Production of Methyl Ketones in E. Coli

The fatty acid synthesis pathways provide a very rich framework for the synthesis of molecules with various chemical structures and carbon chain lengths. The goal of this study is to design and construct efficient Escherichia coli host strains and expression systems and to examine various operating conditions to achieve high level production of fatty acid-like molecules from glucose. In particular, the current study foucses on the production of methy ketone.

Methyl ketone synthase (MKS), an enzyme found in tomato which catalyzes the hydrolysis and decarboxylation reaction of ß-ketoacyl–acyl-carrier-proteins (intermediates in fatty acid biosynthesis), was expressed in E. coli. To examine the effect of expression level on overall culture performance, the expression of MKS was put under the control of a trp or lac promoter system. Various mutant E. coli strains were also constructed to direct carbon fluxes from glucose to the desired product by eliminating competing pathways. These mutant strains were confirmed using genomic PCR techniques. Fermentation products were analyzed using HPLC and methyl ketones were measured using GC-FID and confirmed using GC-MS. Results from these studies will be presented and discussed.