Characterization and expression of xylose reductase gene ( xyl1) of Candida guilliermondii and xylitol production by recombinant Pichia pastoris

Dietary and medical benefits of xylitol, a naturally occurring sugar alcohol, as an alternative sugar has been well documented. The traditional chemical production of xylitol from xylose requires high temperature and pressure and yields several chemical contaminants which need further purification. The bioconversion of xylose has been exploited as an alternative process of xylitol production. Few yeasts are able to convert xylose to xylitol by xylose reductase. The enzyme requiring NAD(P)H for catalytic activity was isolated and identified. To understand the characteristics of the enzyme, the nucleotide sequence of coding region of xylose reductase gene (xyl1) of Candida guilliermondii was determined. The coding sequence of the xyl1 was cloned into Pichia pastoris under methanol inducible AOX1 promoter. The expression and fermentation of recombinants were investigated.; The coding sequence of xyl1 of C. guilliermondii, the most efficient xylitol producing yeasts, was successfully amplified. The nucleotide sequence of xyl1 of C. guilliermondii was 71.43% homologous to those of Pichia stipitis, the most ethanol producing yeast. Fewer homology (70.63%) of deduced amino acid sequence of the two yeast was found. However, all essential cysteine and histidine amino acid residues for cofactor specificity were found. The dissimilarity of cofactor preference of the enzyme of the two yeast may attribute to the difference in position of histidine residues.; The xyl1 coding sequence was highly expressed as functional enzyme in vitro and in vivo. Methanol induced more xylose reductase in recombinant Pichia pastoris than xylose in C. guilliermondii and P. stipitis. The recombinants were able to ferment xylose with xylitol as a major product. As C. guilliermondii, aeration decreased xylitol accumulation in the recombinant. The pre-existing xylose reductase did not increase xylitol production. A proportional utilization of NADPH/NADH was a rate limiting step for xylitol production.