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Debaryomyces hansenii--an extremophilic yeast with biotechnological potential.

We illuminate the ecological, physiological and genetic characteristics of the yeast Debaryomyces hansenii in the view of our belief that this metabolically versatile, non-pathogenic, osmotolerant and oleaginous microorganism represents an attractive target for fundamental and applied biotechnological research. To this end, we give a broad overview of extant biotechnological procedures using D. hansenii, e.g. in the manufacture of various foods, and propose research into the heterologous synthesis of a range of fine chemicals.

Biotechnology↗

DhARO4, an amino acid biosynthetic gene, is stimulated by high salinity in Debaryomyces hansenii.

The highly halotolerant yeast Debaryomyces hansenii when grown in the presence of 2M NaCl, increased the expression of ARO4 which is involved in the biosynthesis of aromatic amino acids. The function of the isolated gene was verified by complementation of a Saccharomyces cerevisiae null mutant, aro4Delta, restoring the specific activity of the enzyme (a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase) to wild-type levels. DhARO4 transcript expression under high salinity was stimulated at the beginning of the exponential growth phase. As the DhARO4 promoter region presents putative GCRE and CRE sequences, its expression was evaluated under conditions of NaCl stress and amino acid starvation, showing similar expression levels for either condition. The combined effect of both stressors resulted in a further increase in transcript levels over the singly added stressors, indicating independent stimulatory events. Our results support the hypothesis that high salinity and amino acid availability are physiologically interconnected.

3-Deoxy-7-Phosphoheptulonate Synthase↗

Cloning and sequencing the genomic encoding region of copper-zinc superoxide dismutase enzyme from several marine strains of the genus Debaryomyces (Lodder & Kreger-van Rij).

Copper-zinc superoxide dismutase (SODC) is a cytosolic enzyme which catalyses the dismutation of the superoxide radical. Due to its physiological importance, the encoding genes have been cloned from several species of higher eukaryotes. However, genes from moulds and yeast have not been studied extensively. In this paper, the encoding region of this gene (sod1) has been cloned from several strains of marine yeast belonging to the genus Debaryomyces (dvv sod1, dvy sod1 and dh sod1-61) through genomic DNA-PCR amplification. Fragments of 480-486 nucleotides were obtained, which contain information for products of 153-156 amino acids with calculated molecular masses of 15.8-16.6 kDa. The deduced amino acid sequence shows that D. vanrijiae enzymes present three additional amino acids not closely related to the active site conformation. In addition, in D. vanrijiae var. vanrijiae (strain 020), one histidine residue is apparently replaced by a proline; the incidence and function of other aromatic or heterocyclic amino acids is discussed. Homology and phylogenetic trees were constructed from amino-acid sequence multi-alignment analyses; the interrelationships among fungi are discussed. The sod-1 sequences reported in this paper were deposited in the public data library of the NCBI under Accession Nos AF301019, AF327449 and AF327448.

Amino Acid Sequence↗

A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae.

The PBS2 gene encodes a MAP kinase kinase that plays a pivotal role in osmosensing signal-transduction pathway in the yeast Saccharomyces cerevisiae. Mutation in the PBS2 gene has a pleotropic effect. Besides being osmosensitive, pbs2 mutants show altered sensitivity to polymyxin B and calcofluor. Recent studies revealed that Pbs2p plays a different role in osmoadaptation and calcofluor sensitivity. We have isolated a gene homologous to PBS2 from the highly salt-tolerant yeast Debaryomyces hansenii by phenotypic complementation. DNA sequencing of the clone revealed that the gene encoded a protein of 683 amino acid residues. Like Pbs2p, this protein also has a proline-rich motif. Further characterization revealed that this gene could complement polymyxin B sensitivity but did not affect calcofluor sensitivity. Thus, it appeared that Pbs2p also has an independent role in these two physiological processes. The GenBank Accession No. of this sequence is AF371315.

Amino Acid Sequence↗

A minisatellite sequence in the upstream region of the DURA3 gene from the halotolerant yeast Debaryomyces hansenii.

The URA3 gene of Debaryomyces hansenii, encoding orotidine 5'-phosphate decarboxylase enzyme, was isolated by complementation in the yeast Saccharomyces cerevisiae. The deduced amino acid sequence is highly similar to Ura3 proteins from other yeast and fungal species. Analysis of the region upstream of the coding sequence revealed the presence of AG-rich minisatellite DNA sequences. In addition, upstream of the DURA3 sequence, we have found the 3'-terminal of a gene encoding a GEA2-like protein.

