Casein Digestion by Debaryomyces hansenii Isolated from Cheese

To understand the possible proteolytic contribution of yeast during cheese ripening, Debaryomyces hansenii 212 was isolated from commercial blue-veined cheese and incubated in a medium containing casein. Growth and casein degradation were recognized at the cheese-ripening temperature. Proteolytic activity was found in the intracellular fraction, and the enzyme, which was attached to the cell wall, primarily acted on β-casein. The cytosol contained more than 90% of the total proteolytic activity which was responsible for the degradation of both α_s- and β-casein. These results suggest that the contribution of yeast to cheese ripening would depend on the susceptibility to cell lysis in addition to its proteolytic activity.     

Isolation of a psychrotolerant Debaryomyces hansenii strain from fermented tea plant (Camellia sinensis) leaves

Abstract

A psychrotolerant, halotolerant and alkalophilic yeast was isolated from fermented leaves of Camellia sinensis Kuntze, the tea plant. The yeast strain, named Tea-Y1, was both phenotypically and genotypically identified as belonging to the species Debaryomyces hansenii. This assignment was confirmed by scanning and transmission electron microscopy. The analysis of growth curves demonstrated the ability this yeast strain to grow in a temperature range between 4°C and 28°C, with an optimum of 23°C. The ecology of this yeast in the C. sinensis phyllosphere, as well as its possible role in tea fermentation and storage, with particular reference to iced tea, are discussed.     

Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. The molecular mechanisms underlying its extreme osmotolerance and halotolerance have drawn considerable attention in the recent past. However, progress in this regard has been limited due to lack of availability of a transformation system and molecular tools to study the functions of the genes in D. hansenii . Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain and the DhHIS4 gene as the selectable marker. By screening the D. hansenii genomic library, we have isolated several autonomous replication sequences that can be used for constructing a replicating vector. Moreover, our study is the first to demonstrate gene disruption in D. hansenii by homologous recombination.     

表面活性剂对D-阿拉伯糖醇发酵的影响

为提高D-阿拉伯糖醇的产量,研究不同类型表面活性剂对德巴利汉逊酵母(Debaryomyces hansenii)发酵生产D-阿拉伯糖醇的影响。结果表明:阳离子和阴离子表面活性剂对D-阿拉伯糖醇的生成几乎没有影响,部分非离子表面活性剂对D-阿拉伯糖醇的生产有促进作用,其中Trition X-100的影响最为显著。在不同发酵时间加入不同浓度的Trition X-100均对D-阿拉伯糖醇的生产有促进作用,当发酵24 h添加30 g/LTrition X-100时,D-阿拉伯糖醇的产量达到最高(92.9 g/L),相比于对照增加了27.2%。     

Oxidative stress sensitivity in Debaryomyces hansenii

Debaryomyces hansenii is an osmotolerant and halotolerant yeast of increasing interest for fundamental and applied research. In this work, we have performed a first study on the effect of oxidative stress on the performance of this yeast. We have used Saccharomyces cerevisiae as a well-known reference yeast. We show that D. hansenii is much more susceptible than S. cerevisiae to cadmium chloride, hydrogen peroxide or 1,4-dithiothreitol. These substances induced the formation of reactive oxygen species (ROS) in both yeasts, the amounts measured being significantly higher in the case of D. hansenii . We also show that NaCl exerted a protective effect against oxidative stress in Debaryomyces , but that this was not the case in Saccharomyces because sodium protected that yeast only when toxicity was induced with cadmium. On the basis of the present results, we raised the hypothesis that the sensitivity to oxidative stress in D. hansenii is related to the high amounts of ROS formed in that yeast and that observations such as low glutathione amounts, low basal superoxide dismutase and peroxidase activities, decrease in ATP levels produced in the presence of ROS inducers and high cadmium accumulation are determinants directly or indirectly involved in the sensitivity process.     

