(686e) Installing the Wood-Ljungdahl Pathway in Clostridium Acetobutylicum

The ability to express the genes necessary for instating a functional Wood-Ljungdahl (WL) pathway into C. acetobutylicum constitutes a major advance in that the WL pathway has never before been instated into a heterologous organism.  In the specific case of C. acetobutylicum, it would allow one to grow this organism to high cell densities and then switch the available carbon substrate from a sugar to CO_­2/H₂, CO/CO₂, or a mixotrophic culture with both sugars and gases, thereby enabling the production of carboxylic acids and solvents from syngas in a flexible and scalable fermentation system.  From the perspective of biological fundamentals, the ability to install this complex, primordial pathway into a heterologous host would constitute a major advance in cell/metabolic engineering that would open new horizons for pathway engineering and synthetic approaches in the genus Clostridium. Comparative genetic analysis of three sequenced acetogens, C. ljungdahlii, C. carboxidivorans, and C. difficile, showed that the WL pathway genes of these clostridial acetogens are concentrated in a highly conserved, 18 kilobase region on their respective genomes.  By examining the C. acetobutylicum genomes, we focused on expressing 11 core genes coding for enzymes and accessory proteins.  To achieve this goal, we developed a system of two co-existing plasmids combined with a method to integrate some of these genes, starting with the formate dehydrogenase gene, into the chromosome.  We will present the new chromosomal integration method and the step-by-step verification of the expression and function of the cloned genes/proteins as well as culture experiments to test the functional installation of the WL pathway in C. acetobutylicum.