Gene loss and cis-regulatory novelty shaped core histone gene evolution in the apiculate yeast Hanseniaspora uvarum.

Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation-mediated by the trans-regulator Spt10-is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage.

Insights into the identification and evolutionary conservation of key genes in the transcriptional circuits of meiosis initiation and commitment in budding yeast.

Initiation of meiosis in budding yeast does not commit the cells for meiosis. Thus, two distinct signaling cascades may differentially regulate meiosis initiation and commitment in budding yeast. To distinguish between the role of these signaling cascades, we reconstructed protein-protein interaction networks and gene regulatory networks with upregulated genes in meiosis initiation and commitment. Analyzing the integrated networks, we identified four master regulators (MRs) [Ume6p, Msn2p, Met31p, Ino2p], three transcription factors (TFs), and 279 target genes (TGs) unique for meiosis initiation, and three MRs [Ndt80p, Aro80p, Rds2p], 11 TFs, and 948 TGs unique for meiosis commitment. Functional enrichment analysis of these distinct members from the transcriptional cascades for meiosis initiation and commitment revealed that nutritional cues rewire gene expression for initiating meiosis and chromosomal recombination commits cells to meiosis. As meiotic chromosomal recombination is highly conserved in eukaryotes, we compared the evolutionary rate of unique members in the transcriptional cascade of two meiotic phases of Saccharomyces cerevisiae with members of the phylum Ascomycota, revealing that the transcriptional cascade governing chromosomal recombination during meiosis commitment has experienced greater purifying selection pressure (P value = 0.0013, 0.0382, 0.0448, 0.0369, 0.02967, 0.04937, 0.03046, 0.03357 and < 0.00001 for Ashbya gossypii, Yarrowia lipolytica, Debaryomyces hansenii, Aspergillus fumigatus, Neurospora crassa, Kluyveromyces lactis, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, and Schizosaccharomyces octosporus, respectively). This study demarcates crucial players driving meiosis initiation and commitment and demonstrates their differential rate of evolution in budding yeast.

Debaryomyces hansenii: an old acquaintance for a fresh start in the era of the green biotechnology.

The halophilic yeast Debaryomyces hansenii has been studied for several decades, serving as eukaryotic model for understanding salt and osmotic tolerance. Nevertheless, lack of consensus among different studies is found and, sometimes, contradictory information derived from studies performed in very diverse conditions. These two factors hampered its establishment as the key biotechnological player that was called to be in the past decade. On top of that, very limited (often deficient) engineering tools are available for this yeast. Fortunately Debaryomyces is again gaining momentum and recent advances using highly instrumented lab scale bioreactors, together with advanced -omics and HT-robotics, have revealed a new set of interesting results. Those forecast a very promising future for D. hansenii in the era of the so-called green biotechnology. Moreover, novel genetic tools enabling precise gene editing on this yeast are now available. In this review, we highlight the most recent developments, which include the identification of a novel gene implicated in salt tolerance, a newly proposed survival mechanism for D. hansenii at very high salt and limiting nutrient concentrations, and its utilization as production host in biotechnological processes.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Characterization of the microbiota and chemical properties of pork loins during dry aging.

Dry aging (DA) allows for the storage of meat without packaging at 0 to 3°C for several weeks. It enhances the production of pleasant flavors, tenderness, and juiciness in meat. Due to the long storage period and roles of indigenous microbiota in the maturation of several meat products, the microbiota of DA meat is of interest in terms of microbial contributions and food hygiene but has not yet been characterized in detail. This study identified the microbiota of pork loins during DA using culturing and culture-independent meta-16S rRNA gene sequencing and elucidated its characteristics. The amounts of free amino acids and profiles of aroma-active compounds were also monitored by high-performance liquid chromatography and gas chromatography, respectively. The meta-16S rRNA gene sequencing revealed that Pseudomonas spp. generally dominated the microbiota throughout DA; however, the culturing analysis showed marked changes in the species composition during DA. Acinetobacter spp. were the second most dominant bacteria before DA in the culture-independent analysis but became a minor population during DA. The cell numbers of yeasts showed an increased tendency during DA, and Debaryomyces hansenii was the only microorganism isolated from all meat samples throughout DA. Well-known foodborne pathogens were not observed in two microbiota analyses. The amounts of free amino acids were increased by DA, and the number of aroma-active compounds and their flavor dilution values markedly changed during DA. Most microbial isolates showed positive reactions with proteolytic and lipolytic activities, suggesting their contribution to tenderness and aroma production in DA meats.