Maintenance and growth requirements in the metabolism of Debaryomyces hansenii performing xylose-to-xylitol bioconversion in corncob hemicellulose hydrolyzate

In order to improve the biotechnological production of xylitol, the metabolism of Debaryomyces hansenii NRRL Y-7426 in corncob hemicellulose hydrolyzate has been investigated under different conditions, where either maintenance or growth requirements predominated. For this purpose, the experimental results of two sets of batch bioconversions carried out alternatively varying the starting xylose concentration in the hydrolyzate (65.6 < or = S(0) < or = 154.7 g L(-1)) or the initial biomass level (3.0 < or = X(0) < or = 54.6 g(DM) L(-1)) were used to fit a metabolic model consisting of carbon material and ATP balances based on five main activities, namely fermentative assimilation of pentoses, semi-aerobic pentose-to-pentitol bioconversion, biomass growth on pentoses, catabolic oxidation of pentoses, and acetic acid and NADH regeneration by the electron transport system. Such an approach allowed separately evaluating the main bioenergetic constants of this microbial system, that is, the specific rates of ATP and xylose consumption due to maintenance (m(ATP) = 21.0 mmol(ATP) C-mol(DM) (-1)h(-1); m(Xyl) = 6.5 C-mmol(Xyl) C-mol(DM) (-1)h(-1)) and the true yields of biomass on ATP (Y(ATP) (max) = 0.83 C-mol(DM) mol(ATP) (-1)) and on xylose (Y(Xyl) (max) = 0.93 C-mol(DM) C-mol(Xyl) (-1)). The results of this study highlighted that the system, at very high S(0) and X(0) values, dramatically increased its energy requirements for cell maintenance, owing to the occurrence of stressing conditions. In particular, for S(0) > 130 g L(-1), these activities required an ATP consumption of about 2.1 mol(ATP) L(-1), that is, a value about seven- to eightfold that observed at low substrate concentration. Such a condition led to an increase in the fraction of ATP addressed to cell maintenance from 47% to 81%. On the other hand, the very high percentage of ATP addressed to maintenance (> 96%) at very high cell concentration (X(0) > or = 25 g(DM) L(-1)) was likely due to the insufficient substrate to sustain the growth.     

Mechanisms underlying the halotolerant way of Debaryomyces hansenii

The yeast Debaryomyces hansenii is usually found in salty environments such as the sea and salted food. It is capable of accumulating sodium without being intoxicated even when potassium is present at low concentration in the environment. In addition, sodium improves growth and protects D. hansenii in the presence of additional stress factors such as high temperature and extreme pH. An array of advantageous factors, as compared with Saccharomyces cerevisiae, is putatively involved in the increased halotolerance of D. hansenii: glycerol, the main compatible solute, is kept inside the cell by an active glycerol-Na+ symporter; potassium uptake is not inhibited by sodium; sodium protein targets in D. hansenii seem to be more resistant. The whole genome of D. hansenii has been sequenced and is now available at http://cbi.labri.fr/Genolevures/ and, so far, no genes specifically responsible for the halotolerant behaviour of D. hansenii have been found.     

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 °C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.     

The influence of hexoses addition on the fermentation of d-xylose in Debaryomyces hansenii under continuous cultivation

The effect of hexoses (glucose and galactose) addition to the feed xylose mineral medium of Debaryomyces hansenii chemostat cultures grown at a constant dilution rate of 0.055 h⁻¹ was studied. Xylitol was the major product detected amongst all tested conditions. The maximal values for xylitol yield and volumetric productivity (0.56 gg⁻¹ xylose and 0.21 gl⁻¹h⁻¹, respectively) were obtained for a glucose/xylose feeding ratio of 10%, showing that the addition of small amounts of glucose, but not galactose, enhanced the xylitol production. A xylitol yield increase of 30%, compared with the sole xylose-containing feed medium, was observed. It was found that the oxygen requirement for D. hansenii growth is lower under glucose compared with xylose. Ethanol and glycerol were only produced for glucose/xylose feeding ratio above 30%. The byproducts accumulation was correlated with glucose metabolism, because a direct relationship between the increase of ethanol (and glycerol) concentration and the increase of glucose in the feed medium was found.     

Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of substituted acetophenones

Chiral secondary alcohols are convenient mediator for the synthesis of biologically active compounds and natural products. In this study fifteen yeast strains belonging to three food originated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to 1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to the corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high conversion rates. This is the first report on the enantioselective reduction of acetophenone by D. hansenii P1 from past?rma, a fermented Turkish meat product. The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess. Compound 2g can be used for the synthesis of (+)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced acetophenone derivatives. This study showed that this yeast can be used industrially to produce enantiomerically pure chiral secondary alcohols, which can be easily converted to different functional groups.     

