Fermentative capacity of Kluyveromyces marxianus and Saccharomyces cerevisiae after oxidative stress

Volatile compound production during alcoholic fermentation has been studied in the production of many beverages. Temperature, yeast strain, nutrients and pH have been identified as important factors in the production of volatile compounds. In addition, other factors could influence this production during the fermentation process as well. Oxidative stress could occur during yeast biomass production because oxygen is an essential nutrient that is added to the growth medium. The fermentation parameters and the volatile compound production of one Saccharomyces cerevisiae strain (MC4) and two Kluyveromyces marxianus strains (OFF1 and SLP1) were evaluated in relation to fermentation parameters after oxidative stress induced by hydrogen peroxide or menadione. These yeasts were compared with S. cerevisiae W303–1A and showed significant differences in ethanol production, ethanol yield and maximum ethanol production rate. K. marxianus (OFF1) showed better fermentative capacity after oxidative stress. The higher alcohol production decreased after oxidative stress by >35% after 72 h fermentation time, and the amyl alcohol decreased at a higher level (>60%); however, the isobutanol production increased after oxidative stress between 1.5 and 4 times. The yeasts produced significant concentrations of esters however ethyl lactate, ethyl caprylate and the ethyl caproate were not detected in the control fermentation, while in the stress fermentation they accounted for up to 3 mg/L. These results demonstrate that oxidative stress can play an important role in the final aroma profile; but it is necessary to guarantee adequate yeast growth to obtain the volatile compounds desired. Copyright © 2017 The Institute of Brewing & Distilling     

耐高糖酵母筛选及其高糖胁迫机制的研究进展

耐高糖酵母菌在浓醪酒精发酵过程中起着重要的作用。目前,国内外对耐高酒精度酵母菌有较为广泛深入的研究,而对耐高糖酵母菌的研究还尚有不足。该文介绍了耐高糖酵母的筛选及其高糖胁迫应急机制的研究进展,并对耐高糖酵母的发展趋势进行展望,指出酒精发酵所需酵母菌应生理耐受性较好,能耐高糖度、高酒度、高渗压等,能够抵抗极端不良环境,在工业生产中能够有效提高乙醇的产量。通过筛选耐高糖酵母菌,旨在为酵母菌高糖胁迫机理的研究提供参考。     

Fission yeast translation initiation factor 3 subunit eIF3h is not essential for global translation initiation,but deletion of eif3h+ affects spore formation

The fission yeast Schizosaccharomyces pombe homologue of the p40/eIF3h subunit of mammalian translation initiation factor eIF3 has been characterized in this study. We show that this protein physically associates with the 40S ribosomal particles as a constituent of the multimeric eIF3 protein complex, which consists of all five known eIF3 core subunits (eIF3a, eIF3b, eIF3c, eIF3g and eIF3i) as well as the five non‐core subunits (eIF3d, eIF3e, eIF3f, eIF3h and eIF3m) that constitute an eIF3 holocomplex in fission yeast. However, affinity purification of eIF3 from fission yeast cells expressing TAP‐tagged eIF3h suggests the presence of distinct forms of eIF3 that differ in their composition of the non‐core subunits. Further characterization of eIF3h shows that strains lacking eif3h⁺ (eif3hΔ) are viable and show no gross defects, either in vegetative growth or in the rate of in vivo protein synthesis. Polysome profile analysis shows no apparent defects in translation initiation. Furthermore, deletion of eif3h⁺ does not affect the ability of the other eIF3 subunits to remain associated with one another in a tight protein complex similar to the situation in wild‐type cells. Additionally, we show that human eIF3h can functionally substitute fission yeast eIF3h in complementing in vivo a genetic deletion of eif3h⁺. Interestingly, mutant eif3hΔ cells show several prominent phenotypic properties. They are hypersensitive to caffeine and highly defective in meiosis, producing either no spores or incomplete tetrads with a very high frequency. The implications of these results in relation to the functions of eIF3h in Sz. pombe are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Svf1 inhibits reactive oxygen species generation and promotes survival under conditions of oxidative stress in Saccharomyces cerevisiae

Aberrant regulation of apoptosis, or programmed cell death, contributes to the aetiology of several diseases, including cancers, immunodeficiencies and neurodegenerative illnesses. We hypothesized that key features of mammalian cell death regulation may be conserved in single celled organisms such as the budding yeast Saccharomyces cerevisiae. We previously identified the yeast gene SVF1 in a screen for mutations that could be functionally complemented by exogenous expression of the human anti-apoptotic gene Bcl-x(L). Anti-apoptotic Bcl-2 family members have been shown to promote redox stability through upregulation of antioxidant pathways in mammalian cells. Here we demonstrate that the Svf1 protein is required for yeast survival under conditions of oxidative stress, including cold stress. Cells lacking SVF1 are hypersensitive to conditions associated with increased reactive oxygen species (ROS) generation and to direct chemical precursors of ROS, and demonstrate increased levels of ROS under these conditions. Hypersensitivity to oxidative stress can be reversed by treatment with the antioxidant N-acetylcysteine or expression of exogenous SVF1, although exogenous expression of Bcl-x(L) did not protect cells from cold stress. Exogenous SVF1 expression in mammalian cells confers resistance to H(2)O(2) exposure. Our data are consistent with previous observations suggesting a key role of oxidative stress response in mammalian apoptotic regulation and validate the use of S. cerevisiae as a model for studying programmed cell death.     

