We describe here a useful metabolic engineering tool to optimize expression levels for multiple genes, Multiple-Gene Promoter Shuffling (MGPS). This method approaches an optimized gene over expression level by fusing varied strength promoters to genes of interest for a particular pathway. The selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation by shuffling the promoters for gnd2 and hxk2 with the genes for transaldolase (TAL1), transketolase (TKL1) and pyruvate kinase (PYK1) in the Saccharomyces cerevisiae strain FPL-YSX3. This host strain has integrated xylose metabolizing genes including xylose reductase, xylitol dehydrogenase and xylulose kinase. The optimal expression levels for TAL1, TKL1 and PYK1 were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be GND2-TAL1-HXK2-TKL1-HXK2-PYK1. The MGPS method could easily be adapted into other eukaryotic and prokaryotic organisms, to optimize expression of genes for industrial fermentations.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|