title: Creation of a shikimate pathway variant.
abstract: The competition between the Escherichia coli carbohydrate phosphotransferase system and 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase for phosphoenolpyruvate limits the concentration and yield of natural products microbially synthesized via the shikimate pathway. To circumvent this competition for phosphoenolpyruvate, a shikimate pathway variant has been created. 2-Keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases encoded by Escherichia coli dgoA and Klebsiella pneumoniae dgoA are subjected to directed evolution. The evolved KDPGal aldolase isozymes exhibit 4-8-fold higher specific activities relative to that for native KDPGal aldolase with respect to catalyzing the condensation of pyruvate and d-erythrose 4-phosphate to produce DAHP. To probe the ability of the created shikimate pathway variant to support microbial growth and metabolism, growth rates and synthesis of 3-dehydroshikimate are examined for E. coli constructs that lack phosphoenolpruvate-based DAHP synthase activity and rely on evolved KDPGal aldolase for biosynthesis of shikimate pathway intermediates and products.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Ningqing
authors.initials: N
authors.lastname: Ran
authors.forename: K M
authors.initials: KM
authors.lastname: Draths
authors.forename: J W
authors.initials: JW
authors.lastname: Frost
mesh_terms: D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D012765:Shikimic Acid / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012765:Shikimic Acid; D000446:Aldehyde-Lyases; C022366:6-phospho-2-keto-3-deoxy-galactonate aldolase