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.     

Cellular mechanism for the improvement of multiple stress tolerance in brewer's yeast by potassium ion supplementation

The ethanol fermentation efficiency was affected by multiple stress tolerance of yeast during brewing and bioethanol industry. The effect of KCl on the multiple stress tolerance of yeast cells was examined. Results showed that KCl addition significantly enhanced the tolerance of yeast cells to osmotic and ethanol stress, which correlated with the decreased membrane permeability, the increased intracellular ergosterol and ATP content, and the improved activity of complex II and complex III in yeast cells. Biomass and viability of yeast cells under osmotic and ethanol stress were increased significantly by KCl addition. Supplementation of 4 and 10 g L⁻¹ KCl exhibited the best promotion activity for yeast cells present in medium with 500 g L⁻¹ sucrose and 10% (v v⁻¹) ethanol, respectively. These results suggested that exogenous potassium addition might be an effective strategy to improve yeast tolerance and fermentation efficiency during industrial very-high-gravity (VHG) fermentation.     

Endocytosis and vacuolar morphology in Saccharomyces cerevisiae are altered in response to ethanol stress or heat shock

The vital lipophilic dye N‐(3‐triethylammoniumpropyl)‐4‐[6‐(4‐(diethylamino)phenyl]hexatrienyl) pyridinium dibromide (FM 4‐64) was used to study the effect of ethanol stress and heat shock on endocytosis in the yeast Saccharomyces cerevisiae. Yeast cells stained with FM 4‐64 were placed in a culture chamber and the internalization of the dye was monitored by fluorescence microscopy during perfusion of the cells with fresh growth medium. In the absence of ethanol in the perfusion medium, the internalization of FM 4‐64 from the plasma membrane to the vacuolar membrane by yeast cells harvested from the exponential phase of growth was completed in 30 min. The presence of 6% (v/v) ethanol in the perfusion medium had no obvious effect on the internalization of FM 4‐64 from the plasma membrane, but did lead to an accumulation of the dye in endocytic intermediates. Consequently, vacuolar membrane staining was delayed. Cells stained with FM 4‐64 and subjected to heat shock displayed a similar effect, with endocytic intermediates becoming more prominent with the severity of the heat shock. For both ethanol stress and heat shock, vacuolar morphology altered from segregated structures to a single, large organelle. The findings of this study reinforce previous observations that ethanol stress and heat shock induce similar responses in yeast. Copyright © 1999 John Wiley & Sons, Ltd.     

Positioning of cell growth and division after osmotic stress requires a map kinase pathway

The yeast Saccharomyces cerevisiae has a genetic program for selecting and assembling a bud site on the cell cortex. Yeast cells confine their growth to the emerging bud, a process directed by cortical patches of actin filaments within the bud. We have investigated how cells regulate budding in response to osmotic stress, focusing on the role of the high osmolarity glycerol response (HOG) pathway in mediating this regulation. An increase in external osmolarity induces a growth arrest in which actin filaments are lost from the bud. This is followed by a recovery phase in which actin filaments return to their original locations and growth of the original bud resumes. After recovery from osmotic stress, haploid cells retain an axial pattern of bud site selection while diploids change their bipolar budding pattern to an increased bias for forming a bud on the opposite side of the cell from the previous bud site. Mutants lacking the mitogen-activated protein (MAP) kinase encoded by HOG1 or the MAP kinase kinase encoded by PBS2 (previously HOG4) show a similar growth arrest after osmotic stress. However, in the recovery phase, the mutant cells (a) do not restart growth of the original bud but rather start a new bud, (b) fail to restore actin filaments to the original bud but move them to the new one, and (c) show a more random budding pattern. These defects are elicited by an increase in osmolarity and not by other environmental stresses (e.g., heat shock or change in carbon source) that also cause a temporary growth arrest and shift in actin distribution. Thus, the HOG pathway is required for repositioning of the actin cytoskeleton and the normal spatial patterns of cell growth after recovery from osmotic stress.     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株。在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失。因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选标记,通过胞内同源重组,实现TOR1基因的敲除,最终成功构建突变株AY12a-tor1Δ。对酵母进行耐受性的测定,发现AY12a-tor 1Δ具有一定的耐高温性能,在高渗条件下也有一定的耐受性,同时具有一定的氧化环境耐受性。同时将突变株与AY12a进行模拟白酒发酵(玉米浓醪发酵),并对发酵完成后的酒度、残糖、48 h细胞存活率、CO 2失重及发酵时间进行测定。发酵数据显示突变株AY12a-tor 1Δ乙醇产量有所上升,残糖含量下降,48 h细胞存活率没有下降,发酵时间有所延长。     

