A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate.

Nitronate monooxygenase (NMO; E.C. 1.13.12.16) oxidizes alkyl nitronates to aldehydes and nitrite. Although the biochemistry of the enzyme from fungal sources has been studied extensively, the physiological role is unknown. The ability of NMO to detoxify propionate-3-nitronate was tested by measuring growth of recombinant Escherichia coli containing the gene encoding for the enzyme in either the absence or presence of the nitronate and its conjugate acid 3-nitropropionate. The mixture propionate-3-nitronate/3-nitropropionate is toxic to E. coli cells lacking expression of NMO, but the toxicity is overcome through either induction of the gene for NMO or through addition of exogenous enzyme to the cultures. Both Williopsis saturnus and Neurospora crassa were able to grow in the presence of 0.4mM propionate-3-nitronate and 19.6mM 3-nitropropionate, while a knockout mutant of N. crassa lacking NMO was inhibited by concentrations of propionate-3-nitronate and 3-nitropropionate >0.3 and 600µM, respectively. These results strongly support the conclusion that NMO functions to protect the fungi from the environmental occurrence of the metabolic toxin.

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2.

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0 kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16 °C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378 bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6 kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.

Disruption of the gene encoding ß-1, 3-glucanase in marine-derived Williopsis saturnus WC91-2 enhances its killer toxin activity.

As the ß-1, 3-glucanase produced by the marine-derived Williopsis saturnus WC91-2 could inhibit the activity of the killer toxin produced by the same yeast, the WsEXG1 gene encoding exo-ß-1, 3-glucanase in W. saturnus WC91-2 was disrupted. The disruptant WC91-2-2 only produced a trace amount of ß-1, 3-glucanase but had much higher activity of killer toxin than W. saturnus WC91-2. After the disruption of the WsEXG1 gene, the expression of the gene was significantly decreased from 100% in the cells of W. saturnus WC91-2 to 27% in the cells of the disruptant WC91-2-2 while the expression of the killer toxin gene in W. saturnus WC91-2 and the disruptant WC91-2-2 was almost the same. During 2-l fermentation, the disruptant WC91-2-2 could produce the highest amount of killer toxin (the size of the inhibition zone was 22 ± 0.7 mm) within 36 h when the cell growth reached the middle of the log phase.

Cloning of exo-ß-1,3-glucanase gene from a marine yeast Williopsis saturnus and its overexpression in Yarrowia lipolytica.

The exo-ß-1,3-glucanase structural gene (WsEXG1 gene, accession number: FJ875997.2) was isolated from both the genomic DNA and cDNA of the marine yeast Williopsis saturnus WC91-2 by inverse PCR and RT-PCR. An open reading frame of 1,254 bp encoding a 417 amino acid protein (isoelectric point: 4.5) with calculated molecular weight of 46.2 kDa was characterized. The promoter of the gene (intronless) was located from -28 to -77 and had one TATA box while its terminator contained the sequence AAGAACAATAAACAA from +1,386 to +1,401. The protein had the Family 5 glycoside hydrolase signature IGLELLNEPL and a fragment with the sequence of NLCGEWSAA, where the Glu-310 (E) was considered to be the catalytic nucleophile. The WsEXG1 gene was overexpressed in Yarrowia lipolytica Po1h and the recombinant WsEXG1 was purified and characterized. The molecular weight of the purified rWsEXG1 was 46.0 kDa. The optimal pH and temperature of the purified rWsEXG1 were 5.0°C and 40°C, respectively. The purified rWsEXG1 had high exo-ß-1,3-glucanase activity. Therefore, the recombinant ß-1,3-glucanase may have highly potential applications in food and pharmaceutical industries.

Purification and molecular characterization of exo-beta-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2.

The extracellular beta-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific beta-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40 degrees C, respectively. The enzyme was significantly stimulated by Li+, Ni2+, and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1,10-phenanthroline. The Km and Vmax values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the beta-1,3-glucanases from other yeasts.

Candida mengyuniae sp. nov., a metsulfuron-methyl-resistant yeast.

A metsulfuron-methyl-resistant yeast strain, JHL(T), was isolated from metsulfuron-methyl-contaminated soil collected in Jiangsu Province, China. Through morphological and physiological analysis as well as a molecular phylogenetic analysis based on the 26S rRNA gene D1/D2 region and internal transcribed spacer (ITS), this strain, which forms a clade with Candida vartiovaarae and a teleomorphic species, Williopsis saturnus, was revealed to represent a novel species in the genus Candida. The name Candida mengyuniae sp. nov. (type strain JHL(T)=CGMCC 2.3681(T)=CBS 10845(T)) is proposed for this novel species.