Purification and characterization of the d-xylose isomerase gene from Escherichia coli

A DNA fragment containing both the Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene and the d-xylulokinase (ATP: d-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The d-xylose isomerase gene was separated from the d-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The d-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the d-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first d-xylose isomerase gene that has been isolated and extensively purified from any organism. d-Xylose isomerase, the enzyme product of the d-xylose isomerase gene, is responsible for the conversion of d-xylose to d-xylulose, as well as d-glucose to d-fructose. It is widely believed that yeast cannot ferment d-xylose to ethanol primarily because of the lack of d-xylose isomerase in yeast. d-Xylose isomerase (also known as d-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the d-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting d-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology.