Enhanced xylitol production rate by engineering glycerol metabolic path-way and pentose phosphate pathway in Candida tropicalis

Abstract

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. Extensive research has been carried out on the dietary and technological properties of xylitol because of its growing market and high added value.

Glycerol can be used as a primary carbon source by yeasts, little is known regarding glycerol metabolism in C. tropicalis. In this study, gk gene which encodes glycerol kinase was disrupted from the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis strain BSXDH-3. The resulting gk knockout C. tropicalis strain, termed "IA", was incapable to grow on glycerol. The cells growth on glycerol was resumed by co-expressing Scheffersomyces stipitis gcy1, 2 and 3 genes, which respectively encode NADP+-dependent alcohol dehydrogenase 1, 2 and 3, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "IA-3", than in BSXDH-3. Rates of xylitol production at 16 and 24 hr for IA-3 were 0.85 g l-1 h-1 and 1.28 g l-1 h-1 respectively; these values are 30% and 18% higher than corresponding rates for BSXDH-3.

The oxidative pentose phosphate pathway (PPP) is a major basis of NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes, which respectively encode glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l-1 h-1...