Influence of prefoldin subunit 4 on the tolerance of Kluyveromyces marxianus to lignocellulosic biomass-derived inhibitors.

BACKGROUND: Kluyveromyces marxianus is a potentially excellent host for microbial cell factories using lignocellulosic biomass, due to its thermotolerance, high growth rate, and wide substrate spectrum. However, its tolerance to inhibitors derived from lignocellulosic biomass pretreatment needs to be improved. The prefoldin complex assists the folding of cytoskeleton which relates to the stress tolerance, moreover, several subunits of prefoldin have been verified to be involved in gene expression regulation. With the presence of inhibitors, the expression of a gene coding the subunit 4 of prefoldin (KmPFD4), a possible transcription factor, was significantly changed. Therefore, KmPFD4 was selected to evaluate its functions in inhibitors tolerance. RESULTS: In this study, the disruption of the prefoldin subunit 4 gene (KmPFD4) led to increased concentration of intracellular reactive oxygen species (ROS) and disturbed the assembly of actin and tubulin in the presence of inhibitors, resulting in reduced inhibitor tolerance. Nuclear localization of KmPFD4 indicated that it could regulate gene expression. Transcriptomic analysis showed that upregulated gene expression related to ROS elimination, ATP production, and NAD+ synthesis, which is a response to the presence of inhibitors, disappeared in KmPFD4-disrupted cells. Thus, KmPFD4 impacts inhibitor tolerance by maintaining integration of the cytoskeleton and directly or indirectly affecting the expression of genes in response to inhibitors. Finally, overexpression of KmPFD4 enhanced ethanol fermentation with a 46.27% improvement in productivity in presence of the inhibitors. CONCLUSION: This study demonstrated that KmPFD4 plays a positive role in the inhibitor tolerance and can be applied for the development of inhibitor-tolerant platform strains.

Polysaccharide Hydrogels for the Protection of Dairy-Related Microorganisms in Adverse Environmental Conditions.

Adverse environmental conditions are severely limiting the use of microorganisms in food systems, such as probiotic delivery, where low pH causes a rapid decrease in the survival of ingested bacteria, and mixed-culture fermentation, where stepwise changes and/or metabolites of individual microbial groups can hinder overall growth and production. In our study, model probiotic lactic acid bacteria (L. plantarum ATCC 8014, L. rhamnosus GG) and yeasts native to dairy mixed cultures (K. marxianus ZIM 1868) were entrapped in an optimized (cell, alginate and hardening solution concentration, electrostatic working parameters) Ca-alginate system. Encapsulated cultures were examined for short-term survival in the absence of nutrients (lactic acid bacteria) and long-term performance in acidified conditions (yeasts). In particular, the use of encapsulated yeasts in these conditions has not been previously examined. Electrostatic manufacturing allowed for the preparation of well-defined alginate microbeads (180-260 µm diameter), high cell-entrapment (95%) and viability (90%), and uniform distribution of the encapsulated cells throughout the hydrogel matrix. The entrapped L. plantarum maintained improved viabilities during 180 min at pH 2.0 (19% higher when compared to the free culture), whereas, L. rhamnosus appeared to be less robust. The encapsulated K. marxianus exhibited double product yields in lactose- and lactic acid-modified MRS growth media (compared to an unfavorable growth environment for freely suspended cells). Even within a conventional encapsulation system, the pH responsive features of alginate provided superior protection and production of encapsulated yeasts, allowing several applications in lacto-fermented or acidified growth environments, further options for process optimization, and novel carrier design strategies based on inhibitor charge expulsion.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

Potassium and Sodium Salt Stress Characterization in the Yeasts Saccharomyces cerevisiae, Kluyveromyces marxianus, and Rhodotorula toruloides.

