title: Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli.
abstract: We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.
authors.affiliation: Graduate School of Science, Technology, and Innovation and ‡Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Graduate School of Science, Technology, and Innovation and ‡Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
authors.forename: Kou
authors.initials: K
authors.lastname: Furuta
authors.affiliation: Graduate School of Science, Technology, and Innovation and ‡Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Graduate School of Science, Technology, and Innovation and ‡Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University , 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D000085:Acetates / Q000378:metabolism; D000123:Acetyltransferases / Q000378:metabolism; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D060847:Metabolic Engineering; D010709:Phosphate Acetyltransferase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
chemical_list: D000085:Acetates; D001426:Bacterial Proteins; D007544:Isopropyl Thiogalactoside; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D010709:Phosphate Acetyltransferase; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: Escherichia coli; enzyme ligation; metabolic engineering; sortase A