1a. Objectives (from AD-416):
To characterize cellulose degradation by three different species of cellulose-degrading ruminal bacteria, using transcriptomic and electron microscopic methods.
1b. Approach (from AD-416):
We will investigate the cellulolytic strategies of three ruminal bacteria, including the cell-associated complexed cellulase system of Ruminococcus albus 7, the cell-associated non-complexed cellulase system of Fibrobacter succinogenes S85, and the cell-free cellulase system of the actinobacterium Micromonospora ruminantium. Specifically, we will identify the genetic networks that underpin cellulose degradation using transcriptomics, employ functional genomics to predict protein-protein interactions associated with cellulases, and further validate these predictions using cell-free expression. We will use these approaches to characterize monocultures of these bacteria grown on bioenergy feedstocks, in addition to co-cultures of these bacteria with the hemicellulolytic bacteria Prevotella ruminicola 23 or Butyrivibrio proteoclasticus B316. By investigating these different strategies of cellulose degradation, we will gain insights into the fundamental properties that may unify their cellulolytic activity. Moreover, because these bacteria are thought to have co-evolved in the same environment, their interactions with hemicellulolytic bacteria may serve as useful models that could potentially translate into effective approaches for industrial applications.
3. Progress Report:
This project relates to Objective 4 of the parent project: Develop technologies to enable commercially viable consolidated bioprocessing (CBP) of lignocellulosic biomass to fuel ethanol and adhesive co-products. In cows, both Ruminococcus (R.)albus and Fibrobacter (F.) succinogenes primarily degrade cellulose for energy and produce acetate as a byproduct which can be used by the host cow. R. albus is one of the few organisms known to ferment cellulose to ethanol in vitro when incubated at rumen temperatures. On the other hand, F. succinogenes is the only cellulolytic bacterium known to produce succinate as its primary end product. In collaboration with a non-ARS institution, an ARS scientist in Madison, Wisconsin obtained the complete genome sequence of both R. albus strain 7 and F. succinogenes S85. These are currently the only completed genomes of any ruminal cellulolytic bacteria. The mechanism of cellulose degradation by R. albus 7 is not well-defined, but is thought to involve special proteins for attachment, unique carbohydrate-binding regions, a thick outer matrix made up of polysaccharides and proteins, and unique cell-surface structures that bind and degrade cellulose. The genome sequence of R. albus 7 was compared to those of other members of the Clostridiales family known to produce cellulosomes (unique protein structures for degrading cellulose), and to the genomes of nonfiber-degrading anaerobic bacteria. Unlike other members of these groups, R. albus 7 did not contain genes for typical cellulosome components. The fiber-degrading capabilities of R. albus 7 were further investigated, using a combination of fermentation analyses and whole transcriptome sequence-based (RNA-seq) gene analyses. R. albus 7 was found to be capable of fermenting a wide range of polysaccharides (not just cellulose) to ethanol. When grown on cellulose or on the soluble sugar, cellobiose, in vitro to obtain the same bacterial growth rate, R. albus 7 utilized a carbohydrate-degrading strategy that involves overexpression of two unique genetic regions that are not overexpressed in other cellulose-degrading bacteria. These findings indicate this unusually efficient cellulose degrader employs novel strategies of carbohydrate degradation, which may suggest new approaches to enhance industrial fermentation of cellulose to ethanol. The ARS scientist also began characterization of the cellulose degradation strategy of F. succinogenes S85. Its cellulose-degrading system involves neither cellulosomes nor excreted cellulases. In order to explore the effects of growth rate and growth substrate on this strain, cultures were grown in controlled in vitro systems on cellulose and on cellobiose, each at three different growth rates. Samples from these cultures have been prepared for RNA-seq analysis to quantitatively characterize gene expression to determine if the type of carbohydrate affects the degree to which each of its 3,085 genes are expressed.