The reaction sequence used was similar to the butyrate
fermentation of Clostridium to the point of butyryl-CoA
formation. Two acetyl-CoA are condensed to acetoacetylCoA,
and then converted in three steps to butyryl-CoA.
However, the final step was the hydrolysis of butyryl-CoA to
HS-CoA and butyrate by the TesB thioesterase, which differs
from the clostridial pathway. The enzymes used for butyrate
production in E. coli came from diverse sources and were
mainly chosen based on studies aimed at optimizing 1-
butanol production, which proceeds via a similar pathway
to the point of butyryl-CoA