Amino Acid Sequence↗

Isolation and sequence analysis of the small subunit ribosomal RNA gene from the euryhaline yeast Debaryomyces hansenii.

The small subunit ribosomal RNA gene (SSU rDNA) from the euryhaline yeast Debaryomyces hansenii has been isolated and sequenced. After appropriate alignment of this sequence with SSU rDNA sequences from 30 other taxa, phylogenetic reconstruction using distance matrix and maximum parsimony methods indicates that D. hansenii is most closely affiliated with Candida albicans, and occurs in the cluster of the yeasts Saccharomyces cerevisiae, Torulaspora delbruekii, Candida glabrata, and Kluyveromyces lactis. It appears that the capacity to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analyses.

Base Sequence↗

Ploidy, ascus formation and recombination in Torulaspora (Debaryomyces) hansenii.

X-ray inactivation studies on the type strain of Torulaspora hansenii carried out to determine ploidy, provided proof that the species has a haplontic life cycle, a fact which hitherto has only been presumed. Observations on the genesis of the ascus by light microscopy and transmission electron microscopy provide no evidence for, what some earlier workers in this field have presumed to be, heterogamous conjugation between a mother cell and its bud. They do, however, show that asci, bearing obliquely-attached, vestigal, bud-like appendages, arise from some cells to form single, non-abstricting, and frequently, recurving protuberances which enlarge. These could, conceivably, be responsible for the impression that abstricted buds are connected to the mother-cells by bent copulatory tubes. The formation during sporulation of elongated protuberances and the presence of a medial, electron-dense line within the electron-translucent layer of the walls of ascospores fixed with OsO4 preclude the possibility of using these features to differentiate between the genera Torulaspora and Debaryomyces. Furthermore, recombinant studies, which involved the use of auxotrophic mutants, indicated that during sporulation the fusion of independent cells accounted for only 0.03-0.6% of the asci formed. The conclusion was reached that somatogamous autogamy must be the main agency of diploidization and that the species is largely inbreeding.

Conjugation, Genetic↗

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress.

Changes in glycerol production and two parameters related to energy metabolism i.e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mumol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.

Adenosine Triphosphate↗

Scanning electron microscopy of ascospores of Debaryomyces and Saccharomyces.

Ascospores from species of Debaryomyces and the Torulaspora-group of Saccharomyces were examined by scanning electron microscopy. Ornamentation on ascospores of D. hansenii varied from short to long interconnected ridges or broad based, elongated conical protuberances. A spiral rigde system was detected on the ascospores of D. marama, but wart-like protuberances occurred on those of D. cantarelli, D. castellii, D. coudertii, D. formicarius, D. phaffii, D. vanriji and D. yarrowii. Ascospores of D. halotolerans did not have protuberances and the species appears to be identical with Pichia farinosa. Wart-like protuberances also were found on ascospores of S. delbrueckii, S. microellipsodes, S. rosei, S. inconspicuus, S. fermentati, S. montanus and S. vafer, but the ascospore surface of S. pretoriensis was covered by fine ridges. Short tapered ridges covered the ascospores of S. kloeckerianus.

Cell Wall↗

On the mechanism of salt tolerance. Production of glycerol and heat during growth of Debaryomyces hansenii.

As glycerol was suggested as an osmotic agent in the salt tolerant Debaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells. During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to--1690 kJ per mole glucose consumed in contrast to--1200 and--1130 kJ at 4 mM and 0.68 M NaCl, respectively. The Ym-values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl respectively.

Ascomycota↗

Inulinase of Debaryomyces cantarellii.

Debaryomyces cantarellii excretes into a buffered medium an inulinase of beta-fructofuranosidase type, its synthesis being induced by inulin. The enzyme has a pH optimum at 4 and its optimum temperature is 50 degrees C. Its Km for inulin is 15 mM.

Ascomycota↗

Osmoregulation and the Genetic Induction of Glycerol-3-phosphate Dehydrogenase by NaCl in the Euryhaline Yeast Debaryomyces hansenii.

: In the yeasts Saccharomyces cerevisiae and Debaryomyces hansenii, glycerol-3-phosphate dehydrogenase (NAD+-GPD) is believed to be the enzyme controlling the synthesis of glycerol in osmotically challenged cells. Consistent with this concept, immunoblot analyses demonstrated quantitative increases in NAD+-GPD 30 minutes after the addition of NaCl to actively growing D. hansenii cells; Northern blot analyses showed increases in GPD-messenger RNA within 15 minutes after the addition of salt. There appears to be an upper limit of salt concentration above which the cells appear to be less responsive. The results we describe support the concept of transcriptional regulation of NAD+-GPD in D. hansenii.

