Integration of fermentation and energy conservation pathways in thermophilic, lignocellulolytic clostridia and related anaerobes.

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I have been working (Genome Canada project, end date Sept. 2014) with anaerobic bacteria capable of converting forestry and agricultural plant-waste biomass into biofuels such as ethanol and hydrogen, mainly Thermoanaerobacter thermohydrosulfuricus and Clostridium thermocellum. Their ability to degrade cellulose and/or hemicellulose to ethanol has made these “organisms-of-interest” for biofuels production from these types of waste biomass. However, the flow carbon and electrons to ethanol competes with fermentation branches that produce less desirable end-products. The genome sequence of these organisms is known. However, the products of few key genes have been characterized biochemically, and the assignment of function mainly being proposed on the basis of sequence homology. Also, some expected reactions appear to be lacking or non-conventional. Proteomic and transcriptomic ('omic) analyses performed by my group for these organisms have shown that several gene products are either over- or under-expressed under the growth conditions tested. This leads to questions as to the actual function of key gene products and co-factors involved in the fermentation of components of this biomass. Such knowledge is crucial for accurate modelling and rational target selection for genetic engineering to improved ethanol yields from cellulose. Based on 'omics analyses, this NSERC Discovery research program proposes to study the integration of fermentation pathways and energy conservation in these organisms. Using expression vectors in E. coli, proteins corresponding to putative ATP and pyrophosphate dependent catabolic genes will be purified and characterized to confirm enzymatic function and co-factor use. Together with my students, I will also study the effect of specific gene knockouts on the flow of carbon and energy, as well as on the regulation of protein expression involved in specific fermentative branches. For this, T. thermohydrosulfuricus will be used since it is amenable for the development of techniques for gene insertion and knockout. This organism’s genome contains sequences consistent with different types of hydrogenases, membrane- and soluble PPiase, ATP and PPi dependent pyruvate dikinase and –phosphofructokinase, as well as multiple alcohol dehydrogenases that may be involved in the conversion of the biomass to ethanol and hydrogen. This apparent functional redundancy makes this a useful model organism in which to study the relative importance of these enzymes using knock-out mutants. Nevertheless, the first step towards such experiments is to develop a genetic system based on procedures developed for other Thermoanaerobacters. We have not been able to observe fermentation to exclusively hydrogen plus CO2 plus acetate in these organisms, most likely because of hydrogen supersaturation of the medium. As a consequence, we have not observed significant expression of key enzymes expected to be aaociated with hydrogen production in either of these organisms, for example the proton-translocating (Ech) hydrogenase or sodium translocating RNF-like ferredoxin-NADH oxidoreductase. Having had success with the analysis of mixed transcriptomes from co-cultures, we propose to perform proteomic analyses during cellobiose fermentation in the presence of hydrogen removing Methanothermobacter thermoautotrophicus. This is expected to drive hydrogen production from our organisms of interest, and provide insights into the regulation of the fermentation branches leading to hydrogen relative to ethanol and other competing fermentation products. At the end of the proposed research program, my students will have gained expertise in biochemisty, molecular biology and bioinformatics, which are all important tools to master for further employment.