Utilization of salt-rich by-products from the dairy industry as feedstock for recombinant protein production by Debaryomyces hansenii.

The dairy industry processes vast amounts of milk and generates high amounts of secondary by-products, which are still rich in nutrients (high Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) levels) but contain high concentrations of salt. The current European legislation only allows disposing of these effluents directly into the waterways with previous treatment, which is laborious and expensive. Therefore, as much as possible, these by-products are reutilized as animal feed material and, if not applicable, used as fertilizers adding phosphorus, potassium, nitrogen, and other nutrients to the soil. Finding biological alternatives to revalue dairy by-products is of crucial interest in order to improve the utilization of dry dairy matter and reduce the environmental impact of every litre of milk produced. Debaryomyces hansenii is a halotolerant non-conventional yeast with high potential for this purpose. It presents some beneficial traits - capacity to metabolize a variety of sugars, tolerance to high osmotic environments, resistance to extreme temperatures and pHs - that make this yeast a well-suited option to grow using complex feedstock, such as industrial waste, instead of the traditional commercial media. In this work, we study for the first time D. hansenii's ability to grow and produce a recombinant protein (YFP) from dairy saline whey by-products. Cultivations at different scales (1.5, 100 and 500 ml) were performed without neither sterilizing the medium nor using pure water. Our results conclude that D. hansenii is able to perform well and produce YFP in the aforementioned salty substrate. Interestingly, it is able to outcompete other microorganisms present in the waste without altering its cell performance or protein production capacity.

Overlapping responses between salt and oxidative stress in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.

Study of the counts, species and characteristics of the yeast population during the manufacture of dry-cured "lacón". Effect of salt level.

The aim of this work was to study the yeast population during the manufacture of dry-cured "lacón" (a Spanish traditional meat product) and the effect of the salting time. For this study, six batches of "lacón" were manufactured with three different salting times (LS (3 days of salting), MS (4 days of salting) and HS (5 days of salting)). Yeast counts increased significantly (P < 0.001) during the whole process from 2.60 to 6.37 log cfu/g. An increased length of salting time did not affect yeast counts throughout the manufacture of dry-cured "lacón", although the highest yeast counts were obtained from LS batches. A total of 226 isolates were obtained from dry-cured "lacón" during drying-ripening stage, of which 151 were yeasts and were identified at the species level using molecular techniques. The total of 151 identified yeasts belonged to 4 different genera: Debaryomyces, Candida, Cryptococcus and Rhodotorula. Debaryomyces hansenii was the most abundant species isolated throughout the whole process as much in the interior as in the exterior of the pieces of three salt levels of "lacón" studied, while Candida zeylanoides was only isolated from the interior of MS and HS batches and from the exterior of LS and HS groups, but at lesser proportion than D. hansenii.

Draft genome sequence of salt-tolerant yeast Debaryomyces hansenii var. hansenii MTCC 234.

Debaryomyces hansenii is one of the most halotolerant species of yeast, and the genome sequence of D. hansenii strain CBS767 is already available. Here we report the 11.46-Mb draft genome of D. hansenii strain MTCC 234, which is even more halotolerant than strain CBS767. Comparative analysis of these sequences would definitely provide further insight into the halotolerance of this yeast.

Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

UNLABELLED: Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. PRACTICAL APPLICATION: Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes.

DhARO4 induction and tyrosine nitration in response to reactive radicals generated by salt stress in Debaryomyces hansenii.

It has been previously reported that growth of Debaryomyces hansenii in 2 M NaCl induced the expression of ARO4. This gene codifies for DhAro4p, involved in the synthesis of the amino acid tyrosine. In this work we studied the activity of DhAro4p upon salt stress; a higher activity was observed in cells grown with 2 M NaCl, but tyrosine levels were not increased. On the other hand, the addition of tyrosine to the saline medium significantly enhanced the growth of D. hansenii. It was found that the oxidized form of tyrosine, 3-nitrotyrosine, increased in the presence of salt. Since NaCl protects against oxidative stress in D. hansenii (Navarrete et al., 2009), we propose that a protective pathway is the de novo synthesis of tyrosine and its immediate oxidation to 3-nitrotyrosine to counteract oxidative stress generated by salt stress, so we measured the production of reactive oxygen species (ROS) and nitric oxide (NO⁻) in D. hansenii after growing in 2 M NaCl. Results showed the presence of NO⁻ and the increased production of ROS; this is probably due to an increased respiratory activity in the cells grown in the presence of salt. Our results demonstrate that upon salt stress D hansenii responds to oxidative stress via the transcriptional activation of specific genes such as DhARO4.

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp.

BACKGROUND: The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. RESULTS: The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. CONCLUSION: Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

Cell wall involvement in the glycerol response to high osmolarity in the halotolerant yeast Debaryomyces hansenii.

Osmotic stress was studied through the induction of the gene coding for glycerol 3-phosphate dehydrogenase (DhGPD1) in the halotolerant yeast Debaryomyces hansenii. This yeast responded to modifications in turgor pressure by stimulating the transcription of DhGPD1 when exposed to solutes that cause turgor stress (NaCl or sorbitol), but did not respond to water stress mediated by ethanol. In contrast to what has been documented to occur in Saccharomyces cerevisiae, D. hansenii protoplasts did not show induction in the transcription of DhGPD1 showing a limitation in their response to solute stress. The results presented indicate that the presence of the cell wall is of significance for the induction of DhGPD1 and hence for osmotic regulation in halotolerant D. hansenii. It appears that the main osmosensor that links high osmolarity with glycerol accumulation may be of a different nature in this yeast.

