An Escherichia coil strain, ZUC99(pATX210), which can produce xylitol from L-arabinose at a high yield, has been created by introducing a new bioconversion pathway into the cells. This pathway consists of three enzymes: L-arabinose isomerase (which converts L-arabinose to L-ribulose), D-psicose 3-epimerase (which converts L-ribulose to L-xylulose), and L-xylulose reductase (which converts L-xylulose to xylitol). The genes encoding these enzymes were cloned behind the araBAD promoter in tandem so that they were polycistronically transcribed from the single promoter, like an operon. Expression of the recombinant enzymes in the active form was successfully achieved in the presence Of L-arabinose. A xylitol production profile of the recombinant strain was evaluated by shake-flask fermentation. ZUC99(pATX210) produced 2.6 g/l xylitol using 4.2 g/l L-arabinose with a xylitol yield of 0.62 g/g L-arabinose in 36 h. It was determined that utilization of glycerol as a co-substrate significantly improved xylitol production and yield. In the presence of 11.8 g/l glycerol, ZUC99(pATX210) produced 9.7 g/l xylitol from 10.5 g/l L-arabinose with a xylitol yield of 0.92 g/g L-arabinose in 36 h. ZUC99(pATX210) also exhibited efficient conversion in fermentor experiments with 1 l medium containing L-arabinose and glycerol. The strain produced 14.5 g/l xylitol from 15.2 g/l L-arabinose with a xylitol yield of 0.95 g/g L-arabinose in 30 h. (c) 2009, The Society for Biotechnology, Japan. All rights reserved.