Improvement of xylitol production by expression of heterologous xylose transporter in Candida tropicalis

Abstract

Xylitol is a five-carbon sugar alcohol that has global markets. It has beneficial health properties and represents an alternative to current conventional sweeteners. It has low calory, but its sweetness level is equal to that of sucrose. Additionally, it has anticariogenic effect that inhibits the growth of the tooth-decaying bacterium. Xylitol can also be used as a chemical building block for the production of high-value chemicals such as xylaric acid and glycols. The asporogenic diploid yeast, Candida tropicalis, is of academic and industrial interest. Especially, C.tropicalis has been extensively studied in the production of xylitol in terms of their distinguished xylose-assimilating ability. The synthesis of xylose reductase (XR) in C. tropicalis is considerably repressed in cells grown on glucose medium because of glucose repression. For this reason, glucose is not an appropriate cosubstrate for supporting cell growth and xylitol production. However, for economic feasibility, inexpensive hemicellulose should be used as a raw material and the hemicellulose is hydrolyzed by mineral acid to produce hydrolysate that is mainly composed of glucose and xylose. A gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was isolated from the genomic DNA of C. tropicalis ATCC 20913 to construct a constitutive expression system without glucose repression. The endogenous XR gene (xyl1) was placed under the control of GAPDH promoter and integrated into the genome of C. tropicalis N43 which is xyl2-disrupted and xyl1-partially disrupted mutant. Expression of XR was confirmed by determining enzyme activity in cells grown on a medium containing glucose as a carbon source. The resulting recombinant yeast, C. tropicalis JA4, showed higher XR activity (465±77 mU/mg of proteins) whereas that of BSXDH-3, the control strain, was not detectable. Glucose uptake inhibits xylose utilization in xylose-assimilating yeasts such as engineered Saccharomyces cerevisiae and Pich...