Tolerance to Oxidative Stress in Budding Yeast by Heterologous Expression of Catalases A and T from Debaryomyces hansenii.

The function of catalases A and T from the budding yeast Saccharomyces cerevisiae (ScCta1 and ScCtt1) is to decompose hydrogen peroxide (H2O2) to mitigate oxidative stress. Catalase orthologs are widely found in yeast, suggesting that scavenging H2O2 is crucial to avoid the oxidative damage caused by reactive oxygen species (ROS). However, the function of catalase orthologs has not yet been experimentally characterized in vivo. Here, we heterologously expressed Debaryomyces hansenii DhCTA1 and DhCTT1 genes, encoding ScCta1 and ScCtt1 orthologs, respectively, in a S. cerevisiae acatalasemic strain (cta1Δ ctt1Δ). We performed a physiological analysis evaluating growth, catalase activity, and H2O2 tolerance of the strains grown with glucose or ethanol as carbon source, as well as under NaCl stress. We found that both genes complement the catalase function in S. cerevisiae. Particularly, the strain harboring DhCTT1 showed improved growth when ethanol was used as carbon source both in the absence or presence of salt stress. This phenotype is attributed to the high catalase activity of DhCtt1 detected at the exponential growth phase, which prevents intracellular ROS accumulation and confers oxidative stress resistance.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Screening, identification, and low-energy ion modified breeding of a yeast strain producing high level of 3-hydroxypropionic acid.

3-Hydroxypropionic acid (3HP) is an important platform chemical with a wide range of applications. The biological preparation of this compound is safe and low cost. In this study, orchard soil and human waste were used as raw materials to screen microbial strains that could produce 3HP in selective medium containing varying amounts of propionic acid. A yeast strain that can use propionic acid as substrate and produce 48.96 g/L 3HP was screened. Morphological observation, physiological and biochemical identification, and 26s rDNA sequencing identified the IS451 strain as Debaryomyces hansenii. The low-energy ion N+ , with the energy of 10 keV and a dose of 70 × 2.6 × 1013  ions/cm2 , was implanted into the IS451 strain. The mutant strain WT39, whose 3HP titer reached 62.42 g/L, was obtained. The strain exhibited genetic stability and tolerance to high concentrations of propionic acid and was considered to have broad application prospects.

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.

Extensive loss of cell-cycle and DNA repair genes in an ancient lineage of bipolar budding yeasts.

Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine-Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 (WHI5), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [MAD1], Mitotic Arrest-Deficient 2 [MAD2]) and DNA-damage-checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [MEC3], RADiation sensitive 9 [RAD9]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 (MAG1), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 (PHR1), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [POL4] and POL32) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [RIF1], Replication Factor A 3 [RFA3], Cell Division Cycle 13 [CDC13], Pbp1p Binding Protein [PBP2]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.

Salinity and high pH affect energy pathways and growth in Debaryomyces hansenii.

The physiological behavior of Debaryomyces hansenii in response to saline stress and elevated pH was studied. The combination of 1 M NaCl salt and pH 8.0 was required to produce significant changes in the lag phase of growth and a consequent effect on viability. pH 8.0 in the absence or presence of 1 M NaCl produced changes in physiological functions such as respiration, acidification, rubidium transport, transmembrane potential, and fermentation. Our data indicated a stimulation of the H+-ATPase of the plasma membrane at pH 8.0, which increased the transmembrane potential and favored the entry of Na+; this effect was intensified in the presence of NaCl, so the increased energy expenditure resulting from H+ pumping and the extrusion of excess Na+ affected viability. The gene expression pattern studied by microarrays of cells incubated under saline conditions and high pH revealed a down-regulation in genes related to energy-producing pathways and in some genes involved in the cell cycle and DNA transcription, confirming our experimental hypothesis. Although D. hansenii can tolerate high pH and high salt concentrations, its physiological behavior, is better at pH 6.0 and in the absence of sodium; thus, it is an alkali-halotolerant yeast and not a halophilic yeast as previously proposed by other authors.