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is a vital cell factory, but more diverse cell factories are essential for the sustainable use of natural resources. Here, we benchmarked nonconventional yeasts Kluyveromyces marxianus and Rhodotorula toruloides against S. cerevisiae strains CEN.PK and W303 for their responses to potassium and sodium salt stress. We found an inverse relationship between the maximum growth rate and the median cell volume that was responsive to salt stress. The supplementation of K+ to CEN.PK cultures reduced Na+ toxicity and increased the specific growth rate 4-fold. The higher K+ and Na+ concentrations impaired ethanol and acetate metabolism in CEN.PK and acetate metabolism in W303. In R. toruloides cultures, these salt supplementations induced a trade-off between glucose utilization and cellular aggregate formation. Their combined use increased the beta-carotene yield by 60% compared with that of the reference. Neural network-based image analysis of exponential-phase cultures showed that the vacuole-to-cell volume ratio increased with increased cell volume for W303 and K. marxianus but not for CEN.PK and R. toruloides in response to salt stress. Our results provide insights into common salt stress responses in yeasts and will help design efficient bioprocesses. IMPORTANCE Characterization of microbial cell factories under industrially relevant conditions is crucial for designing efficient bioprocesses. Salt stress, typical in industrial bioprocesses, impinges upon cell volume and affects productivity. This study presents an open-source neural network-based analysis method to evaluate volumetric changes using yeast optical microscopy images. It allows quantification of cell and vacuole volumes relevant to cellular physiology. On applying salt stress in yeasts, we found that the combined use of K+ and Na+ improves the cellular fitness of Saccharomyces cerevisiae strain CEN.PK and increases the beta-carotene productivity in Rhodotorula toruloides, a commercially important antioxidant and a valuable additive in foods.

Adaptive responses of Kluyveromyces marxianus CCT 7735 to 2-phenylethanol stress: Alterations in membrane fatty-acid composition, ergosterol content, exopolysaccharide production and reduction in reactive oxygen species.

2-phenylethanol (2-PE) is a higher aromatic alcohol with a rose-like aroma used in the cosmetic and food industries as a flavoring and displays potential for application as an antifungal. Biotechnological production of 2-PE from yeast is an interesting alternative due to the non-use of toxic compounds and the generation of few by-products. Kluyveromyces marxianus CCT 7735 is a thermotolerant strain capable of producing high 2-PE titers from L-Phenylalanine; however, like other yeast species, its growth has been strongly inhibited by this alcohol. Herein, we aimed to evaluate the effect of 2-PE on cell growth, cell viability, membrane permeability, glucose uptake, metabolism, and morphology in K. marxianus CCT 7735, as well as its adaptive responses. The stress condition was imposed after 4 h of cultivation by adding 3.0 g.L-1 of 2-PE in exponential growing cells. 2-PE stress impaired yeast growth, glucose uptake, fermentative metabolism, membrane permeability, and cell viability. Moreover, the stress condition provoked changes in both morphology and surface roughness. The reactive oxygen species (ROS) increased immediately on exposure to 2-PE. Changes in membrane fatty-acid composition, ergosterol content, exopolysaccharides production, and reduction of the ROS levels appear to be the result of adaptive responses in K. marxianus. Our results provided insights into a better understanding of the effects of 2-PE on K. marxianus and its adaptive responses.

UPC2 gene deletion modifies sterol homeostasis and susceptibility to metabolic inhibitors in Kluyveromyces lactis.

Kluyveromyces lactis Upc2p is an ortholog of Upc2p/Ecm22p transcription factors involved in regulation of sterol import and sterol homeostasis in Saccharomyces cerevisiae. In this work, we investigated the role of Upc2p in K. lactis. The absence of KlUpc2p significantly reduced the tolerance of mutant cells to antifungal azoles and Li+ cations. Reduced expression of genes from the late ergosterol pathway results in a decreased ergosterol content and altered plasma membrane-associated functions in Klupc2 mutant cells-the plasma membrane is hyperpolarized, and its fluidity is reduced. KlUpc2p contributes to transcriptional upregulation of KlENA1, KlPMA1 and KlYAP1 under azole stress. Our study demonstrates that KlUpc2p is involved in the regulation of ergosterol homeostasis in K. lactis. The analysis of KlPMA1 and KlPDR12 transcripts in wild-type and Klupc2Δ mutant strains showed that KlUpc2p acts as an activator or as a repressor depending upon its target.

