Physiological responses contributing to multiple stress tolerance in Pichia kudriavzevii with potential enhancement for ethanol fermentation.

Economically feasible ethanol production requires efficient hydrolysis of lignocellulosic biomass and high-temperature processing to enable simultaneous saccharification and fermentation. During the lignocellulolysic hydrolysate, the yeast must encounter with a multiple of inhibitors such as heat and furfural. To solve this problem, a potential fermentative yeast strain that tolerated simultaneous multistress and enhance ethanol concentration was investigated. Twenty yeast isolates were classified into two major yeast species, namely Pichia kudriavzevii (twelve isolates) and Candida tropicalis (eight isolates). All P. kudriavzevii isolates were able to grow at high temperature (45 °C) and exhibited stress tolerance toward furfural. Among P. kudriavzevii isolates, NUCG-S3 presented the highest specific growth rate under each stress condition of heat and furfural, and multistress. Morphological changes in P. kudriavzevii isolates (NUCG-S2, NUCG-S3, NUKL-P1, NUKL-P3, and NUOR-J1) showed alteration in mean cell length and width compared to the non-stress condition. Ethanol production by glucose was also determined. The yeast strain, NUCG-S3, gave the highest ethanol concentrations at 99.46 ± 0.82, 62.23 ± 0.96, and 65.80 ± 0.62 g/l (P < 0.05) under temperature of 30 °C, 40 °C, and 42 °C, respectively. The tolerant isolated yeast NUCG-S3 achieved ethanol production of 53.58 ± 3.36 and 48.06 ± 3.31 g/l (P < 0.05) in the presence of 15 mM furfural and multistress (42 °C with 15 mM furfural), respectively. Based on the results of the present study, the novel thermos and furfural-tolerant yeast strain P. kudriavzevii NUCG-S3 showed promise as a highly proficient yeast for high-temperature ethanol fermentation.

The ability of Pichia kudriavzevii to tolerate multiple stresses makes it promising for developing improved bioethanol production processes.

Thermotolerant ethanol fermenting yeasts have been extensively used in industrial bioethanol production. However, little is known about yeast physiology under stress during bioethanol processing. This study investigated the physiological characteristics of the thermotolerant yeast Pichia kudriavzevii, strains NUNS-4, NUNS-5 and NUNS-6, under the multiple stresses of heat, ethanol and sodium chloride. Results showed that NUNS-4, NUNS-5 and NUNS-6 displayed higher growth rates under each stress condition than the reference strain, Saccharomyces cerevisiae TISTR5606. Maximum specific growth rates under stresses of heat (45°C), 15% v/v ethanol and 1·0 M sodium chloride were 0·23 ± 0·04 (NUNS-4), 0·11 ± 0·01 (NUNS-5) and 0·15 ± 0·01 h-1 (NUNS-5), respectively. Morphological features of all yeast studied changed distinctly with the production of granules and vacuoles when exposed to ethanol, and cells were elongated under increased sodium chloride concentration. This study suggests that the three P. kudriavzevii strains are potential candidates to use in industrial-scale fermentation due to a high specific growth rate under multiple stress conditions. Multiple stress-tolerant P. kudriavzevii NUNS strains have received much attention not only for improving large-scale fuel ethanol production, but also for utilizing these strains in other biotechnological industries.

Vba4p, a vacuolar membrane protein, is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae.

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.

Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

Functional expression of Schizosaccharomyces pombe Vba2p in the vacuolar membrane of Saccharomyces cerevisiae.

A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.

Vba5p, a novel plasma membrane protein involved in amino acid uptake and drug sensitivity in Saccharomyces cerevisiae.

Vba5p is closest to Vba3p in the vacuolar transporter for basic amino acids (VBA) family of Saccharomyces cerevisiae. We found that green fluorescence protein (GFP)-tagged Vba5p localized exclusively to the plasma membrane. The uptake of lysine and arginine by whole cells was little affected by deletion of the VBA5 gene, but was stimulated by overexpression of the VBA5 gene. The inhibitory effect of 4-nitroquinoline N-oxide on cell growth was accelerated by expression of the VBA5 gene, and was lessened by the addition of arginine. These results suggest that Vba5p is a plasma membrane protein involved in amino acid uptake and drug sensitivity.

Silencing of CD44 by siRNA suppressed invasion, migration and adhesion to matrix, but not secretion of MMPs, of cholangiocarcinoma cells.

We studied the expression pattern and the role of CD44 in regulating the malignant behavior of two cholangiocarcinoma (CCA) cell lines which expressed different levels of CD44 using the siRNA technique. KKU-100, the high CD44 expresser, exhibited a high degree of in vitro invasiveness, migration and adhesion to Matrigel compared to HuCCA-1. Silencing of CD44 by siRNA did not have a significant effect on cell proliferation. However, in vitro invasiveness, directional migration (chemotaxis) and adhesion to Matrigel were markedly reduced in both cell lines, although chemokinesis and MMP secretion were variable, demonstrating the distinct functional role and requirement for CD44 in different cellular activities and in different cell types. In addition, immunohistochemical analysis suggested that CD44 may be involved in the differentiation process or tumor progression, depending on the macroscopic type of CCA. Taken together, our data indicate that CD44 is an important requirement for the invasive phenotype of CCA cells, although the role that CD44 plays may vary depending on the CCA type and the cellular activity in which it is engaged.