Cheese brines from Danish dairies reveal a complex microbiota comprising several halotolerant bacteria and yeasts.

The Danish Danbo cheese is a surface ripened semi-hard cheese, which before ripening is submerged in brine for up to 24 h. The brining is required in order to obtain the structural and organoleptic properties of the cheeses. Likewise, the content of NaCl in the cheese will influence especially the surface microbiota being of significant importance for flavour development and prevention of microbial spoilage. Even though the microbiota on cheese surfaces have been studied extensively, limited knowledge is available on the occurrence of microorganisms in cheese brine. The aim of the present study was to investigate by both culture-dependent and -independent techniques the brine microbiota in four Danish dairies producing Danbo cheese. The pH of the brines varied from 5.1 to 5.6 with a dry matter content from 20 to 27% (w/w). The content of lactate varied from 4.1 to 10.8 g/L and free amino acids from 65 to 224 mg/L. Bacteria were isolated on five different media with NaCl contents of 0.85-23.0% (w/v) NaCl. The highest count of 6.3 log CFU/mL was obtained on TSA added 4% (w/v) NaCl. For yeasts, the highest count was 3.7 log CFU/mL on MYGP added 8% (w/v) NaCl. A total of 31 bacterial and eight eukaryotic species were isolated including several halotolerant and/or halophilic species. Among bacteria, counts of ≥6.0 log CFU/mL were obtained for Tetragenococcus muriaticus and Psychrobacter celer, while counts between ≥4.5 and < 6.0 log CFU/mL were obtained for Lactococcus lactis, Staphylococcus equorum, Staphylococcus hominis, Chromohalobacter beijerinckii, Chromohalobacter japonicus and Microbacterium maritypicum. Among yeasts, counts of ≥3.5 log CFU/mL were only obtained for Debaryomyces hansenii. By amplicon-based high-throughput sequencing of 16S rRNA gene and ITS2 regions for bacteria and eukaryotes respectively, brines from the same dairy clustered together indicating the uniqueness of the dairy brine microbiota. To a great extent the results obtained by amplicon sequencing fitted with the culture-dependent technique though each of the two methodologies identified unique genera/species. Dairy brine handling procedures as e.g. microfiltration were found to influence the brine microbiota. The current study proves the occurrence of a specific dairy brine microbiota including several halotolerant and/or halophilic species most likely of sea salt origin. The importance of these species during especially the initial stages of cheese ripening and their influence on cheese quality and safety need to be investigated. Likewise, optimised brine handling procedures and microbial cultures are required to ensure an optimal brine microbiota.

Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire.

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while ß-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

Debaryomyces hansenii Strains from Valle De Los Pedroches Iberian Dry Meat Products: Isolation, Identification, Characterization, and Selection for Starter Cultures.

Yeasts, filamentous fungi, and bacteria colonize the surface of fermented sausages during the ripening process. The source of this microbiota is their surrounding environment, and is influenced by the maturing conditions and starter cultures. Debaryomyces hansenii was previously isolated from several dry-cured meat products and associated with the lipolytic and proteolytic changes that occur in these products, influencing their taste and flavor. Therefore, this study isolated the yeast microbiota present in the casing from different meat products ("lomo," "chorizo," and "salchichón") from the Valle de los Pedroches region in southern Spain. D. hansenii was by far the most abundant species in each product, as all 22 selected isolates were identified as D. hansenii by biochemical and/or molecular methods. In contrast, no yeasts were found in the meat batter. These data constitute the first study of the yeasts present in "lomo" sausages and particularly the highly appreciated Valle de los Pedroches "lomo" sausages. Furthermore, the resistance of these isolates to different pHs, temperatures, and saline stress was studied, together with their catabolic characteristics. Based on the results, certain isolates are proposed as valuable candidate starter cultures that could improve both the manufacture and the flavor of such dry-cured meat products, and provide an understanding of new mechanisms involved in stress tolerance. Applied mediumscale industrial tests are currently in progress.

