title: Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme.
abstract: Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD(+) and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major 'flux controlling' enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. Bradley.Stevenson@csiro.au
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Stevenson
authors.forename: Jian-Wei
authors.initials: JW
authors.lastname: Liu
authors.forename: Philip W
authors.initials: PW
authors.lastname: Kuchel
authors.forename: David L
authors.initials: DL
authors.lastname: Ollis
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis* / Q000378:metabolism; D019149:Bioreactors; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000431:Ethanol; D000255:Adenosine Triphosphate; D005947:Glucose