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C. japonicus growing on a homogenized biomass plate

The degradation of polysaccharides is critical in a variety of contexts, specifically environmental, nutritional, and biotechnological. For example, the complex polysaccharides that comprise plant cell walls or crustacean shells are abundant terrestrial and marine sources of carbon for the growth of microorganisms. On the order of 100 billion tons of these polymers are turned over every year as an essential arc in the carbon cycle. Microbial activities in both terrestrial and marine environments are the main driver of this turnover. In addition, the microbial flora in human digestive tracts contributes nutritionally with the breakdown of the plant-derived polysaccharides eaten. It is estimated that 10% of your daily caloric intake comes from the short chain fatty acids that are the byproducts of cellulose degradation in your gut. Finally, polysaccharide degradation is critical biotechnologically because it is currently one of the more expensive parts of biofuel production. If low-cost biofuels are to compete with fossil fuels, we need to understand how these polymers are efficiently degraded.

The polysaccharides found in plant cell walls and crustacean shells are difficult to degrade (recalcitrant), and this is due to several factors: (1) structural and chemical complexity of the polymers, (2) the overall insolubility of the polymers, and (3) toxic moieties of some plant cell wall constituents. Collectively these traits prevent efficient depolymerization and subsequent sugar utilization of polysaccharides by the vast majority of microorganisms in the environment.

Research in the Gardner laboratory uses an interdisciplinary approach including systems biology (transcriptomics and proteomics), classical bacterial genetics (targeted and random gene disruptions), and biochemistry (enzyme purification and assay) to understand the regulation and mechanisms of recalcitrant polysaccharide degradation by bacteria. We use the bacterium Cellvibrio japonicus because of the sophisticated genetic tools to manipulate the microorganism, and because this bacterium has the incredible ability to completely depolymerize both plant cell wall and crustacean shell polysaccharides to obtain carbon and energy. We have four main questions that drive our research:

(1) How do bacteria sense the environment and detect recalcitrant polysaccharides?

The crystalline and hydrophobic nature of the polymers that comprise plant cell wall and crustacean shell polysaccharides make them largely insoluble and inaccessible to most bacteria. Our transcriptomic data shows that C. japonicus is not only able to detect these polymers, but can alter the composition of degradative enzymes produced over time as it depolymerizes the substrate. Currently, our lab is dissecting what signals cause these physiological and metabolic changes necessary for insoluble polysaccharide degradation.

(2) What proteins are needed for complete consumption of recalcitrant polysaccharides?

There are several hundred genes predicted to be involved in degrading plant cell wall and crustacean shell polysaccharides in C. japonicus. However, it is unclear which genes encode proteins with functions that are absolutely critical for degradation. Work in our lab has identified several gene products that are essential for the degradation of the plant polymer cellulose. Through mutational analysis and enzymatic assay we are expanding our understanding of what proteins are essential for plant cell wall and crustacean shell polysaccharide degradation in C. japonicus.

(3) How is the process of recalcitrant polysaccharide detection, degradation, and consumption regulated?

With several hundred proteins to synthesize and export to degrade plant cell wall and crustacean shell polysaccharides, it is unclear how this process is regulated. Our transcriptomic and genetic data suggest that degradation is done in a coordinated manner, and we have identified several pathways critical for plant cell wall degradation. Further analysis of the physiological and metabolic changes that take place during the course of recalcitrant polysaccharide degradation will elucidate how this complex process unfolds. We have begun to use computational biology and bioinformatic approaches to determine suites of co-regulated genes.

(4) How can we use our understanding of recalcitrant polysaccharides degradation for industrial applications?

We have previously engineered an ethanol production pathway into C. japonicus and shown that when grown on the plant polymer cellulose, C. japonicus can produce bio-ethanol.  Current work in our lab uses synthetic biology approaches to examine the possibility of incorporating novel metabolic pathways into bacteria with useful industrial properties. Examples of current work are the production of biofuels beyond bio-ethanol, and synthesis of value added chemicals such as nutritional supplements.