This project addresses the primary obstacle to realizing the significant potential of thermophilic bacteria for ethanol production: low product selectivity (yield of ethanol/yield of products other than ethanol). Three tasks are to be carried out. Task 1 involves cloning Clostridium thermocellum target genes (acetate kinase and/or phosphotransacetylase, lactate dehydrogenase) in Escherichia coli, deleting a portion of the target genes to render the corresponding enzymes non-functional, and preparing plasmid constructs for reintroduction of the deleted genes back into C. thermocellum. Task 2 involves development of an electroporation protocol to introduce deleted genes into C. thermocellum on a non-replicative plasmid. Task 3 utilizes continuous culture to simultaneously verify strain stability, evaluate ethanol tolerance, and examine the metabolic and/or kinetic impact(s) of pathway manipulation. This project is novel in the context of the emerging field of pathway (or metabolic) engineering because it: (1) uses a host organism for which knowledge of genetics is not well developed; (2) uses gene deletion to manipulate flux in a branched pathway rather than overexpression; and (3) does not require a replicative plasmid or expression of plasmid-borne genes.
