9E and Cel9V of Clostridium cellulolyticum, except for the presence of dockerin domains in place of the CBM3730
Summary
The utilization of lignocellulosic biomass for production of liquid fuels is an important and promising alternative energy production. Multiple cellulolytic and hemicellulolytic enzymes are physically linked to a tethering protein scaffold, termed scaffoldin Both catalytic domains and non-catalytic accessory modules are linked to facilitate binding to the substrate and are active against a variety of substrates such as cellulose, xylan, and pectin[10]. Glycoside hydrolases (GH), the enzymes responsible for the cleavage of glycosydic bonds are grouped into families on the basis of amino acid sequence in the Carbohydrate Active enZYme (CAZY) database[16,17]. These enzymes have different structural folds, different mechanisms, and a wide range of substrates[16,18]. Most of the GH9 family enzymes have low or no activity on crystalline cellulose, but have activity on soluble cellulose derivatives, including carboxymethyl cellulose and phosphoric acid swollen cellulose[21,22]
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