Regulatory mutations that allow the growth of Escherichia coli on butanol as carbon source

Starting with adhC mutants of Escherichia coli in which alcohol dehydrogenase (ADH) and acetaldehyde CoA dehydrogenase (ACDH) are expressed constitutively at high levels, we selected mutants with still higher levels of both enzymes. Selection for growth on ethanol in the presence of inhibitors of ADH gave several mutants that had from 2- to 10-fold increases in the levels of both enzymes. These mutations were found to map far from the adhC locus at around 90 min. Such adhR mutants were unable to grow on acetate or ethanol in certain media unless supplemented with extra manganese. This growth disability was suppressed by secondary mutations, one of which, aceX, increased sensitivity to several toxic metals and may perhaps derepress Mn transport. When the adhR mutation expressing the highest ADH and ACDH levels was present together with fadR and atoC mutations (allowing efficient catabolism of acetoacetyl-CoA) and with an aceX mutation, the resulting strains became capable of using n-butanol as sole carbon and energy source. The use of butanol by E. coli illustrates the artificial evolution of a new catabolic pathway, in this case by the selection of four successive regulatory mutations (fadR, adhC, atoC, and adhR) together with the poorly defined aceX mutation. Each stage in the acquisition of this novel pathway confers the ability to use a new growth substrate: decanoic acid (fadR), ethanol (adhC), butyric acid (atoC), and butanol (adhR, when present with aceX).