Relationship between lipid composition, frequency of ethanol-induced respiratory deficient mutants, and ethanol tolerance in Saccharomyces cerevisiae

The frequency of ethanol-induced respiratory deficient mutants and lipid composition in two Saccharomyces cerevisiae strains showing different degrees of ethanol tolerance were investigated. The more ethanol-tolerant strain exhibited a lower frequency of ethanol-induced respiratory deficient mutants than the less ethanol-tolerant strain. In addition, the more ethanol-tolerant strain contained a higher ergosterol/phospholipid ratio, a higher proportion of phosphatidylcholine, a lower proportion of phosphatidylethanolamine, a higher incorporation of long-chain fatty acids in total phospholipids, and a slightly higher proportion of unsaturated fatty acids in total phospholipids than the less ethanol-tolerant strain. These results show a clear relationship between the lipid composition, the frequency of ethanol-induced respiratory deficient mutants, and the ethanol tolerance of S. cerevisiae. A possible explanation of this relationship is discussed.     

Improved yeast tolerance to ethanol-induced death by centrifugates of dense cell suspensions incubated with ethanol

Summary Ethanol-induced death rate was higher for cells ofSaccharomyces bayanus orKluyveromyces marxianus in spanse suspensions (2×10⁴ cells/ml) compared with dense suspensions (2×10⁵–2×10⁷ cells/ml). Specific death rates of sparse suspensions decreased to values similar to dense suspensions if ethanol-induced death experiments were undertaken in the media obtained after harvesting the cells previously incubated with the same lethal concentration of ethanol.     

原玻璃蝇节杆菌D-氨基酸氧化酶及突变体的酶学特性

【目的】研究原玻璃蝇节杆菌(DSM 20168)中D-氨基酸氧化酶的酶学特性。【方法】通过PCR从原玻璃蝇节杆菌(DSM 15035,20168)中克隆获得D-氨基酸氧化酶基因apdaao-1和apdaao-2,构建原核表达载体,以表达质粒pET-ApDAAO-2为模板,采用QuickChange Site-Directed Mutagenesis技术构建定点突变体,经过原核表达及纯化获得重组型和突变体酶蛋白,分析其酶学特性。【结果】通过原核表达及纯化成功获得了2个重组蛋白和4个突变体酶蛋白,SDS-PAGE检测显示其分子量均约为36 kDa;酶学特性分析表明,ApDAAO-2和突变体蛋白的最适反应温度为30℃;ApDAAO-2和T286A的最适反应pH范围为7.0-11.0,其它突变体为8.0-11.0;ApDAAO-2和突变体都具有较广泛的底物特异性,除T256K的最适底物为D-Phe外,其余均为D-Met;动力学参数测定结果显示,以二级表观常数kcat/Km表示,对于底物D-Met或D-Phe,ApDAAO-2和4个突变体的kcat/Km值均比ApDAAO-1和pKDAAO高数倍以上。【结论】ApDAAO-2及突变体具有比ApDAAO-1和pKDAAO更广泛的底物特异性和较高的催化效率,有一定的商业应用价值。     

Paraquat tolerant mutants in Ceratopteris: Genetic characterization and reselection for enhanced tolerance

A selection system in vitro utilizing the haploid phase of the fern Ceratopteris richardii Brongn. has been used to isolate mutations conferring tolerance to the herbicide paraquat. Initial studies characterized two separate recessive nuclear mutations that confer slightly different levels of tolerance. Tolerance is expressed in both the gametophyte and sporophyte generations. Segregation analyses and complementation tests in diploid sporophytes have shown the two mutants to be allelic. One of the mutants was used in a reselection experiment aimed at obtaining plants with enhanced tolerance. This resulted in the recovery of individuals with enhanced tolerance. Gametophytes of one of the reselections showed tolerance levels of 40-fold over the wild type and 2-fold over the mutant parent strain. In addition to being tolerant of paraquat, this strain exhibits significantly increased growth in comparison to the wild type, even in the absence of the herbicide.     

