Escherichia coli was engineered for the production of even-and odd-chain fatty acids (FAs) by fermentation. Co-production of thiolase, hydroxybutyryl-CoA dehydrogenase, crotonase and trans-enoyl-CoA reductase from a synthetic operon allowed the production of butyrate, hexanoate and octanoate. Elimination of native fermentation pathways by genetic deletion (DldhA, DadhE, DackA, Dpta, DfrdC) helped eliminate undesired by-products and increase product yields. Initial butyrate production rates were high (0.7 g l "1 h "1 ) but quickly levelled off and further study suggested this was due to product toxicity and/or acidification of the growth medium. Results also showed that endogenous thioesterases significantly influenced product formation. In particular, deletion of the yciA thioesterase gene substantially increased hexanoate production while decreasing the production of butyrate. E. coli was also engineered to co-produce enzymes for even-chain FA production (described above) together with a coenzyme B 12 -dependent pathway for the production of propionyl-CoA, which allowed the production of odd-chain FAs (pentanoate and heptanoate). The B 12 -dependent pathway used here has the potential to allow the production of odd-chain FAs from a single growth substrate (glucose) in a more energy-efficient manner than the prior methods.

Dependence on petroleum for fuels is a well-known issue in the United States today. However, petroleum is also used to produce a broad range of chemicals that are used in applications such as plastics, fragrances, surfactants, detergents, food additives, and pharmaceuticals. As the world's supply of petroleum dwindles, we must look to another method of procuring these chemicals. Biorenewable chemical production attempts to fill this void. Shortchain fatty acids and ketones are desirable precursors to many of these industrially relevant chemicals. Short-chain fatty acids are precursors to alpha-olefins, which are used as lubricants and surfactants in a variety of industries, including the automotive industry. They can also be used as precursors to fatty alcohols, which have potential applications as biodiesel. Methyl ketones are also a class of chemicals with many industrial applications. Butanone is a common industrial solvent, while 4-hydroxybutanone is used in pesticides, terpenoids, and most importantly, is an intermediate in the production of doxorubicin, an anticancer agent. Here we report the biorenewable production of short-chain fatty acids and methyl ketones from fermentation in Escherichia coli. A series of synthetic constructs were made to produce the desired metabolites utilizing glucose as the feedstock. Butyrate was produced at 9.670 g/L, hexanoate at 1.963 g/L, and octanoate at 0.216 g/L. In addition, 0.201 g/L of valerate was produced. Heptanoate production by fermentation in E. coli was reported for the first time, reaching a titer of 0.008 g/L. 4-hydroxy-2-butanone was produced by fermentation at a titer of 2.5 mM. To our knowledge this is the first report of production of 4-hydroxy-2-butanone by microbial fermentation.