Physiological overlap between osmotolerance and thermotolerance in Saccharomyces cerevisiae

Abstract A convenient enzyme-linked immunoabsorbent assay (ELISA) to titrate serum immunoglobin G to Campylobacter pylori (Cp) is described. It was found that 80% of the serum samples obtained from patients with Cp-associated chronic gastritis ( N = 476) displayed statistically raised IgG anti-Cp, as compared to healthy volunteer controls ( N = 24). Titers in patient sera were statistically higher than the average control value plus twice the standard deviation, thus supporting Cp involvement in these gastric diseases. Also, ELISA inhibition experiments suggested that antigenic variation among various Cp isolates is likely, thus indicating the need for subgrouping of Cp. We note the usefulness of the above IgG anti-Cp ELISA for sero-epidemiological studies of Cp associated chronic gastritis, especially those tracing the source of Cp infections and those determining potentially disease related Cp subgroups.     

Lipid content of Saccharomyces cerevisiae strains with different degrees of ethanol tolerance

Summary We analysed the fatty acid and sterol compositions of various Saccharomyces cerevisiae strains with ethanol tolerance varying from 4% to 12% (v/v) ethanol and at different concentrations of ethanol. The results we obtained agree with the existence of a relationship between membrane fluidity and ethanol tolerance but they do not support a direct role of unsaturated fatty acids in this tolerance. On the other hand, they support the importance of ergosterol in this phenomenon.     

Increased osmotolerance of genetically modified ethanol producing strains of Saccharomyces Sp.

Summary A stable spheroplast fusion product of the polyploid brewing strain Saccharomyces uvarum (cares bergensis) , strain 21 and a genetically constructed diploid Saccharomyces diastaticus, strain 1384 has been shown to have improved ethanol producing capability in defined media (Panchal et al., 1982). This fusion product, strain 1400 was further subjected to fermentations in defined media containing glucose substrate and varying concentrations of the non-metabolized sugars sorbitol or mannitol.While the fermentation efficiencies of all the three strains decreased with increasing osmotic pressure imparted by sorbitol or mannitol, the detrimental effect was least apparent with the fusion product than with either of the fusion partners. This attribute of the stable fusion product has major significance in relation to its potential for industrial ethanol production.     

Enhanced thermotolerance and ethanol tolerance in Saccharomyces cerevisiae mutated by high-energy pulse electron beam and protoplast fusion

To increase thermotolerance and ethanol tolerance in Saccharomyces cerevisiae strain YZ1, the strategies of high-energy pulse electron beam (HEPE) and three rounds of protoplast fusion were explored. The YF31 strain had the characteristics of resistant to high-temperature, high-ethanol tolerance, rapid growth and high yield. The YF31 could grow on plate cultures up to 47?°C, containing 237.5?g?L^?1 of ethanol. In particular, the mutant strain YF31 generated 94.2?±?4.8?g?L^?1 ethanol from 200?g glucose L^?1 at 42?°C, which was 2.48 times the production of the wild strain YZ1. Results demonstrated that the variant phenotypes from the strains screening by HEPE irradiation could be used as parent stock for yeast regeneration and the protoplast fusion technology is sufficiently powerful in combining suitable characteristics in a single strain for ethanol fermentation.     

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy‐demanding growth‐related processes. Studies using genome‐wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.     

Effect of 5,7-unsaturated sterols on ethanol tolerance in Saccharomyces cerevisiae.

Structural membrane lipids are known to contribute to the high ethanol resistance of Saccharomyces cerevisiae (2, 4, 17). By manipulating the yeast cellular sterol level by changing the carbon-to-nitrogen source ratio in the chemostat growth medium, high delta 5,7-sterol levels were found to increase the resistance of yeast populations to ethanol-induced death. The resistance of the erg2 (delta 8----delta 7-sterol isomerase) mutant to ethanol-induced death was generally comparable with that of the delta 5,7-sterol-synthesizing strain. In contrast, the sensitivity of anaerobic growth to inhibition by ethanol was higher in the erg2 mutant in comparison with the delta 5,7-sterol-synthesizing strains but a high level of those sterols increased the vulnerability of anaerobic growth to ethanol inhibition.     

