Journal
JOURNAL OF BIOTECHNOLOGY
Volume 157, Issue 1, Pages 113-123Publisher
ELSEVIER
DOI: 10.1016/j.jbiotec.2011.09.019
Keywords
Adenosine triphosphate; Glucose; Fermentation; Cell-free; Embden-Meyerhof pathway; P-31 NMR spectroscopy
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Funding
- Australian Research Council
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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. (C) 2011 Elsevier B.V. All rights reserved.
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