Investigation the cytotoxicity and photo-induced toxicity of carbon dot on yeast cell.

Carbon dots (CDs) as a new fluorescent material with excellent water solubility, chemical inertness, and easy surface modification are a good candidate for bioimaging and biosensing due to their low toxicity and good biocompatibility. Although carbon is not an intrinsically toxic substance, carbon nanomaterials such as CDs may cause risks to human health and the potentially hazardous effects of CDs on various living systems must be completely determined. So far, cytotoxicity studies of CDs have focused on human cells and are mainly conducted on limited cell lines. In the present study, toxicity assessment of CDs was evaluated on yeast cells Pichia pastoris as a unicellular eukaryotic model. Results revealed dose-dependent toxicity of CDs on yeast cells and less relative cell growth in 25 mg/ml of CDs as compared to the control group. CDs binding curve confirmed the interaction between CDs and surface of yeast cells. SEM images showed that the CDs caused cell shrinkage and hole formation on the surface of yeast cells and also induced slightly cell deformation. It was demonstrated that CDs could generate the ROS dose-dependently. Finally, results showed the growth inhibition and ROS generation effects of CDs were enhanced at light exposure, as an important environmental factor. These findings could have important implications for applications of CDs.

Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108 ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

The CsHSP17.2 molecular chaperone is essential for thermotolerance in Camellia sinensis.

Small heat shock proteins (sHSPs) play important roles in responses to heat stress. However, the functions of sHSPs in tea plants (Camellia sinensis) remain uncharacterized. A novel sHSP gene, designated CsHSP17.2, was isolated from tea plants. Subcellular localization analyses indicated that the CsHSP17.2 protein was present in the cytosol and the nucleus. CsHSP17.2 expression was significantly up-regulated by heat stress but was unaffected by low temperature. The CsHSP17.2 transcript levels increased following salt and polyethylene glycol 6000 treatments but decreased in the presence of abscisic acid. The molecular chaperone activity of CsHSP17.2 was demonstrated in vitro. Transgenic Escherichia coli and Pichia pastoris expressing CsHSP17.2 exhibited enhanced thermotolerance. The transgenic Arabidopsis thaliana exhibited higher maximum photochemical efficiencies, greater soluble protein proline contents, higher germination rates and higher hypocotyl elongation length than the wild-type controls. The expression levels of several HS-responsive genes increased in transgenic A. thaliana plants. Additionally, the CsHSP17.2 promoter is highly responsive to high-temperature stress in A. thaliana. Our results suggest that CsHSP17.2 may act as a molecular chaperone to mediate heat tolerance by maintaining maximum photochemical efficiency and protein synthesis, enhancing the scavenging of reactive oxygen species and inducing the expression of HS-responsive genes.

Strain improvement of Pichia kudriavzevii TY13 for raised phytase production and reduced phosphate repression.

In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate-degrading capacity. The mutant strain TY1322 shows a biomass-specific phytate degradation of 1.26 mmol g-1  h-1 after 8 h of cultivation in a high-phosphate medium, which is about 8 times higher compared with the wild-type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46°C) and in the presence of ox bile (2% w/v), indicating this strain's ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55°C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non-genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification.

Hansenula polymorpha Hac1p Is Critical to Protein N-Glycosylation Activity Modulation, as Revealed by Functional and Transcriptomic Analyses.

Aggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular protective response to ER stress, the unfolded protein response (UPR), which is mediated by a basic leucine zipper (bZIP) transcription factor, Hac1p/Xbp1. In this study, we identified and studied the molecular functions of a HAC1 homolog from the thermotolerant yeast Hansenula polymorpha (HpHAC1). We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose interaction with the 5' untranslated region is responsible for the translational inhibition of the HpHAC1 mRNA. The H. polymorpha hac1-null (Hphac1Δ) mutant strain grew slowly, even under normal growth conditions, and was less thermotolerant than the wild-type (WT) strain. The mutant strain was also more sensitive to cell wall-perturbing agents and to the UPR-inducing agents dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the WT and Hphac1Δ strains treated with DTT and TM, we identified HpHAC1-dependent core UPR targets, which included genes involved in protein secretion and processing, particularly those required for N-linked protein glycosylation. Notably, different glycosylation and processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the WT and Hphac1Δ strains. Moreover, overexpression of active HpHac1p significantly increased the N-linked glycosylation efficiency and TM resistance. Collectively, our results suggest that the function of HpHac1p is important not only for UPR induction but also for efficient glycosylation in H. polymorpha.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris.

