Expanding the chemical diversity of microbial fermentation products enables green production of fuel, chemicals, and pharmaceuticals. In recent years, coenzyme A (CoA) dependent chain elongation, resembling the reversed β-oxidation pathway, has attracted interest for its use in producing higher alcohols, fatty acids, and polyhydroxyalkanoate. To expand the chemical diversity of this pathway, we metabolically engineered Escherichia coli to produce 2-pentanone, which is not a natural fermentation product of E. coli. We describe the first demonstration of 2-pentanone synthesis in E. coli by coupling the CoA-dependent chain elongation with the acetone production pathway. By bioprospecting for enzymes capable of efficient hydrolysis of 3-keto-hexanoyl-CoA, production of 2-pentanone increased 20 fold, reaching a titer of 240 mg/L. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3167–3175, 2013
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