Characterization of the AXDH gene and the encoded xylitol dehydrogenase from the dimorphic yeast Arxula adeninivorans

Abstract

The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter-GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 degrees C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD+ or NADH as coenzymes.

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

“…Enzyme activity greatly decreased at temperatures above 35°C and was not detectable at temperature above 50°C ( Figure 5). Similar pH and temperature optima for oxidative and reductive reactions were found for XDH from P. stipitis, G. mastodermitis, and A. adeniniVorans (11,13,27).…”

“…This is in good agreement with the predicted molecular mass of 40.8 kDa (39.3-kDa XDH with a 1.5-kDa His-tag). The XDHs from other yeast and fungal strains have similar molecular masses, i.e., 38-41 kDa (10)(11)(12)(13)27).…”

“…XDH in another xylose-utilizing yeast, Pichia stipitis, was encoded by XYL2 (8). XYL2 genes from some yeasts and fungi, such as Saccharomyces cereVisiae, Hypocrea jecorina, Aspergillus oryzae, Arxular adeniniVorans, and Galactocandida mastodermitis, have been cloned (9)(10)(11)(12)(13).…”

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

“…These unusual properties make A. adeninivorans an ideal host for heterologous gene expression as well as a useful source of genes of biotechnological significance (Wartmann et al, 1998(Wartmann et al, , 2002a(Wartmann et al, , 2003a(Wartmann et al, , 2003bBöer et al, 2004Böer et al, , 2005Böer et al, , 2007. Enzymes with wide substrate spectra and high specific activities are of special interest for biotechnological applications.…”

Characterization and expression analysis of a gene cluster for nitrate assimilation from the yeast Arxula adeninivorans

“…An expression platform based on A. adeninivorans has been developed in recent years, and has been used to synthesize recombinant proteins such as human serum albumin (HSA), lipase, invertase, xylitol dehydrogenase, transaldolase, interleukin-6, α-amylase, acid phosphatase and phytase, and for production of polyhydroxyalkanoates (Wartmann et al, 2002(Wartmann et al, , 2003a(Wartmann et al, , 2003bBöer et al, 2004bBöer et al, , 2005aBöer et al, , 2005bBöer et al, , 2005cBöer et al, , 2007Terentiev et al, 2004;Steinborn et al, 2005Steinborn et al, , 2007El Fiki et al, 2007;Kaur et al, 2007). The system involves the use of vectors with multiple cloning sites for the integration of selection markers, expression cassettes bearing strong constitutive or inducible promoters, autonomously replicating sequences and chaperone cassettes, which are inserted between rDNA-targeting sequences (Steinborn et al, 2007).…”