Our research group is located at the Genoscope, the french national genomics center, which is part of the Genopole in Evry, France. We are affiliated with the Université de Paris Saclay system and have had students from the Université d'Evry, AgroParisTech, Université de Lyon, and the Université Pierre et Marie Curie.

Evolving clostridia as plant-fermenting biocatalysts

Advances in high-throughput screening of biomolecules and genome engineering promise to revolutionize medicine, agriculture, and production of renewable energy. Our research focuses on developing these technologies to study and engineer a group of plant-fermenting bacteria with important applications in human and environmental health, the clostridia. These gram-positive anaerobes are dominant members of the human gut microbiome (El Kaoutari et al, 2013), which ferments indigestible plant fiber to provide 5-10% of calories (McNeil, 1984) and protects against ailments such as inflammatory bowel and Crohn's disease. As such, Clostridia are gut microorganisms that interact with the human host in ways that are mutually beneficial, thereby enhancing our genetic and metabolic attributes. Clostridia also contribute significantly to recycling of plant biomass in soil, a key part of the global carbon cycle that balances atmospheric CO2. As humans currently only use 2% of cellulosic biomass (Pauly & Keegstra, 2008), it is a vast potential industrial feedstock and clostridia are top candidates to transform biomass into fuels and commodities (Lynd et al, 2002). Our goals are to create new approaches to study these bacteria and translate this knowledge into useful applications in medicine, environmental management, and renewable energy.

Our primary model is Clostridium phytofermentans (Warnick et al, 2002, Tolonen et al, 2013), an anaerobic, mesophile that ferments diverse plant polysaccharides (cellulose, hemicelluloses, pectins, and starch) to ethanol and hydrogen. The C. phytofermentans genome encodes 171 carbohydrate-active enzymes (CAZymes), highlighting the elaborate set of enzymes needed to breakdown diverse plant components. We combine computational and experimental methods including DNA sequencing (Petit et al, 2015; Tolonen and Zuroff, et al, 2015), RNA sequencing (Boutard et al, 2014), and mass spectrometry (Tolonen et al, 2011; Tolonen & Haas, 2014) to study how these bacteria ferment plant polymers and interact with their environment (Fig 1). In parallel, we are developing tools (Fig 2) to explore the genetic basis of plant fermentation (Tolonen et al, 2009; Tolonen et al, 2015) and to create strains with novel properties.

Fig 1 Systems biology approaches identify key enzymes and enable the reconstruction of metabolic pathways used to ferment plant biomass (A from Tolonen et al, 2011; B from Boutard et al, 2014).

Fig 2 The genetic basis of important adaptions in clostridia. Plasmid DNA can be efficiently transferred to C. phytofermentans: a replicating plasmid pQexp enables heterologous gene expression and A pQint bearing a designed group II intron can be B retargeted to insert anywhere in the chromosome. C Gene inactivation made a mutant strain (AT02-1) that grows normally on most carbon sources, but has completely lost the ability to degrade cellulose (Fig 2C), revealing the central role of this gene in cellulose degradation. (Images from Tolonen et al, 2009).