title: Screening for enhanced triacetic acid lactone production by recombinant Escherichia coli expressing a designed triacetic acid lactone reporter.
abstract: Triacetic acid lactone (TAL) is a signature byproduct of polyketide synthases (PKSs) and a valuable synthetic precursor. We have developed an endogenous TAL reporter by engineering the Escherichia coli regulatory protein AraC to activate gene expression in response to TAL. The reporter enabled in vivo directed evolution of Gerbera hybrida 2-pyrone synthase activity in E. coli . Two rounds of mutagenesis and high-throughput screening yielded a variant conferring ~20-fold increased TAL production. The catalytic efficiency (kcat/Km) of the variant toward the substrate malonyl-CoA was improved 19-fold. This study broadens the utility of engineered AraC variants as customized molecular reporters. In addition, the TAL reporter can find applications in other basic PKS activity screens.
authors.affiliation: Department of Chemical Engineering, Pennsylvania State University, 226A Fenske Laboratory, University Park, Pennsylvania 16802, USA.
authors.forename: Shuang-Yan
authors.initials: SY
authors.lastname: Tang
authors.forename: Shuai
authors.initials: S
authors.lastname: Qian
authors.forename: Olubolaji
authors.initials: O
authors.lastname: Akinterinwa
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Frei
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Gredell
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Cirino
mesh_terms: D051777:AraC Transcription Factor / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D015202:Protein Engineering; D011753:Pyrones / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051777:AraC Transcription Factor; D011753:Pyrones; D011994:Recombinant Proteins; C030720:triacetic acid lactone