Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12.

The genes xylA and xylB were cloned together with their promoter region from the chromosome of Klebsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xylA encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated M(r) of 49,793). The gene xylB encodes the enzyme xylulokinase (XK or XylB) with a calculated M(r) of 51,783 (483 amino acids). The two genes successfully complemented xyl mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylAKp 5' upstream region in high copy number (but lacking an active xylB gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5' upstream regions of xylA on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.