title: Engineering Escherichia coli for production of C₁₂-C₁₄ polyhydroxyalkanoate from glucose.
abstract: Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C₁₂ and C₁₄ fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW--the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.
authors.affiliation: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Agnew
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Stevermer
authors.forename: J Tyler
authors.initials: JT
authors.lastname: Youngquist
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D054813:Polyhydroxyalkanoates / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D005947:Glucose