D-arabitol metabolism in Candida albicans: construction and analysis of mutants lacking D-arabitol dehydrogenase

Abstract

Candida albicans produces large amounts of the acyclic pentitol D-arabitol in culture and in infected animals and humans, and most strains also grow on minimal D-arabitol medium. An earlier study showed that the major metabolic precursor of D-arabitol in C. albicans was D-ribulose-5-PO 4 from the pentose pathway, that C. albicans contained an NAD-dependent D-arabitol dehydrogenase (ArDH), and that the ArDH structural gene (ARD) encoded a 31-kDa short-chain dehydrogenase that catalyzed the reaction D-arabitol ؉ NAD ‫>؍<‬ D-ribulose ؉ NADH. In the present study, we disrupted both ARD chromosomal alleles in C. albicans and analyzed the resulting mutants. The ard null mutation was verified by Southern hybridization, and the null mutant's inability to produce ArDH was verified by Western immunoblotting. The ard null mutant grew well on minimal glucose medium, but it was unable to grow on minimal D-arabitol The human pathogen Candida albicans produces large amounts of D-arabitol in culture and in infected mammalian hosts (3,7,15,27), and most strains also grow on minimal The evidence supporting the conclusions that fungi metabolize D-arabitol via the pathways described above is indirect, consisting mostly of 14 C and 13 C metabolic labeling data and the presence in cell extracts of enzymes with the expected catalytic activities. To our knowledge, it has not yet been shown for any fungus that a defined mutation or a specific enzyme inhibitor interferes with either biosynthesis or utilization of D-arabitol. Therefore, we sought to ascertain more directly the metabolic functions of ArDH in C. albicans. Specifically, we tested the hypothesis that ArDH catalyzes the last step in D-arabitol biosynthesis and the first step in D-arabitol utilization by disrupting both chromosomal alleles of ARD and by analyzing the resulting null mutants. MATERIALS AND METHODSStrains, media, and plasmids. C. albicans 1161 (arg4 lys1 ser57 MPA1 ura3 gal1) was obtained from S. Scherer (University of Minnesota) (9) and was cultured in yeast extract-peptone supplemented with 1% glucose (YEPD), 1% D-arabitol (YEP/D-arabitol), 1% D-arabinose (YEP/D-arabinose), or no sugar (YEP) (10) or in 0.67% yeast nitrogen base without amino acids (Difco, Detroit, Mich.) supplemented with arginine, lysine, and serine to which 1% glucose (minimal glucose), 1% D-arabitol (minimal D-arabitol), or 1% D-arabinose (minimal D-arabinose) was added. C. albicans transformants were selected on minimal glucose medium plus 1 M sorbitol, and uracil auxotrophs (Ura Ϫ ) were selected on minimal glucose medium supplemented with 625 mg of 5-fluoroorotic acid (FOA) and 100 mg of uridine per liter (FOA medium). Agar (1.5 to 2%) was included as needed.Escherichia coli strains DH5␣ (Gibco BRL, Gaithersburg, Md.) and JM109 (29) were used as plasmid hosts. Cultures were grown in Luria-Bertani medium (20), supplemented with ampicillin (50 g/ml) and/or agar (2%) as needed. E.

Metabolic regulation in model ascomycetes – adjusting similar genomes to different lifestyles

“…C. albicans is much more flexible in its sugar use and can grow efficiently on a variety of 5-carbon sugars, such as xylose [53] and arabinose [54], that are not exploited by normal strains of S. cerevisiae [55]. The failure to use xylose is not due to missing enzymatic capacity in S. cerevisiae because functional enzymes exist for all metabolic processing steps of the 5-carbon sugar, and thus the inability to use xylose is presumably linked to the metabolic regulatory circuitry, perhaps through redox-potential balance [53].…”

Expression of bacterial mtlD in Saccharomyces cerevisiae results in mannitol synthesis and protects a glycerol-defective mutant from high-salt and oxidative stress

“…Therefore, it may be possible to exploit the phenotypic effects in S. cerevisiae of new polyol biosynthetic pathways to clone the genes or cDNAs encoding key polyol biosynthetic enzymes in other fungi. For example, it may be possible to isolate the genes or cDNAs encoding mannitol-1-phosphate dehydrogenase in mannitol-producing fungi such as C. neoformans or Aspergillus species and the corresponding enzyme (Darabinitol-5-phosphate dehydrogenase) in D-arabinitol-producing fungi such as C. albicans (27) by their abilities to confer osmotolerance on gpd1 mutants such as S. cerevisiae UTL-7AG3 or S. cerevisiae YPH 252 gpd1⌬::leu2.…”

“…For example, Candida albicans produces the five-carbon polyol D-arabinitol in culture and in infected animals and humans (13,27), and Cryptococcus neoformans and Aspergillus fumigatus produce the six-carbon polyol mannitol in culture and in infected animals (26,28). Little is known about the functions of polyols other than glycerol in fungi.…”

Enhanced d-arabitol production by Zygosaccharomyces rouxii JM-C46: isolation of strains and process of repeated-batch fermentation

“…Xylitol has many known applications in the food and pharmaceutical industry, particularly as a sugar substitute for diabetics, an anticariogenic agent and a natural food sweetener [11]. Similar to xylitol, as the catabolism of d-arabitol by Escherichia coli involves the formation of d-arabitol phosphate which induces the synthesis of compounds that X. Qi (*) · Y. Luo · X. Wang · J. Zhu · J. Lin · H. Zhang · F. Chen · W. Sun School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China e-mail: qxh@ujs.edu.cn 1 3 an NADH/NAD-dependent pentitol dehydrogenase [17,18]. The two possible routes are summarized in Fig.…”