Dielectric property measurements as a method to determine the physiological state of Kluyveromyces marxianus and Saccharomyces cerevisiae stressed with furan aldehydes.

Cell physiology parameters are essential aspects of biological processes; however, they are difficult to determine on-line. Dielectric spectroscopy allows the on-line estimation of viable cells and can provide important information about cell physiology during culture. In this study, we investigated the dielectric property variations in Kluyveromyces marxianus SLP1 and Saccharomyces cerevisiae ERD yeasts stressed by 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde during aerobic growth. The dielectric properties of cell permittivity, specific membrane capacitance (Cm), and intracellular conductivity (σIn) were considerably affected by furan aldehydes in the same way that the cell population, viability, cell size, substrate consumption, organic acid production, and respiratory parameters were. The yeasts stressed with furan aldehydes exhibited three physiological states (φ): adaptation, replicating, and nonreplicating states. During the adaptation state, there were small and stable signs of permittivity, Cm, and σIn; additionally, no cell growth was observed. During the replicating state, cell growth was restored, and the cell viability increased; in addition, the permittivity and σIn increased rapidly and reached their maximum values, while the Cm decreased. In the nonreplicating state, the permittivity and σIn were stable, and Cm decreased to its minimum value. Our results demonstrated that knowing dielectric properties allowed us to obtain information about the physiological state of the cells under control and stressed conditions. Since the permittivity, Cm, and σIn are directly associated with the physiological state of the yeast, these results should contribute to a better understanding of the stress response of yeasts and open the possibility to on-line monitor and control the physiological state of the cell in the near future.

Effects of intrinsic microbial stress factors on viability and physiological condition of yeasts isolated from spontaneously fermented cereal doughs.

Fermented cereal doughs constitute a predominant part of West African diets. The environment of fermented doughs can be hostile for microbial survival due to high levels of microbial metabolites such as weak carboxylic organic acids and ethanol. In order to get a better understanding of the intrinsic factors affecting the microbial successions of yeasts during dough fermentation, survival and physiological responses of the yeasts associated with West African fermented cereal doughs were investigated at exposure to relevant concentrations of microbial inhibitory compounds. Three strains each of the predominant species, i.e. Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kudriavzevii as well as the opportunistic pathogen Candida glabrata were studied. The strains were exposed to individual stress factors of cereal doughs, i.e. (i) pH 3.4, (ii) 3% (v/v) ethanol (EtOHpH3.4), (iii) 285 mM lactic acid (LApH3.4) and (iv) 150 mM acetic acid (AApH3.4) as well as to combinations of these stress factors, i.e. (v) (LA + AA)pH 3.4 and (vi) (LA + AA+EtOH)pH 3.4. Growth and single cell viability were studied by flow cytometry using combined SYTO 13 and propidium iodide (PI) staining. Intracellular pH (pHi), plasma membrane integrity and micro-colony development of stressed cells were studied by fluorescence microscopy using PI and carboxyfluorescein diacetate succinimidyl ester (CFDA-se). Viability of the yeast strains was not affected by pH 3.4 and 3% (v/v) ethanol (EtOHpH3.4). 285 mM lactic acid (LApH3.4) reduced the specific growth rate (µmax) from 0.27-0.41 h-1 to 0.11-0.26 h-1 and the viability from 100% to 2.6-41.7% at 72 h of exposure in most yeast strains, except for two strains of C. glabrata. 150 mM acetic acid (AApH3.4) as well as the combinations (LA + AA)pH 3.4 and (LA + AA+EtOH)pH 3.4 reduced µmax to 0.0 h-1 and induced significant cell death for all the yeast strains. Exposed to (LA + AA+EtOH)pH 3.4, the most resistant yeast strains belonged to S. cerevisiae followed by P. kudriavzevii, whereas C. glabrata and K. marxianus were more sensitive. Strain variations were observed within all four species. When transferred to non-stress conditions, i.e. MYGP, pH 5.6, after exposure to (LA + AA+EtOH)pH 3.4 for 6 h, 45% of the single cells of the most resistant S. cerevisiae strain kept their plasma membrane integrity, recovered their pHi to near physiological range (pHi = 6.1-7.4) and resumed proliferation after 3-24 h of lag phase. The results obtained are valuable in order to change processing conditions of the dough to favor the survival of preferable yeast species, i.e. S. cerevisiae and K. marxianus and inhibit opportunistic pathogen yeast species as C. glabrata.

Inhibitory effect of four novel synthetic peptides on food spoilage yeasts.

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 µg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 µg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.

Effect of thermal treatment of whey contaminated with antibiotics on the growth of Kluyveromyces marxianus.

The objective of the studies reported in this research communication was to investigate the use of whey contaminated with antibiotics such as cephalosporins, quinolones and tetracyclines as a nutrient medium for the growth of Kluyveromyces marxianus with particular attention to the effect of thermal treatment used to overcome the inhibitory effects of antibiotic concentrations close to the Maximum Residue Limits. The heat treatments at 120 °C for 40 min, 120 °C for 83 min, and 120 °C for 91 min caused total inactivation of cephalosporins, tetracyclines and quinolone residues in whey respectively.

Stb5p is involved in Kluyveromyces lactis response to 4-nitroquinoline-N-oxide stress.

In yeast, the STB5 gene encodes a transcriptional factor belonging to binuclear cluster class (Zn2Cys6) of transcriptional regulators specific to ascomycetes. In this study, we prepared the Kluyveromyces lactis stb5Δ strain and assessed its responses to different stresses. We showed that KlSTB5 gene is able to complement the deficiencies of Saccharomyces cerevisiae stb5Δ mutant. The results of phenotypic analysis suggested that KlSTB5 gene deletion did not sensitize K. lactis cells to oxidative stress inducing compounds but led to Klstb5Δ resistance to 4-nitroquinoline-N-oxide and hygromycin B. Expression analysis indicated that the loss of KlSTB5 gene function induced the transcription of drug efflux pump encoding genes that might contribute to increased 4-nitroquinoline-N-oxide and hygromycin B tolerance. Our results show that KlStb5p functions as negative regulator of some ABC transporter genes in K. lactis.

Survival of Kluyveromyces marxianus with stigmasterol as subjected to freezing stress.

Kluyveromyces marxianus is an aerobic yeast and is interested to be applied in many industries. This research was aimed to study the effect of the sterol alternative to ergosterol on the freezing stress of K. marxianus UBU1-11, a thermotolerant yeast. The 0-9 mgL-1 stigmasterol were added to the YM broth and applied for culturing. The growth of all conditions were not interfered by the addition of stigmasterol. The intra-cellular sterol content was detected in the medium with 5 mgL-1 stigmasterol and higher, where the maximum content was 0.32 mg g-1 cell dry weight. After frozen and thawed, the cultures contained stigmasterol had significantly higher viability than those without. It was found that the amount of stigmasterol contained in cells did not affect the number of survival. The stigmasterol provided a significant protection to the yeast cell when subjected to slow freezing. It also increased the survival rate of the culture subjected to subzero temperature storage.

Erg6 gene is essential for stress adaptation in Kluyveromyces lactis.

We investigated the effect of Kluyveromyces lactis ERG6 gene deletion on plasma membrane function and showed increased susceptibility of mutant cells to salt stress, cationic drugs and weak organic acids. Contrary to Saccharomyces cerevisiae, Klerg6 mutant cells exhibited increased tolerance to tunicamycin. The content of cell wall polysacharides did not significantly vary between wild-type and mutant cells. Although the expression of the NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGPD1) in the Klerg6 mutant cells was only half of that in the parental strain, it was induced in the presence of calcofluor white. Also, cells exposed to this drug accumulated glycerol. The absence of KlErg6p led to plasma membrane hyperpolarization but had no statistically significant influence on the plasma membrane fluidity. We propose that the phenotype of Klerg6 mutant cells to a large extent was a result of the reduced activity of specific plasma membrane proteins that require proper lipid composition for full activity.

Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease.

The term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene ATP2C1 the orthologous of the yeast gene PMR1. We observed that K. lactis cells defective for PMR1 gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with PMR1 deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in K. lactis. These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target NQO1. In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD.

Kluyveromyces marxianus, an Attractive Yeast for Ethanolic Fermentation in the Presence of Imidazolium Ionic Liquids.

Imidazolium ionic liquids (ILs) are promising solvents for lignocellulosic biomass (LCB) pretreatment and allow the achievement of higher ethanolic yields after enzymatic hydrolysis and ethanolic fermentation. However, residual ILs entrapped in pretreated biomass are often toxic for fermentative microorganisms, but interaction mechanisms between ILs and cells are still unknown. Here we studied the effects of 1-ethyl-3-methylimidazolium acetate [Emim][OAc] and 1-ethyl-3-methylimidazolium methylphosphonate [Emim][MeO(H)PO2] on Kluyveromyces marxianus, a thermotolerant ethanologenic yeast. Morphological impacts induced by ILs on K. marxianus were characterized by Scanning Electron Microscopy analysis and showed wrinkled, softened, and holed shapes. In Yeast-Malt-Dextrose (YMD) medium, K. marxianus tolerated IL additions up to 2% for [Emim][OAc] and 6% for [Emim][MeO(H)PO2]. Below these thresholds, some IL concentrations enhanced ethanolic yields up to +34% by switching the metabolic status from respiratory to fermentative. Finally, K. marxianus fermentation was applied on several substrates pretreated with [Emim][OAc] or [Emim][MeO(H)PO2] and enzymatically hydrolyzed: a model long fiber cellulose and two industrial LCBs, softwood (spruce) and hardwood (oak) sawdusts. The maximum ethanolic yields obtained were 1.8 to 3.9 times higher when substrates were pretreated with imidazolium ILs. Therefore K. marxianus is an interesting fermentative yeast in a second-generation bioethanol process implying IL pretreatment.

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state.

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing ß-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K. marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase.

Candida infanticola and Candida spencermartinsiae yeasts: Possible emerging species in cancer patients.

Opportunistic infections due to Candida species occur frequently in intensive care settings. We investigated the prevalence of Candida species among 65 clinical specimens obtained from 200 cancer patients by phenotypic and molecular (ITS sequencing and AFLP) methods. Among the 65 yeast isolates, Candida albicans was the most commonly isolated species (n = 34, 52.3%), whereas other Candida species comprised 47.7% (n = 31) and consisted of Candida glabrata (n = 14, 21.5%), Candida tropicalis (n = 5, 7.7%) and uncommon Candida species (n = 12, 18.5%) such as Candida pelliculosa (n = 3, 4.6%), Pichia kudriavzevii (= Candida krusei, n = 2, 3.1%), Candida orthopsilosis (n = 2, 3.1%), Candida parapsilosis (n = 1, 1.5%), Candida infanticola (n = 2, 3.1%), Candida spencermartinsiae (n = 1, 1.5%), and Kluyveromyces marxianus (=Candida kefyr, n = 1, 1.5%). Candida infanticola and Candida spencermartinsiae were recovered from oral lesions of cancer patients. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) easily confirmed these isolates as less common Candida isolates (4.6%). The in vitro antifungal susceptibilities of C. spencermartinsiae and the two strains of C. infanticola were determined according to CLSI guidelines (M27-A3). MIC results among these isolates showed they were susceptible to isavuconazole, posaconazole and voriconazole, however, fluconazole and caspofungin had high MIC values. These Candida species that may occur more commonly in infections remain unnoticed using commonly used phenotypical methods in routine microbiology laboratories. MALDI-TOF MS proved to be a more fast and robust diagnostic technique for identification of the yeasts isolated from different clinical specimens of cancer patients.

