Our research focuses on the systems biology and genetic engineering of Clostridia which ferment plant biomass. Clostridia is a group of Gram-positive anaerobic bacteria which play an important role in the environmental carbon cycle and human health. We are studying their molecular biology using high-throughput methods including genome sequencing, expression profiling (transcriptome, proteome and metabolomic) and IT modeling. In parallel, we are developing methods of genome engineering in Clostridia in order to test the predictions deriving from our systems biology studies and create strains with new properties of interest. Our objective is to elucidate the biology of the bacteria and transfer that knowledge to applications in medicine, environmental management and renewable energies.

An interdisciplinary approach to the study of plant fermentation 

Bacteria which ferment plant biomass are important for the environmental carbon cycle, human nutrition and the industrial production of biofuels and renewable products. For example, plant biomass recycling by microorganisms is a major component of the global carbon cycle (Leschine 1995);  intestinal bacteria ferment non-digestible fiber from plants to yield short-chain fatty acids, which constitute 60 to 85% of the calories in ruminants and 5 to 10% in humans (McNeil, 1984). However, only 2% of the cellulose biomass is currently used by man;  the biomass constitutes a vast potential resource that industrial microorganisms could convert into biofuels and bioproducts (Pauly & Keegstra, 2008). Among those microorganisms, Clostridia have the particular interest of being a dominant group in the human intestinal microbiome (El Kaoutari et al, 2013) and they are among the best candidates for industrial processing of cellulose biomass (Lynd et al, 2002). We are developing an integrated approach to elucidate how Clostridia evolved to ferment plant biomass effectively.

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Artistic representation of cellulose degradation 

Systems biology of cellulose fermentation 

Our research is mainly focused on the microorganism model Clostridium phytofermentans, a mesophilic inhabitant of forest soils which ferments components of the biomass (cellulose, hemicelluloses, pectins and starch) to yield ethanol and hydrogen (Warnick et al, 2002;  Tolonen et al, 2013). The genome of C. phytofermentans codes for 171 enzymes that degrade polysaccharides (CAZymes) drawing attention to the complex set of enzymes necessary to transform biomass into sugars. We are looking to elucidate the systems that C. phytofermentans uses to ferment biomass by combining high-throughput measurements of gene expression such as proteomics (Tolonen and Haas, 2014), transcriptomics (Boutard et al, 2014​) and metabolomics with analyses of growth and fermentation, and microscopy (figure 1A). The approach enables identification of key enzymes which enable us to reconstitute the metabolic pathways involved in plant polymer fermentation (Figure 1B). We are currently applying the set of 'omic' data with a view to constructing IT forecasting models to orient our genomic engineering approaches in order to create strains that ferment biomass more effectively.

Fig 1:

A. Systems biology approach for the study of cellulose bioconversion. The cultures metabolizing different biomass substrates are studied with respect to growth, biomass consumption, fermentation products, cell morphology and gene expression. Image by Tolonen et al, 2011. 

B. The physiological and molecular data are integrated in order to identify the key enzymes in the degradation and fermentation of biomass. Image by Boutard et al, 2014.

Genomic engineering of Clostridia

While Clostridia have been studied since the 19th century (Duerre, 2005), a lack of methods for their genetic manipulation has prevented their use in biotechnology. We are developing methods to experimentally manipulate gene expression in Clostridia. Plasmid DNA can be transferred to C. phytofermentans and other Clostridia by inter-species conjugation with E. coli. We have observed that transfer of a replication plasmid pQexp enables the expression of heterologous genes. A plasmid pQint bearing a group II intron (figure 2A) may be targeted for insertion anywhere in the chromosome (figure 2B). In an initial study (Tolonen et al, 2009), we inactivated gene cphy3367 to create a mutant strain (AT02-1) which grows normally on most carbon sources but has completely lost the ability to degrade cellulose (figure 2C). While C. phytofermentans expresses numerous CAZymes on cellulose, a single enzyme is therefore essential for cellulose degradation. We recently observed that mutant strains of C. phytofermentans that have lost 1 of the 2 CAZymes most strongly expressed during growth on cellulose metabolize cellulose and other plant substrates normally but have a reduced ability to hydrolyze chitin substrates and fungi (Tolonen et al, 2014). These enzymes inhibit fungal growth by synergy to hydrolyze chitin, a principal component of the fungal cell wall. C. phytofermentans thus increases its growth on cellulose by lysing fungi with its most strongly expressed hydrolytic enzymes. Currently, we are focusing on the development of genetic engineering tools for high-throughput screening of the mutant and large-scale genome modification of Clostridia.

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Fig 2

A. A plasmid for the inactivation of the genes of C. phytofermentans by a group II intron.

B. The group II intron may be re-targeted by 2-stage PCR for insertion anywhere in the genome. 

C. Inactivation of gene cphy3367 in strain AT02-1 showed that cellulose degradation requires a single enzyme. Image by Tolonen et al, 2009​.