Saccharomyces cerevisiae strains with different degrees of ethanol tolerance exhibit different adaptive responses to produced ethanol

Two Saccharomyces cerevisiae strains with different degrees of ethanol tolerance adapted differently to produced ethanol. Adaptation in the less ethanol-tolerant strain was high and resulted in a reduced formation of ethanol-induced respiratory deficient mutants and an increased ergosterol content of the cells. Adaptation in the more ethanol-tolerant strain was less pronounced. Journal of Industrial Microbiology & Biotechnology (2000) 24, 75–78. Received 22 June 1999/ Accepted in revised form 06 October 1999     

Induction of heat shock proteins and thermotolerance by ethanol in Saccharomyces cerevisiae

The temperature sensitivity of $\text{Saccharomyces}\text{cerevisiae}$ and the conditions of moderate heat pretreatment required to induce thermotolerance are established. Ethanol is identified as an inducer of heat shock proteins and an inducer of thermotolerance.     

三种选育高乙醇耐受性工业酿酒酵母方法的比较

由于乙醇耐性受多基因控制,因此需要从全基因组水平进行改造以期得到高乙醇耐受的突变体。文中分别使用紫外诱变、等离子体诱变及人工转录因子3种方法对工业酿酒酵母Sc4126进行改造,获得了乙醇耐性提高的突变体,并比较了3种方法的正突变率。人工转录因子文库转化的方法获得了最多数量的乙醇耐性突变体,高出紫外诱变和等离子体诱变方法1~2个数量级,且遗传稳定。研究结果表明,人工转录因子技术可以用于对工业酿酒酵母快速进行基因组工程改造。     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae.

We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different thermal killing characteristics. When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance. Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state. Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature. cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state. Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1. It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C. We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C. The results suggest a relationship between the heat shock phenomenon and the cell cycle in S. cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.     

Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance.     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

利用SPT3的定向进化提高工业酿酒酵母乙醇耐受性

利用对转录因子的定向进化可对多基因控制的性状进行有效的代谢工程改造。本研究对酿酒酵母负责胁迫相关基因转录的SAGA复合体成分SPT3编码基因进行易错PCR随机突变,并研究了SPT3的定向进化对酿酒酵母乙醇耐性的影响。将SPT3的易错PCR产物连接改造的pYES2.0表达载体并转化酿酒酵母Saccharomyces cerevisiae4126,构建了突变体文库。通过筛选在高浓度乙醇中耐受性提高的突变株,获得了一株在10%(V/V)乙醇中生长较好的突变株M25。该突变株利用125g/L的葡萄糖进行乙醇发酵时,终点乙醇产量比对照菌株提高了11.7%。由此表明,SPT3是对酿酒酵母乙醇耐性进行代谢工程改造的一个重要的转录因子。     

Relationship between ethanol tolerance and fatty acyl composition of Saccharomyces cerevisiae

Summary The effect of ethanol on exponential phase cultures of S. cerevisiae has been examined using l-alanine uptake and proton efflux as indices of ethanol tolerance. Preincubation with 2 M ethanol inhibited l-alanine uptake, proton efflux and fermentation rates. However, the effect of ethanol varied in yeast cells enriched with different fatty acyl residues. It was observed that cells enriched with polyunsaturated fatty acids acquired greater tolerance to ethanol as compared to monounsaturated fatty acids. By varying the degree of unsaturation of supplemented fatty acid, a sequential insertion of double bonds in yeast membrane lipid was achieved. Results demonstrated that S. cerevisiae became more resistant to ethanol with an increase in the degree of unsaturation and that membrane fluidity could be an important determinant of ethanol tolerance.     

Role of phospholipid head groups in ethanol tolerance of Saccharomyces cerevisiae

Pre-incubation of cells of Saccharomyces cerevisiae with 2 M-ethanol led to decreased rates of L-alanine uptake, H+ efflux and fermentation rate. However, these responses were modified in yeast cells with altered phospholipid composition. Using L-alanine transport and H+ efflux as indices of ethanol tolerance, it was observed that cells enriched with phosphatidylserine had greater tolerance to ethanol. This resulted from altered charge of membrane phospholipids rather than changes in membrane fluidity. It is suggested that the anion:zwitterion ratio of phospholipids may be one of the important determinants of ethanol tolerance in S. cerevisiae.     

Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains

The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains.Non-standard abbreviations InGP Indole-3-glycerolphosphate - PRA N(5-phosphoribosyl)-anthranilate     

Effect of growth rate on ethanol tolerance ofSaccharomyces cerevisiae

Δ^5,7 Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium. The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of Δ^5,7. Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance. Differences between the Δ^5,7 and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.