Fermentative metabolism impedes p53-dependent apoptosis in a Crabtree-positive but not in Crabtree-negative yeast.

Tumour cells distinguish from normal cells by fermenting glucose to lactate in presence of sufficient oxygen and functional mitochondria (Warburg effect). Crabtree effect was invoked to explain the biochemical basis of Warburg effect by suggesting that excess glucose suppresses mitochondrial respiration. It is known that the Warburg effect and Crabtree effect are displayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstrated that expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, we demonstrate that p53 expression in S. cerevisiae (Crabtree-positive yeast) causes increase in ROS levels and apoptosis when cells are growing on non-fermentable carbon sources but not on fermentable carbon sources, a feature similar to tumour cells. In contrast, in Kluyveromyces lactis (Crabtree-negative yeast) p53 causes increase in ROS levels and apoptosis regardless of the carbon source. Interestingly, the increased ROS levels and apoptosis are correlated to increased oxygen uptake in both S. cerevisiae and K. lactis. Based on these results, we suggest that at least in yeast, fermentation per se does not prevent the escape from apoptosis. Rather, the Crabtree effect plays a crucial role in determining whether the cells should undergo apoptosis or not.

[Emerging pathogen: Candida kefyr (Kluvyeromyces marxianus)]. / Önem kazanan patojen: Candida kefyr (Kluvyeromyces marxianus).

In the central microbiology laboratory of Gazi University Hospital Candida kefyr was isolated from different clinical samples as 5.3% in 2016 and in 2017 this rate increased to 9.3% which was nearly two-fold and this has drawn our attention. The aim of this study was to evaluate the special characteristics, antifungal susceptibility and virulence properties of C.keyfr species. Germ tube, corn meal-tween 80 agar morphology and carbohydrate assimilation profiles on ID32C yeast identification system were used for the diagnosis of Candida species. In this study, DNA sequencing was performed using ITS1 and ITS4 primers amplifying fungal gene between 5.8S and 18S regions of rRNA. Antifungal susceptibility was performed using M27A microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI). Minimum inhibitory concentration (MIC) values for amphotericin B, fluconazole, voriconazole and itraconazole were determined. MIC distribution, MIC50 and MIC90 values and geometric mean (GM) were detected. The existence of virulence factors caseinase, secreted aspartyl proteinase, esterase and phospholipase were investigated in vitro. A total of 865 Candida species were isolated from different clinical samples in the central microbiology laboratory of Gazi University Hospital in 2016. Among them, 46 (5.3%) were C.kefyr. In the first four months of 2017, 30 (9.3%) C.kefyr were identified among 320 Candida isolates. Ten isolates which have shown atypical morphology on corn meal agar were selected. Among these 10 isolates, nine of them were identified as C.kefyr by using ID32C system and DNA sequencing method. Amphotericin B MIC value was 2 µg/ml for one isolate, and fluconazole MIC value was 8 µg/ml for another isolate among 46 isolates. Among the 30 isolates of the year 2017, one of them presented MIC value for fluconazole as 8 µg/ml. No marked antifungal resistance was detected in our isolate group. Caseinase was positive in one C.kefyr isolate, and phospholipase were positive in eight of nine isolates. As a result, the reason of increase in the incidence of this Candida species, which does not show significant resistance and presents mostly phospholipase activity as a virulence factor, should be investigated in more detail.

Identification and Characterization of Oleaginous Yeast Isolated from Kefir and Its Ability to Accumulate Intracellular Fats in Deproteinated Potato Wastewater with Different Carbon Sources.

The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua, Debaryomyces hansenii, Kluyveromyces marxianus, Kazachstania unispora, and Zygotorulaspora florentina. We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L-1. Kazachstania unispora was able to accumulate the high amount of palmitoleic acid.