Xylitol production with high yield by xylitol dehydrogenase gene-disrupted mutant of candida tropicalis

Abstract

A gene encoding xylitol dehydrogenase (XDH) of Candida tropicalis ATCC 20913 was cloned and characterized. The 1,095-bp coding sequence of the XYL2 gene encodes a polypeptide of 364 amino acids, with a molecular mass of 39.4 kDa. The XYL2 gene of Candida tropicalis was disrupted by sequential gene replacement using Ura-blasting method. The XYL2-disrupted mutant, BSXDH-3, could not grow on a minimal medium containing D-xylose as a sole carbon source. Enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using xylitol fermentation medium containing glucose as a co-substrate. Since glucose was not sufficient as a co-substrate, various carbon sources were screened for efficient cofactor regeneration. Glycerol was selected as the best co-substrate and, subsequently, the optimization of fermentation condition was carried out. As the result of experiments, BSXDH-3 produced xylitol with a volumetric productivity of 3.23 $g1^{-1}h^{-1}$ a specific productivity of 0.76 g$g1^{-1}h^{-1}$, and a xylitol yield of 98 %. This is the first report of gene disruption of C. tropicalis for enhancing the efficiency of xylitol production and will be a turning point of process development for xylitol production.