Molecular Cloning and Characterization of NAD⁺‐Dependent Xylitol Dehydrogenase from Candida tropicalis ATCC 20913

Abstract

Induction of xylitol dehydrogenase of Candida tropicalis ATCC 20913 by various carbon sources was investigated. The enzyme activity was induced when the yeast was grown on l‐arabinose and d‐xylose. A novel gene encoding the enzyme was cloned and characterized. The 1,095‐bp coding sequence of the gene encodes a polypeptide of 364 amino acids, with a molecular mass of 39.4 kDa. Sequence analysis of the putative protein showed it to be a member of the zinc‐containing alcohol dehydrogenase family and to have homology to xylitol dehydrogenase genes from other yeasts and fungi. The recombinant xylitol dehydrogenase expressed in Escherichia coli oxidized polyols such as xylitol and d‐sorbitol and reduced ketoses such as d‐xylulose and d‐fructose. It required exclusively NAD or NADH as a cofactor.

Codon optimization, not gene content, predicts XYLose metabolism in budding yeasts

“…Given the seeming ecological irrelevance of xylose utilization in the Saccharomycetaceae, the diversification and retention of XYL 2 genes in this group lacks a clear explanation unless the primary function of XYL2 homologs in this family is not in xylose degradation. Several lines of evidence in the literature that support this notion: 1) there is ample evidence that budding yeast XDH enzymes are promiscuous across polyols (48– 51), 2) the XYL2 reverse reaction (reduction of xylulose to xylitol) is more energetically favorable by an order of magnitude (52), and 3) the strongest phylogenetic signal of XYL gene loss we observed was in the W/S clade of yeasts, which is a group of fructose-specializing yeasts that have evolved a novel means of reducing fructose to maintain redox balance (53). Taken together, these data are suggestive of an alternative role of the XYL pathway, and XYL2 in particular.…”

The Positive Effect of the Decreased NADPH-Preferring Activity of Xylose Reductase fromPichia stipitison Ethanol Production Using Xylose-Fermenting RecombinantSaccharomyces cerevisiae

“…Tel: +81-774-38-3517; Fax: +81-774-38-3524; E-mail: kmak@iae.kyoto-u.ac.jp Abbreviations: XR, xylose reductase; XDH, xylitol dehydrogenase; PsXR, XR from Pichia stipitis; PsXDH, XDH from Pichia stipitis; WT, wildtype enzyme; CpXR, XR from Candida parapsilosis; AKR, aldo-keto reductase; CtXR, XR from Candida tenuis Candida tropicalis, which contains typical NAD þ -dependent XDH. 9) In fact, XR (AKR2B5) is a unique member with dual coenzyme specificity in the aldo-keto reductase (AKR) superfamily, 10) but significant NADPH preference is found in most XRs, similarly to most AKR superfamily enzymes. However, CpXR utilizes NADH 100-fold higher than NADPH in catalytic efficiency.…”

Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in Candida tropicalis

“…-dependent activity (Watanabe et al 2005), and XDH from P. stipitis and from C. tropicalis share high amino acid sequence identity (72%) (Ko et al 2006a). Accordingly, it was anticipated that the approach used to engineer P. stipitis XDH could be applied to C. tropicalis XDH with similar results.…”