Journal Article↗

A physiological and enzymatic study of Debaryomyces hansenii growth on xylose- and oxygen-limited chemostats.

The effect of changing growth rate and oxygen transfer rate (OTR) on Debaryomyces hansenii physiology was studied using xylose-limited and oxygen-limited chemostat cultures, respectively, and complemented with enzymatic assays. Under xylose-limited chemostat (oxygen-excess), neither ethanol nor xylitol was produced over the entire range of dilution rate ( D). The maximal volumetric biomass productivity was 2.5 g x l(-1) x h(-1) at D =0.25 h(-1) and cell yield was constant at all values of D. The respiratory rates and xylose consumption rate increased linearly with growth rate but, above 0.17 h(-1), oxygen consumption rate had a steeper increase compared to carbon dioxide production rate. Enzymatic analysis of xylose metabolism suggests that internal fluxes are redirected as a function of growth rate. For values of D up to 0.17 h(-1), the xylose reductase (XR) titre is lower than the xylitol dehydrogenase (XDH) titre, whereas above 0.17 h(-1) XR activity is about twice that of XDH and the NADPH-producing enzymes sharply increase their titres indicating an internal metabolic flux shift to meet higher NADPH metabolic requirements. Moreover, the enzymes around the pyruvate node also exhibited different patterns if D was above or below 0.17 h(-1). Under oxygen-limited chemostat (xylose-excess) the metabolism changed drastically and, due to oxidative phosphorylation limitation, cell yield decreased to 0.16 g g(-1) for an OTR of 1.4 mmol l(-1) h(-1) and xylitol became the major extracellular product along with minor amounts of glycerol. The enzymatic analysis revealed that isocitrate dehydrogenase is not regulated by oxygen, whereas XR, XDH and the NADPH-producing enzymes changed their levels according to oxygen availability.

Culture Media↗

Intracellular pH homeostasis plays a role in the NaCl tolerance of Debaryomyces hansenii strains.

The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30-35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pK(a,i)) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK(a,i) close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK(a,i) to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.

Culture Media↗

Purification, characterization, and potential applications of formate oxidase from Debaryomyces vanrijiae MH201.

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of alphaalpha, alphabeta, and betabeta subunits with apparently similar MM. The preparation acted on formate with K (m) and V (max) values of 11.7 mM and 262 micromol min(-1) mg(-1), respectively, at pH 4.5 and 25 degrees C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35 degrees C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4 degrees C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml(-1) of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml(-1) of a microbial aldehyde oxidase and 100 U ml(-1) of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.

Aldehyde Oxidase↗

Biodegradation of biphenyl by the ascomycetous yeast Debaryomyces vanrijiae.

Cells of the yeast strain Debaryomyces vanrijiae SBUG 770, grown with glucose, converted biphenyl to 4-hydroxybiphenyl as the major metabolite. In addition, 2-hydroxybiphenyl was formed in minor amounts. No further degradation of these substances was detected. However, these monohydroxylated derivatives were oxidised by alkane-grown cells in the presence of the co-metabolic substrate, tetradecane. Under these conditions 2-hydroxybiphenyl was oxidised to 2,5-dihydroxybiphenyl, and 4-hydroxybiphenyl was rapidly metabolised by formation of two major metabolites. One was identified as 3,4-dihydroxybiphenyl. Characterisation of the second product as 4-phenylmuconolactone points to a further metabolism of the initially formed dihydroxylated biphenyl via ortho-ring fission.

Ascomycota↗

Growth under alkaline conditions of the salt-tolerant yeast Debaryomyces hansenii IFO10939.

A salt-tolerant yeast Debaryomyces hansenii IFO 10939, which is able to grow at pH 10.0, was isolated and characterized. IFO 10939 had the ability of maintaining intracellular pH. The in vivo activation of plasma membrane ATPase was observed in cells grown at pH 6.2 during conditioning in buffer at pH 9.0. Alkalification of growth medium exhibited a significant increase in acetate and propionate production. The results suggested that the regulation of intracellular pH was involved in plasma membrane ATPase pumping protons out of the cells and weak acid formation for the source of the protons in cells growing at high pH.

Adenosine Triphosphatases↗