Biotransformations of propenylbenzenes by an Arthrobacter sp. and its t-anethole blocked mutants

Propenylbenzenes are often used as starting materials in the chemical synthesis of aroma compounds and fine chemicals. In the present study, we demonstrate the ability of an Arthrobacter sp. to transform various structures of propenylbenzenes derived from essential oils to flavor, fragrance, and fine chemicals. Arthrobacter strain TA13 and its t-anethole blocked mutants (incapable of growing on t-anethole) converted isoeugenol to vanillin and vanillic acid; and safrole to hydroxychavicol. High conversion efficiencies were achieved in the biotransformations of isosafrole to piperonylic acid, and eugenol to a mixture of ferulic acid and vanillic acid. In addition, anisic acid was produced in high yields from t-anethole, anisyl alcohol, or anisaldehyde. The accumulation of the corresponding aromatic acids from the tested propenylbenzenes is due to the lack of m-demethylase activity in strain TA13 that prevents further cleavage of the benzene ring. Interestingly, in the transformation of eugenol (a 2-propenylbenzene) the side chain was initially oxidized to the corresponding cinamic acid derivative (ferulic acid) while the 1-propenylbenzenes gave substituted benzoic acids, suggesting two different chain shortening mechanisms.     

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.     

The use of free and immobilised Arthrobacter simplex in organic solvent/aqueous two-liquid-phase reactors

Summary The use of free and immobilised Arthrobacter simplex (NCIB 8929) for steroid ¹-dehydrogenation in two-liquid-phase, stirred-tank reactors has been compared. Product formation is related to the logarithm of the water-octanol partition coefficient (log P) of the organic solvent employed, but the relationship is different for the two forms of the biocatalyst. No reaction was seen with either biocatalyst in media containing solvents of log P2.5. For free bacteria, product formation rose linearly with log P thereafter to a maximum at a value of 9.8. With immobilised bacteria, product formation reached a maximum with a solvent of log P=4.0 and remained constant with solvents of higher log P value. Consequently extended reactor operation was possible with immobilised bacteria, and the production of high quality (> 95% purity) steroid product was demonstrated. Offprint requests to: M. D. Lilly     

Microwave attenuation of ethanol-induced hypothermia: ethanol tolerance, time course, exposure duration, and dose response studies

Four experiments were conducted to quantify the reported attenuation by microwave (MW) irradiation of ethanol-induced hypothermia. In one experiment rats were irradiated (continuous wave 2.45 GHz, specific absorption rate = 0.3 W/kg) or sham irradiated for 45 min, injected with 3.6 g/kg, 20% (v/v) ethanol (EtOH) or saline (NaCl) i.p.. Colonic temperature was monitored at 20-min intervals for 2 h. This procedure was repeated for 8 days to determine the rate of tolerance development to the hypothermic effect of ethanol. While MW irradiation did significantly attenuate EtOH-induced hypothermia, it did not enhance or retard the rate of tolerance development. To determine the duration of irradiation necessary to attenuate EtOH-induced hypothermia, groups of rats were irradiated or sham irradiated for 5, 15, 30, or 60 min prior to EtOH injection and subsequent temperature measurements. The attenuation was apparent only after 60 min of irradiation. To determine the duration of the attenuation effect after irradiation, rats were injected with EtOH or NaCl at 0, 30, 60, 120, or 480 min after 45 min of irradiation or sham irradiation. The attenuation effect was apparent among rats injected 0 to 30 min after irradiation and for the first 40 min for groups injected at 120 min. Additional rats were injected with NaCl or 0.9, 1.8, or 2.7 g/kg of EtOH i.p. following 45 min of irradiation or sham irradiation to determine if the attenuation effect depends on the dose of EtOH administered. Attenuation of EtOH-induced hypothermia was more apparent at lower doses of EtOH than at higher doses. These results indicate that the effect is an acute response to irradiation, and rule out several other potential explanations.     

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

The genetic relationships between ethanol preference,acute ethanol sensitivity,and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.     

