Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme.

Bradley J Stevenson, Jian-Wei Liu, Philip W Kuchel, David L Ollis

Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia. Bradley.Stevenson@csiro.au

Journal of biotechnology 2012 Jan

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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. Copyright © 2011 Elsevier B.V. All rights reserved.

Citation

Bradley J Stevenson, Jian-Wei Liu, Philip W Kuchel, David L Ollis. Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme. Journal of biotechnology. 2012 Jan;157(1):113-23