The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. I n cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640mU/mg), phosphoribose isomerase (1600mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action Of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO2 and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly.

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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.