The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose-1) and a maximum growth rate of 0.21 h -1. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose. Copyright © 2009, American Society for Microbiology. All Rights Reserved.