ABSTRACT We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. 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 mixture 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 for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.