Cloning and Over-Expression of the E. coli D-Xylose Isomerase Gene in Various Microorganisms
D-Xylose isomerase, also known as D-glucose isomerase, catalyzes the conversion of D-xylose to D-xylulose and D-glucose to D-fructose. Both reactions have important industrial applications. Recently, we have succeeded in the identification of a ColEl-hybrid plasmid containing the Escherichia coli xylose isomerase gene (xylA) from the Clarke and Carbon E. coli gene bank. Subsequently, through subcloning and restriction mapping, xylA was found to be located on a 1.6 kb Bglll fragment. We found that the intact E. coli xylA gene was not expressed in yeast (Saccharomyces cerevisiae) or Bacillus subtilis. Fusion of the xylA structural gene to the yeast TRP5 or ADH1 promoter did not result in the expression of the hybrid gene in yeast. However, through lacZ fusion, we have been able to demonstrate that the TRP5-xylA hybrid gene did contain the necessary genetic elements to direct the synthesis of a protein in yeast. On the contrary, fusion of the xylA structural gene to a promoter functional in B. subtilis resulted in the expression of the xylA structural gene in the latter organism.
KeywordsRibosomal Binding Site Xylose Isomerase lacZ Fusion xylA Gene Trp5 Promoter
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