P134: Metabolic engineering of E. coli β-oxidation and overexpression of a plant thioesterase for endogenous production of defined mcl-PHA polyester from an unrelated carbon source

Polyhydroxyalkanoates (PHA) are a class of naturally occurring microbial polyesters whose metabolism and material properties have been well characterized owing to their renewable nature and potential to substitute for petroleum derived plastics such as polypropylene. Polyesters with monomers consisting of ≥6 carbons are known as medium chain-length (mcl)-PHA and are industrially relevant, allowing for greater variation in the material properties of the polymer. Production of mcl-PHA is often achieved through feeding of exogenous fatty acids (FA) and related compounds and generally results in a polymer with a heterogeneous monomer composition due to the iterative nature of fatty acid metabolism. However, this method of production is limited by the cost of the FA feedstock and in many cases, the lack of control over the composition of the final product. To overcome these obstacles, we have engineered a strain of Escherichia coli that is capable of growth on a non-FA feedstock (e.g., glucose, acetate) for production of mcl-PHA with a pre-determined monomer composition. Endogenous FA production on glucose was achieved by overexpression of a plant thioesterase (BTE) which is capable of generating a defined pool of free FAs consisting mainly of dodecanoic acid (C12). The resulting free FAs were routed through an engineered β-oxidation cycle toward mcl-PHA via overexpression of PHA biosynthesis genes phaJ and phaC from Pseudomonas aeruginosa. Strain design was informed through experimental analysis of a β-oxidation knockout library which led to insight into the role of fad genes in the presence of exogenous and endogenous free FAs.