Screening of Debaryomyces hansenii Strains for Flavor Production under a Reduced Concentration of Nitrifying Preservatives Used in Meat Products.

A total of 15 Debaryomyces hansenii strains from different food origins were genetically characterized and tested on a culture medium resembling the composition of fermented sausages but different concentrations of nitrifying preservatives. Genetic typing of the D. hansenii strains revealed two levels of discrimination: isolation source or strain specific. Different abilities to proliferate on culture media containing different concentrations of nitrate and nitrite, as sole nitrogen sources and in the presence of amino acids, were observed within D. hansenii strains. Overall metabolism of amino acids and generation of aroma compounds were related to the strain origin of isolation. The best producers of branched aldehydes and ethyl ester compounds were strains isolated from pork sausages. Strains from cheese and llama sausages were good producers of ester compounds and branched alcohols, while vegetable strains produced mainly acid compounds. Nitrate and nitrite reduction affected in different ways the production of volatiles by D. hansenii.

Functional analysis of Debaryomyces hansenii Rpn4 on a genetic background of Saccharomyces cerevisiae.

The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

Transcriptomic analysis of cobalt stress in the marine yeast Debaryomyces hansenii.

The yeast Debaryomyces hansenii overproduces riboflavin upon exposure to subtoxic levels of cobalt (Co(+2)). However, mechanisms for survival have yet to be studied and have been hindered by D. hansenii's high genetic heterogeneity among strains. In this study, we used transcriptomic analyses and RNA-seq in order to identify differentially expressed genes in D. hansenii in response to cobalt exposure. Highly upregulated genes under this condition were identified to primarily comprise DNA damage and repair genes, oxidative stress response genes, and genes for cell wall integrity and growth. The main response of D. hansenii to heavy metal stress is the activation of non-enzymatic oxidative stress response mechanisms and control of biological production of reactive oxygen species. Our results indicate that D. hansenii does not seem to be pre-adapted to survive high concentrations of heavy metals. These organisms appear to possess genetic survival and detoxification mechanisms that enable the cells to recover from heavy metal stress.

The Debaryomyces hansenii carboxylate transporters Jen1 homologues are functional in Saccharomyces cerevisiae.

We have functionally characterized the four Saccharomyces cerevisiae (Sc) Jen1 homologues of Debaryomyces hansenii (Dh) by heterologous expression in S. cerevisiae. Debaryomyces hansenii cells display mediated transport for the uptake of lactate, acetate, succinate and malate. DHJEN genes expression was detected by RT-PCR in all carbon sources assayed, namely lactate, succinate, citrate, glycerol and glucose. The heterologous expression in the S. cerevisiae W303-1A jen1Δ ady2Δ strain demonstrated that the D. hansenii JEN genes encode four carboxylate transporters. DH27 gene encodes an acetate transporter (Km 0.94 ± 0.17 mM; Vmax 0.43 ± 0.03 nmol s(-1) mg(-1)), DH17 encodes a malate transporter (Km 0.27 ± 0.04 mM; Vmax 0.11 ± 0.01 nmol s(-1) mg(-1)) and both DH18 and DH24 encode succinate transporters with the following kinetic parameters, respectively, Km 0.31 ± 0.06 mM; Vmax 0.83 ± 0.04 nmol s(-1) mg(-1)and Km 0.16 ± 0.02 mM; Vmax 0.19 ± 0.02 nmol s(-1) mg(-1). Surprisingly, no lactate transporter was found, although D. hansenii presents a mediated transport for this acid. This work advanced the current knowledge on yeast carboxylate transporters by characterizing four new plasma membrane transporters in D. hansenii.

Identification of key residues in Debaryomyces hansenii carbonyl reductase for highly productive preparation of (S)-aryl halohydrins.

Key residues of Debaryomyces hansenii carbonyl reductase in the determination of the reducing activity towards aryl haloketones were identified through combinatorial mutation of conserved residues. This study provides a green and efficient biocatalyst for the synthesis of (S)-aryl halohydrins.