Adrenal glucocorticoid function in rats with various ethanol-induced sleeping time following ethanol administration

The effect of single and chronic ethanol (Eth) administration (25 % solution, 3.5 g/kg) on functional activity of the hypophyseal-adrenal system in rats with different sensitivity to the hypnotic action of ethanol (short-sleep - SS; non-sleep--NS, long-sleep--LS, intermediate group--IG), was studied. It has been shown that, after a single Eth administration, the concentration of corticosterone (K) in LS rat plasma was 1.5-fold higher than that in the NS animals although it did not differ from the K level in SS and Ig those. After repeated ethanol load, the corticosterone contents in the NS rat blood plasma was 3.5-fold and 4.9-fold lower compared to the control and LS groups, respectively. The data obtained indicate that the SS and LS animals had initially different basal blood plasma glucocorticoid level. The SS animals showed a decreased blood plasma K, whereas the LS ones--an increased one. The features of the glucocorticoid status are suggested to be a factor determining the sensitivity of rats to the ethanol hypnotic effect.     

The genetic relationships between ethanol preference, acute ethanol sensitivity, and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.     

A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate

Glyphosate or Roundup^® is the most extensively used herbicide for broad-spectrum control of weeds. Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. Applying the staggered extension process, we randomly mutated and recombined the aroA genes of Salmonella typhimurium and Escherichia coli to obtain four variants that exhibit significantly enhanced tolerance to glyphosate. All four mutants are chimeras of the two parental genes and, in addition, three of them carry one or more de novo point mutations. None of the amino acid substitutions in the mutants was in a position previously known to be important for catalysis or substrate binding. Kinetic analysis of EPSPS activity from these mutants indicated that the tolerance was attributed to a 2–10-fold increased specific activity, 0.4–8-fold reduced affinity to glyphosate, and 2.5–19-fold decreased K_m for phosphoenolpyruvate. Such mutants will be instrumental for the structural and function study of the enzyme and for the generation of transgenic crops resistant to the herbicide.     

Transcriptional changes associated with ethanol tolerance in Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanol production; however, fermentation productivity is negatively affected by the impact of ethanol accumulation on yeast metabolic rate and viability. This study used microarray and statistical two-way ANOVA analysis to compare and evaluate gene expression profiles of two previously generated ethanol-tolerant mutants, CM1 and SM1, with their parent, Saccharomyces cerevisiae W303-1A, in the presence and absence of ethanol stress. Although sharing the same parentage, the mutants were created differently: SM1 by adaptive evolution involving long-term exposure to ethanol stress and CM1 using chemical mutagenesis followed by adaptive evolution-based screening. Compared to the parent, differences in the expression levels of genes associated with a number of gene ontology categories in the mutants suggest that their improved ethanol stress response is a consequence of increased mitochondrial and NADH oxidation activities, stimulating glycolysis and other energy-yielding pathways. This leads to increased activity of energy-demanding processes associated with the production of proteins and plasma membrane components, which are necessary for acclimation to ethanol stress. It is suggested that a key function of the ethanol stress response is restoration of the NAD⁺/NADH redox balance, which increases glyceraldehyde-3-phosphate dehydrogenase activity, and higher glycolytic flux in the ethanol-stressed cell. Both mutants achieved this by a constitutive increase in carbon flux in the glycerol pathway as a means of increasing NADH oxidation.     

The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans

Myosin isoforms A and B are located at the surface of the central and polar regions, respectively, of thick filaments in body muscle cells of Caenorhabditis elegans, whereas paramyosin and a distinct core structure comprise the backbones of these filaments. Thick filaments and related structures were isolated from nematode mutants that have altered thick filament protein compositions. These mutant filaments and their complexes with specific antibodies were studied by electron microscopy to determine the distribution of the two myosins. The compartmentation of the two myosin isoforms in body wall muscle thick filaments depends not only upon the intrinsic properties of the myosins but their interactions with other components such as paramyosin and their relative quantities determined by synthesis.     

