Construction of C5~C8 Carboxylic Acids Producing Metabolic Pathway in Escherichia coli

C5~C8 carboxylic acids producing bacterium, Megasphaera genus, was isolated from rumen of cattle. The isolate produced hexanoic acid mainly as well, produced pentanoic acid(C5), heptanoic acid (C7), and octanoic acid (C8) according to short chain fatty acid added as an electron acceptor. In order to investigate genes and metabolic pathway related to carbon elongation of fatty acids such as pentanoic acid, hexanoic acid, heptanoic acid, and octanoic acid, whole genome and RNA expression level of the isolate were analyzed by pyrosequencing method. Metabolic pathways were postulated and several genes related to fatty acid production were selected. Among them, shown high RNA expression level, 7 genes, acetoacetyl CoA acetyl transferase, hydroxybutyryl CoA dehydrogenase, hydroxybutyryl CoA dehydratase, butyryl CoA dehydrogenase, electron transfer alpha unit, electron transfer beta unit, and acetyl CoA transferase were cloned using two plasmids containing different origin and expressed into genetically modified Escherichia coli BL21 (DE3). Transformed E. coli was cultivated in rich medium containing 20 g/L glucose, anaerobically. In addition to this, effect of external electron acceptors such as acetate, propionate, and butyrate was evaluated at a same time. Expression of inserted genes was confirmed by SDS page and metabolites were analyzed by GC and GC-MS. The metabolites were compared with control condition and produced metabolites were quantified.