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein.

This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol-inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P. thermomethanolica BCC16875 is Man(8-12) GlcNAc(2) , which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α-1,6- and α-1,2-linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial yeasts makes this microorganism an attractive host for large-scale fermentation.

Combined effect of selected non-thermal technologies on Escherichia coli and Pichia fermentans inactivation in an apple and cranberry juice blend and on product shelf life.

The combination of novel, non-thermal technologies for preservation purposes is a recent trend in food processing research. In the present study, non-thermal hurdles such as ultraviolet light (UV) (5.3 J/cm²), high intensity light pulses (HILP) (3.3 J/cm²), pulsed electric fields (PEF) (34 kV/cm, 18 Hz, 93 µs) or manothermosonication (MTS) (4bar, 43 °C, 750 W, 20 kHz) were examined. The objective was to establish the potential of these technologies, applied individually or in paired sequences, to inactivate Escherichia coli and Pichia fermentans inoculated in a fresh blend of apple and cranberry juice. The shelf-life evaluation of selected non-thermally treated samples was conducted over 35 days and compared to pasteurised samples and untreated juices. All treatments applied individually significantly reduced (1.8-6.0 log cfu/ml) microbial counts compared to the untreated sample (p<0.01). Furthermore, UV treatment produced significantly greater inactivation (p<0.05) for E. coli compared to P. fermentans. Combinations of non-thermal hurdles consisting of UV or HILP followed by either PEF or MTS resulted in comparable reductions for both microorganisms (p ≥ 0.05) to those observed in thermally pasteurised samples (approx. 6 log cfu/ml). Thermally pasteurised samples had a shelf life exceeding 35 days, while that of UV+PEF and HILP+PEF-treated samples was 14 and 21 days, respectively. These results indicate that combinations of these non-thermal technologies could successfully reduce levels of E. coli and P. fermentans in apple and cranberry juice, although optimisation is required in order to further extend shelf life.

A UV-induced mutant of Pichia stipitis with increased ethanol production from xylose and selection of a spontaneous mutant with increased ethanol tolerance.

In the fermentation process of lignocellulosic biomass (such as wood and rice straw), efficient conversion of pentose (mainly xylose) into ethanol is important. Mutants of Pichia stipitis NBRC1687 were obtained after UV mutagenesis and selection of large colonies on ethanol-containing medium. One mutant, PXF58, produced 4.3% ethanol from 11.4% xylose while the parent strain only produced 3.1%. The ethanol productivities of PXF58 from glucose and fructose were about were about 1.4-fold higher than those of the parent strain. After continuous cultivation of PXF58 in YNB (yeast nitrogen base) medium containing 2% xylose and 5-7% ethanol, an ethanol-tolerant mutant, PET41, was obtained. Strain PET41 was able to produce 4.4% ethanol when first supplied with xylose then with glucose. This isolate might be thus useful for two-phase fermentation in which xylan is saccharified by xylanase to produce xylose, and glucan is saccharified later by cellulase and ß-glucosidase to produce glucose.

Dual fluorochrome flow cytometric assessment of yeast viability.

A novel staining protocol is reported for the assessment of viability in yeast, specifically the biocontrol yeast, Pichia anomala. Employing both the red fluorescent membrane potential sensitive oxonol stain DiBAC(4)(5) (Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol), a structural analog of the commonly used DiBAC(4)(3) (Bis-(1,3-dibutylbarbituric acid)trimethine oxonol), with one of the esterase dependent green fluorogenic probes such as CFDA-AM (5-Carboxyfluorescein diacetate, acetoxymethyl ester) or Calcein-AM (Calcein acetoxymethyl ester), a two-color flow cytometric method was developed, which yields rapid quantitative information on the vitality and vigor of yeast cell cultures. The method was validated by cell sorting and analysis of live, heat killed, and UV-treated yeast.

Construction of Hansenula polymorpha strains with improved thermotolerance.

The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high-temperature resistance and high-temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat-shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12-fold increased tolerance to heat-shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8-fold improvement of ethanol production from xylose at 50 degrees C. Although the maximum ethanol concentration achieved from xylose was 0.9 g L(-1), our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high-temperature ethanol producers from this pentose.

Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor.

Mutants of Pichia stipitis NRRL Y-7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars.

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis.

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl(2). One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30 degrees C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.

