Directed evolution of glycolytic enzymes : glyceraldehyde-3-phosphate dehydrogenases

Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) performs a vital step in glycolysis. An in vitro fermentative system is being developed to convert glucose to ethanol. The system is not cofactor neutral, so will eventually stall through production of adenosine triphosphate (ATP). The Gapdh catalysed step can be modified by use of a non-phosphorylating variant (GapN) of the enzyme. GapN catalyses the Gapdh oxidation and the next step of the pathway without the production of ATP through incorporation of phosphate that normally occurs. This results in the pathway producing ethanol and carbon dioxide from glucose, with complete cofactor recycling. This also gives an overall thermodynamic advantage to the pathway. A GapN enzyme from the thermophile Thermoproteus tenax was examined for suitability to this role, and was modified to increase activity under mesophilic conditions through application of directed evolution techniques. Methods for evolving this enzyme were developed and are described. Two mutants from the evolution are characterised. Both possess a threefold enhanced activity at 30{u00B0}C and one and a half to tenfold improvement at 50{u00B0}C. One suggests a change in optimal active temperature, while the other has improved activity under all conditions tested. The first heat activation of GapN was observed in both the wild type and the mutants, with a sevenfold improvement in wild-type activity and a fourfold and modest improvement in the mutants. This phenomenon is not commonly reported outside of heat-response proteins. Alternative screening methods were investigated for the evolution. Primarily an E. coli strain lacking the gene gapA, that encodes Gapdh, was to be used for screening. There were significant difficulties knocking out gapA that are now believed to be caused by non-glycolytic roles of Gapdh in the cell. A second evolution was performed with a mutant of the E. coli Gapdh possessing a cysteine 149 to alanine mutation. This mutation causes a GapN like activity in Gapdh, though it is understood to occur by a different mechanism and to use a different isomer of the substrate for activity. Following two rounds of evolution an enzyme with a wild-type cysteine at position 149 was selected. This suggests potential problems with the C149A mutant as an evolution target. A third Gapdh variant from Zymomonas mobilis was investigated for possible use in the pathway, or as a possible target for evolution. This variant has not been previously characterised. The first kinetic characterisation and crystal structures are reported. The enzyme is kinetically interesting for its strong substrate binding of all substrates with Km at low micromolar ranges and a turnover of 16 s-1 that could be attributed to the environment of Z. mobilis. The best crystallographic data collected have given a structural solution to 3.6 A