Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme |
| |
Authors: | Stevenson Bradley J Liu Jian-Wei Kuchel Philip W Ollis David L |
| |
Affiliation: | a Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia b Singapore Bioimaging Consortium, Biomedical Sciences Institutes, 11 Biopolis Way, #02-02 Helios, Singapore 138667, Singapore |
| |
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. |
| |
Keywords: | 13BPG, 1,3-bisphosphoglycerate 2PG, 2-phosphoglycerate 3PG, 3-phosphoglycerate Adh, alcohol dehydrogenase CFPS, cell-free protein synthesis DHAP, dihydroxyacetone phosphate DTT, dithiothreitol Eno, enolase F6P, fructose 6-phosphate Fba, fructose-1,6-bisphosphate aldolase FBP, fructose 1,6-bisphosphate G6P, glucose 6-phosphate GAP, glyceraldehyde 3-phosphate Gapdh, glyceraldehyde-3-phosphate dehydrogenase Glk, glucokinase Gpi, glucose-6-phosphate isomerase PEP, phospho-enol-pyruvate Pdc, pyruvate decarboxylase PIPES, piperazine-N,N&prime -bis(2-ethanesulfonic acid) Pfk, 6-phosphofructokinase Pgk, phosphoglycerate kinase Pgm, phosphoglycerate mutase Pi, inorganic phosphate Pyk, pyruvate kinase TEP, triethylphosphate Tpi, triose-phosphate isomerase TPP, thiamine pyrophosphate |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|