Engineering a homobutanol fermentation pathway in Escherichia coli

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B using genes from the bacterium Clostridium acetobutylicum ATCC 824. The pathway genes that encode for acetoacetyl-CoA (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned into pUC19 resulting in plasmid pEAG13. All of the competing fermentation pathways were eliminated in E. coli B by chromosomal gene deletions. The resulting strain E. coli EG03 ( DeltafrdABCD DeltaldhA DeltaackA DeltapflB Delta adhE DeltapdhR ::pflBp6-acEF-lpd DeltamgsA) lost the ability to grow and ferment glucose anaerobically. When pEAG13 was transformed into E. coli EG03, a homobutanol pathway was established, which converted glucose to butanol (~16 mM).