Development of tolerance to ethanol-induced suppression of breathing movements and brain activity in the near-term fetal sheep during short-term maternal administration of ethanol

The effect of short-term maternal ethanol administration on the ethanol-induced suppression of fetal breathing movements, electrocortical (ECoG) activity, and electroocular (EOG) activity was determined in the near-term fetal sheep. Twelve conscious instrumented pregnant ewes (between 125 and 139 days of gestation; term, 147 days) received 1-h intravenous infusion of 1 g ethanol/kg total body weight daily for six days (n = 6) or an equivalent volume of normal saline daily for six days (n = 6). On the seventh day, the ethanol- and saline-pretreated animals were administered 1 g ethanol/kg total body weight. A further six ewes received 1-h intravenous infusion of 1 g ethanol/kg total body weight (n = 3) or an equivalent volume of normal saline (n = 3) daily for thirteen days with both groups receiving 1 g ethanol/kg total body weight on day fourteen. Fetal ECoG and EOG activities, and fetal breathing movements were monitored continuously over the post- operative and experimental periods. Saline infusion had no significant effect on the parameters studied. Fetal breathing movements were suppressed for 8 h after the first ethanol dose, and were not significantly suppressed after fourteen days of once-daily, maternal ethanol administration. Low-voltage ECoG and EOG activities were suppressed for 3 h after the first ethanol dose, and were not significantly suppressed after seven days of repeated ethanol administration. Maternal and fetal blood gases and acid-base balance were not significantly affected by maternal ethanol administration. These data demonstrate that short-term maternal administration of ethanol results in the development of tolerance to ethanol in the mature fetus.     

The microsomal ethanol oxidizing system mediates metabolic tolerance to ethanol in deermice lacking alcohol dehydrogenase

Metabolic tolerance to ethanol has been attributed to enhanced mitochondrial reoxidation of reducing equivalents produced in the alcohol dehydrogenase (ADH) pathway or to non-ADH mechanisms. To resolve this issue, deermice lacking low Km hepatic ADH were fed for 2 weeks a liquid diet containing ethanol or isocaloric carbohydrate and hepatocytes were isolated. Ethanol (50 mM) oxidation increased (9.8 vs 4.5 nmol/min/10(6) cells in controls). To differentiate which of two non-ADH pathways (the microsomal ethanol oxidizing system (MEOS) or catalase) was responsible for the induction, four approaches were used. First, MEOS was assayed in hepatic microsomes and found to be increased (24.4 vs 6.8 nmol/min/mg protein in controls). Second, hepatocyte ethanol metabolism was measured after addition of the catalase inhibitor azide (0.1 mM) and found to be unchanged. By contrast, the competitive MEOS inhibitor, 1-butanol, depressed metabolism in a concentration-dependent manner. A third approach relied on measurement of isotope effects known to be different for MEOS and catalase. From the isotope effect values, MEOS was calculated to contribute 85% or more of total ethanol oxidation by cells from both ethanol-fed and control animals. A fourth approach involved in vivo pretreatment with pyrazole (300 mg/kg/day for 2 days), which reduced peroxidation by catalase to 13% of control values in liver homogenates while inducing MEOS activity to 152% of controls. Hepatocytes from pyrazole-treated deermice showed a 47% increase in ethanol metabolism, paralleling the MEOS induction and contrasting with the catalase suppression. These results indicate that since metabolic tolerance occurs in the absence of ADH, it is not necessarily ADH mediated, and further, that MEOS rather than catalase accounts for basal ethanol metabolism and its increase after chronic ethanol treatment.     

The selection and properties of Penicillium verruculosum mutants with enhanced production of cellulases and xylanases

The paper describes three Penicillium verruculosum 28K mutants with about threefold enhanced production of five industrially important carbohydrases. The two-stage fermentation process that we developed provided a further two- to threefold increase in the production of carbohydrases. Physiological and biochemical studies showed that the synthesis of all five carbohydrases is inducible. Carboxymethylcellulase, xylanase, and -glucanase are synthesized under a common regulatory control, as is evident from the concurrent increase in the synthesis of these enzymes in the presence of microcrystalline cellulose. The synthesis of avicelase and -glucosidase is evidently induced by other cellulose- and hemicellulose-containing compounds present in the fermentation medium and, hence, is regulated independently of the three aforementioned enzymes.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 172–178.Original Russian Text Copyright © 2005 by Soloveva, Okunev, Velkov, Koshelev, Bubnova, Kondrateva, Skomarovskii, Sinitsyn.