title: Altered glucose transport and shikimate pathway product yields in E. coli.
abstract: Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Yi
authors.forename: K M
authors.initials: KM
authors.lastname: Draths
authors.forename: Kai
authors.initials: K
authors.lastname: Li
authors.forename: J W
authors.initials: JW
authors.lastname: Frost
mesh_terms: D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics*
chemical_list: D009097:Multienzyme Complexes; C096387:3-dehydroshikimate; D012765:Shikimic Acid; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D005941:Glucokinase; D005947:Glucose