Production of ribonucleotides by autolysis of Pichia anomala mutant and some physiological activities.

Various mutants of Pichia anomala were isolated by ethyl methanesulfonate (EMS) treatment and UV irradiation through cycloheximide resistance and KCl sensitivity. The selected mutant HA-2 accumulated a higher content of RNA and grew faster than the wild-type strain in yeast extract-malt (YM) broth. Autolysis of the HA-2 mutant at 60 degrees C and pH 7.0 for 6 h was the best condition to obtain maximum yields of 5'-ribonucleotides, inosinic monophosphate (IMP) (6.2 mg/g biomass) and guanylic monophosphate (GMP) (35.5 mg/g biomass). The yield of adenylic monophosphate (AMP) (7.8 mg/g biomass) was optimal at 60 degrees C at pH 6.5 for 6 h. The inhibitory activity of the angiotensin-converting enzyme and the nitrite-scavenging activity for autolysates of the HA-2 mutant were about 13.0% and 47.0% higher than those of native strain, respectively.

Mutant Hansenula polymorpha strain with constitutive alcohol oxidase and peroxisome biosynthesis.

A mutant of the methylotrophic yeast Hansenula polymorpha with constitutive alcohol oxidase (AOX) and peroxisome biosynthesis was obtained after UV treatment followed by cell plating on a medium containing methanol and 2-deoxy-D-glucose (DOG). DOG-resistant colonies of mutants were insensitive to catabolic repression by glucose and methanol. A selection procedure is described that allows the isolation of a mutant exhibiting a constitutive phenotype of AOX involved in methanol utilization. Furthermore, additional features of the constitutive presence of peroxisomes are demonstrated. 562 DOG-resistant colonies were tested, 24 of them demonstrating constitutive AOX formation. Based on quantitative analysis, one of the strains--DOG-13 was selected and its growth, biochemical and ultrastructural characteristics were examined. Its specific enzyme activity when cultivated on a yeast nitrogen base + 1% glucose (YNB + 1% Glucose) was found to reach 145 nmol x min(-1) x mg(-1) protein (compared to zero of the parent strain) after he 20th hour of cultivation. This was confirmed by fine-structure analysis, showing typical peroxisomes, which number and size increased with the enzyme activity. This study demonstrates a constitutive AOX and peroxisome biosynthesis by the mutant strain H. polymorpha DOG-13 obtained.

Optimization of the production of Chondrus crispus hexose oxidase in Pichia pastoris.

Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts.

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7.1 kilobase pairs-long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria-associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria-associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide.

Radiosensitivity of haplont yeast cells irradiated with sparsely and densely ionizing radiations.

Five haploid and three diploid yeast strains of various species (Yarrowia lipolytica, Pichia pinus and Pichia guilliermondii) were irradiated with alpha-particles from 239Pu and gamma-rays from 137Cs or 60Co in the stationary phase of growth. A common feature of these species is that they exhibit a haploid state as a normal vegetative state in natural conditions. It was shown that the transition from the haploid to the diploid state is not accompanied by increased radioresistance, and diploid strains were unable to perform liquid-holding recovery. The absence of diploid-specific recovery in diploid strains was also supported by the fact that the RBE of alpha-particles was almost identical for haploid and the corresponding diploid strains being much smaller than that observed in typical wild-type diploid strains capable of diploid-specific recovery. The results suggest that haplont yeast may have evolved to diplont yeast via the development of a specific repair system conferring specific resistance in the diploid state.

Killer toxin production in Pichia acaciae is associated with linear DNA plasmids.

We have identified a strain of the yeast Pichia acaciae which produces a "killer" toxin active against the yeast Debaryomyces tamarii. The killer phenotype was associated with the presence of two DNA plasmids, pPacl-1 (13.6 kilobase pairs) and pPacl-2 (7.3 kilobase pairs). P. acaciae strains, cured of these plasmids by irradiation with ultraviolet light, lacked killer activity and were sensitive to toxin produced by the parental strain. A partially cured strain, GS-1215, missing only the smaller plasmid, pPacl-2, also exhibited loss of both toxin activity and immunity. Exonuclease studies revealed that both plasmids were linear double-stranded DNA molecules with 5' protected ends. The P. acaciae system differs from that of the well-studied Kluyveromyces lactis "killer" system both in the range of susceptible strains and in the sizes of the plasmids involved. Our studies contradict previous reports that Pichia killer systems are invariably chromosomal.