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations.

The highly NaCl-tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra- and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl-tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species.

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.

Application of central composite design and response surface methodology to the fermentation of olive juice by Lactobacillus plantarum and Debaryomyces hansenii.

The objectives of this study were to investigate the effects of NaCl, calcium acetate and calcium lactate in concentrations corresponding to ionic strengths equivalent to 2-10%, w/v salt brines as well as the 50% replacement of NaCl contained in the above mixture by KCl. A central composite design and response surface methodology were used to optimize the maximum specific growth rate of Lactobacillus plantarum ATCC 8014 and Debaryomyces hansenii 2114. The fermentation was carried out in olive juice obtained from Kalamon black olives at 30 degrees C with initial pH 5.0. Mathematical models describing the combined effects of these factors on the maximum specific growth rate of L. plantarum or D. hansenii were established. Both strains in single cultures showed higher maximum specific growth rate in olive juice supplemented with NaCl/KCl, Ca-acetate and Ca-lactate. But in mixed culture fermentations of olive juice supplemented with NaCl, Ca-acetate and Ca-lactate, higher specific growth rates were obtained. Under the optimum growth conditions determined for the single culture fermentations, i.e. 378.4 mM NaCl, 34.1 mM Ca-acetate and 39.9 mM Ca-lactate, mixed culture fermentation was undertaken by varying the time of inoculation of the yeast strain. When D. hansenii was inoculated 48 h before L. plantarum the maximum specific growth rate of L. plantarum was increased to 0.247 per hour, which was significantly higher compared to L. plantarum alone (0.211 per hour). In mixed culture fermentation of olive juice supplemented with the mixture of NaCl/KCl under similar conditions as above, a maximum specific growth rate of L. plantarum of 0.218 per hour was determined. The optimum conditions determined for mixed culture fermentation are useful in fermentation of black olives Kalamon variety under lower salt content.

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.

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses.

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].

Cloning and expression of two genes coding for sodium pumps in the salt-tolerant yeast Debaryomyces hansenii.

Two genes encoding Na(+)-ATPases from Debaryomyces hansenii were cloned and sequenced. The genes, designated ENA1 from D. hansenii (DhENA1) and DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis. DhENA1 was expressed in the presence of high Na(+) concentrations, while the expression of DhENA2 also required high pH. A mutant of Saccharomyces cerevisiae lacking the Na(+) efflux systems and sensitive to Na(+), when transformed with DhENA1 or DhENA2, recovered Na(+) tolerance and also the ability to extrude Na(+).

Monovalent cation fluxes and physiological changes of Debaryomyces hansenii grown at high concentrations of KCl and NaCl.

Debaryomyces hansenii showed an increased growth in the presence of either 1 M, KCl or 1 M NaCl and a low acidification of the medium, higher for the cells grown in the presence of NaCl. These cells accumulated high concentrations of the cations, and showed a very fast capacity to exchange either Na+ or K+ for the opposite cation. They showed a rapid uptake of 86Rb+ and 22Na+. 86Rb+ transport was saturable, with K(m) and Vmax values higher for cells grown in 1 M NaCl. 22Na+ uptake showed a diffusion component, also higher for the cells grown with NaCl. Changes depended on growth conditions, and not on further incubation, which changed the internal ion concentration. K+ stimulated proton pumping produced a rapid extrusion of protons, and also a decrease of the membrane potential. Cells grown in 1 M KCl showed a higher fermentation rate, but significantly lower respiratory capacity. ATP levels were higher in cells grown in the presence of NaCl; upon incubation with glucose, those grown in the presence of KCl reached values similar to the ones grown in the presence of NaCl. In both, the addition of KCl produced a transient decrease of the ATP levels. As to ion transport mechanisms, D. hansenii appears to have (a) an ATPase functioning as a proton pump, generating a membrane potential difference which drives K+ through a uniporter; (b) a K+/H+ exchange system; and (c) a rapid cation/cation exchange system. Most interesting is that cells grown in different ionic environments change their studied capacities, which are not dependent on the cation content, but on differences in their genetic expression during growth.

Hybrids obtained by protoplast fusion with a salt-tolerant yeast.

An industrial strain of Saccharomyces cerevisiae was fused with an osmotolerant yeast, Debaryomyces hansenii, to obtain hybrids having increased tolerance to elevated salt concentrations. The hybrids were intermediate to parent species in production of ethanol and polyols.

Osmotic significance of glycerol accumulation in exponentially growing yeasts.

Natural-abundance 13C-nuclear magnetic resonance spectroscopy has shown glycerol to be the major osmotically significant low-molecular-weight solute in exponentially growing, salt-stressed cells of the yeasts Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Debaromyces hansenii. Measurement of the intracellular nonosmotic volume (i.e., the fraction of the cell that is osmotically unresponsive) by using the Boyle-van't Hoff relationship (for nonturgid cells, the osmotic volume is directly proportional to the reciprocal of the external osmotic pressure) showed that the nonosmotic volume represented up to 53% of the total cell volume; the highest values were recorded in media with maximum added NaCl. Determinations of intracellular glycerol levels with respect to cell osmotic volumes showed that increases in intracellular glycerol may counterbalance up to 95% of the external osmotic pressure due to added NaCl. The lack of other organic osmotica in 13C-nuclear magnetic resonance spectra indicates that inorganic ions may constitute the remaining component of intracellular osmotic pressure.

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.