SusChEM: Installing the Wood-Ljungdahl pathway in a clostridium platform organism for enhanced metabolite production

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1511660 Papoutsakis, Eleftherios T. The goal of this project is to engineer the industrial organism Clostridium acetobutylicum to make it capable of utilizing carbon dioxide, carbon monoxide and hydrogen, as sole substrates for growth and product formation. This will enable the production of biofuels (e.g. butanol) and other useful chemicals from waste gases in a fermentation system that can be easily scaled for industrial production, and with exceptional efficiencies. From the fundamental point of view, the ability to install this complex, primordial pathway in a different organism constitutes a major advance in synthetic biology that would open new horizons for pathway engineering and synthetic approaches in other industrial microorganisms. The most efficient pathway for non-photosynthetic chemotrophic carbon utilization is the Wood-Ljungdahl pathway (WLP) employed by acetogens, which are Clostridium organisms that fix enormous quantities of carbon dioxide in the geobiosphere. Comparative genomic analysis of the three sequenced genomes of mesophilic acetogens showed that the WLP genes of all three organisms are concentrated in a highly conserved region in their respective genomes. Based on the published literature and work from the investigator's laboratory, the model acetogen C. ljungdahlii was chosen as the source of most of the WLP genes that will be used to install the WLP in C. acetobutylicum. The plan is to integrate a number of genes into the chromosome and express other genes on two large compatible/co-existing plasmids. Chromosomal integrations will be based on a recently developed technology for double-crossover allelic DNA exchange that enables markerless chromosomal gene deletions and DNA integration. The constructed strains will be assessed for the WLP functionality through in vitro assays, growth experiments and carbon-13 based carbon-fixation assays.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.