Phosphate limitation stress induces xylitol overproduction by Debaryomyces hansenii

The physiological responses of xylose-grown Debaryomyces hansenii were studied under different nutritive stress conditions using continuous cultivation at a constant dilution rate of 0.055 h⁻¹. Metabolic steady-state data were obtained for xylose, ammonium, potassium, phosphate and oxygen limitation. For xylose and potassium limitation, fully oxidative metabolism occurred leading to the production of biomass and CO₂ as the only metabolic products. However, potassium-limiting cultivation was the most severe nutritional stress of all tested, exhibiting the highest xylose and O₂ specific consumption rates along with the lowest biomass yield, 0.22 g g⁻¹ xylose. It is suggested that carbon was mainly channelled to meet the cellular energy requirements for potassium uptake. For the other limiting nutritional conditions increasing amounts of extracellular xylitol were found for ammonium, phosphate and oxygen limitation. Although xylitol excretion is not significant for ammonium limitation, the same is not true for phosphate limitation where the xylitol productivity reached 0.10 g l⁻¹ h⁻¹, about half of that found under oxygen-limiting conditions, 0.21 g l⁻¹ h⁻¹. This work is the first evidence that xylitol production by D. hansenii might not only be a consequence of a redox imbalance usually attained under semi-aerobic conditions, but additional physiological mechanisms must be involved, especially under phosphate limitation. Cell yields changed drastically as a function of the limiting nutrient, being 0.22, 0.29, and 0.39 g g⁻¹ xylose for potassium, oxygen and phosphate limitation, respectively, and are a good indicator of the severity of nutritive stress.     

Proteomic changes in Debaryomyces hansenii upon exposure to NaCl stress

The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.     

Xylitol production from barley bran hydrolysates by continuous fermentation with Debaryomyces hansenii

The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l^–1 h^–1 were obtained for dilution rates of 0.008–0.088 h^–1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l^–1 h^–1) was reached at a dilution rate of 0.284 h^–1.     

Differential detection of Debaryomyces hansenii isolated from intermediate-moisture foods by PCR-RFLP of the IGS region of rDNA

The amplification by PCR of the Intergenic Spacer region (IGS) of rDNA followed by Restriction Fragment Length Polymorphism (RFLP) analysis was evaluated as a potential method for the identification of Debaryomyces hansenii among other yeast species that frequently contaminate Intermediate-Moisture Foods (IMFs). For a first rapid differentiation at the species level, the determination of the IGS-PCR fragment size was found to be a useful approach. The digestion of this region with the enzymes HhaI, HapII and MboI resulted in specific patterns that permit the identification of D. hansenii among other yeast species. This method also permitted the discrimination between the D. hansenii varieties (var. hansenii and var. fabryi) as well as the differentiation of D. hansenii from other species of the genus, such as Debaryomyces pseudopolymorphus or Debaryomyces polymorphus var. polymorphus. The IGS-PCR RFLP method was assayed for the differential detection of D. hansenii in contaminated or spoiled IMF products and compared with traditional identification procedures, resulting in a 100% detection rate for D. hansenii.     

Energy conversion coupled to cyanide-resistant respiration in the yeasts Pichia membranifaciens and Debaryomyces hansenii

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.     

Debaryomyces hansenii fermentation for arabitol production

The fermentation process for arabitol production from glycerol was developed using a Debaryomyces hansenii strain recently selected from a broad screening. The high-producing strain produced arabitol as the only detectable polyol from glycerol. In this work, the pH, dissolved oxygen concentration (DO), inoculum size and magnesium concentration, and the nitrogen-to-phosphorus (N/P) ratio were systematically evaluated for effects on cell growth rate and arabitol productivity. Among those evaluated, the medium with N/P = 9, DO of 5% air saturation and pH 3.5 supported the highest arabitol production. Under these optimal conditions, arabitol production of 40 g/L was achieved in 5 days compared to earlier studies with 15 g/L arabitol in 5 days. Volumetric productivity and specific productivity were successfully improved from 0.13 to 0.33 g/L-h and 0.007 to 0.02 g/g-h respectively with arabitol yield of 55% from glycerol.