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Applied and Environmental Microbiology, August 2008, p. 5031-5037, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00924-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Engineering Pseudomonas putida S12 for Efficient Utilization of D-Xylose and L-Arabinose

TNO-Quality of Life, Business Unit Food and Biotechnology Innovations, Julianalaan 67, 2628 BC Delft, The Netherlands,¹ B-Basic, Julianalaan 67, 2628 BC Delft, The Netherlands,² Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands³

Received 23 April 2008/ Accepted 19 June 2008

The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate^–1], where CDW is cell dry weight), and the growth rate was poor (0.01 h^–1). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose^–1]) at a considerably improved growth rate (0.35 h^–1). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved D-xylose-utilizing strain metabolized L-arabinose as efficiently as D-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.

Published ahead of print on 27 June 2008.