Arabitol-free xylitol production with the bacterial L-arabinose pathway engineered Candida tropicalis

Abstract

Xylitol, a five-carbon sugar alcohol, is used as an alternative natural sweetener in the food and confectionary industry. It has an anticariogenic effect with the same order of sweetness as sucrose and can replace sucrose on an equal weight basis. As it does not require insulin for metabolic regulation, it can be used as an insulin-independent sugar substitute for diabetics. Xylose reductase (XR) is the key enzyme in xylitol production, catalyzing the reduction of D-xylose to xylitol. The formation of XR in Candida tropicalis is significantly repressed in cells grown on medium containing glucose as carbon and energy sources, probably due to glucose repression. This is one reason why glucose is not a suitable cosubstrate for supporting cell growth for xylitol production. Although Neurospora crassa XR (NcXR) has high catalytic efficiency, NcXR was not expressed in C. tropicalis because of different codon usage between the two species. The NcXR codons were changed to the codons preferred in C. tropicalis. The codon-optimized NcXR gene (NXRG) was placed under the control of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter derived from C. tropicalis and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. The high level of expression of NXRG was confirmed by determining the enzyme activity in cells grown on glucose medium. The resulting recombinant yeast, C. tropicalis LNG2, showed high XR activity (2.86 U $(mg of protein)^{-1})$, whereas no activity was detected in the parent strain, C. tropicalis BSXDH-3. In xylitol fermentation using glucose as a cosubstrate with xylose, LNG2 produced xylitol at a rate of 1.44 $g L^{-1}h^{-1}$ and a xylitol yield of 96% in 44 h. Compared with a xylitol production rate of 0.83 $g L^{-1}h^{-1}$ and xylitol yield of 59% in BSXDH-3, these values were 73% and 62% higher than those of BSXDH-3, respectively. XRs in most yeasts and filamentous fungi have evolved to have broad substrate ...