酿酒酵母工业菌株胁迫条件耐受性分析

对酿酒酵母（Saccharomyces cerevisiae）工业菌株胁迫条件，包括高浓度酒精、高渗透压、高温、营养饥饿、氧化胁迫、糠醛毒性的耐受性进行了分析，同时测定了抗生素G418对这些菌株的最低抑菌浓度。结果表明，所测定的酵母菌株对这些逆境条件的耐受性有明显差别，表现出良好耐受性的是6508和安琪酵母菌株，同时多倍性的酿酒酵母工业菌株的耐受性均比单倍性实验室菌株高。     

Tryptophan biosynthesis is important for resistance to replicative stress in Saccharomyces cerevisiae

Acute tryptophan depletion is used to induce low levels of serotonin in the brain. This method has been widely used in psychiatric studies to evaluate the effect of low levels of serotonin, and is generally considered a safe and reversible procedure. Here we use the budding yeast Saccharomyces cerevisiae to study the effects of tryptophan depletion on growth rate upon exposure to DNA‐damaging agents. Surprisingly, we found that budding yeast undergoing tryptophan depletion were more sensitive to DNA‐damaging agents such as methyl methanesulphonate (MMS) and hydroxyurea (HU). We found that this defect was independent of several DNA repair pathways, such as homologous recombination, base excision repair and translesion synthesis, and that this damage sensitivity was not due to impaired S‐phase signalling. Upon further analysis, we found that the DNA‐damage sensitivity of tryptophan depletion was likely due to impaired protein synthesis. These studies describe an important source of variance in budding yeast when using tryptophan as an auxotrophic marker, particularly on studies focusing on DNA repair, and suggest that further testing of the effect of tryptophan depletion on DNA repair in mammalian cells is warranted. Copyright © 2016 John Wiley & Sons, Ltd.     

Practical application of mammalian cytochrome P450

Heterologous expression systems play an important role in the analysis of structure-function relationships of mammalian P450s. In addition, these expression systems allow practical application of mammalian P450s. Genetically engineered fused enzymes between mammalian P450 and yeast NADPH-P450 reductase have possible applications in bioconversion processes. Combined use of techniques reported thus far could produce steroid hormones in the recombinant yeast cells harboring four P450 species, CYP11A1, CYP17A1, CYP21B1 and CYP11B1. In an Escherichia coli expression system, the technology of the construction of the mitochondrial P450 electron transport chain has been established. The recombinant E. coli cells expressing CYP27B1, adrenodoxin and NADPH-adrenodoxin reductase would be applicable to a bioconversion process to produce 1alpha,25-dihydroxyvitamin D3. We also demonstrated the usefulness of heterologous expression systems for human liver microsomal P450s for the prediction of drug metabolism in the human body. Microsomal fractions prepared from recombinant yeast, insect and mammalian cells are commercially available and play an important role in preclinical drug development. Application of mammalian P450 to bioremediation with genetic engineering has also been developed. Thus, mammalian P450s appear to have great potential for a wide range of practical applications.     

Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental stresses

Trehalose is believed to play an important role in stress tolerance in the yeast Saccharomyces cerevisiae. In this research, the responses to various environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses, were investigated in a strain with deletion of the NTH1, NTH2, and ATH1 genes encoding trehalases that are involved in trehalose degradation and the triple deletion strains overexpressing TPS1 or TPS2, both of which encode trehalose biosynthesis enzymes in S. cerevisiae. The contents of trehalose constitutively accumulated in the TPS1- and TPS2-overexpressing triple deletion strains were higher than that in the original triple deletion strain. High trehalose accumulation and growth activity were observed in the TPS2-overexpressing triple deletion strain after ethanol stress induction. The same was also observed in the triple deletion and the TPS1- and TPS2-overexpressing triple deletion strains after heat stress induction. In case of freezing stress, all the recombinant strains with high constitutive trehalose content showed high tolerance. However, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicated that high trehalose accumulation can make yeast cells resistant to multiple stresses, but the importance of this accumulation before or after stress induction is varied depending on the type of stress.     

Role of Sho1p adaptor in the pseudohyphal development,drugs sensitivity,osmotolerance and oxidant stress adaptation in the opportunistic yeast Candida lusitaniae

In yeast, external signals such as high osmolarity or oxidant conditions activate the high osmolarity glycerol (HOG) mitogen‐activated protein kinase (MAPK) cascade pathway, which consists of two upstream branches, i.e. Sho1p and Sln1p and common downstream elements, including the Pbs2p MAPK kinase and the Hog1p MAPK. We recently showed that the Candida lusitaniae SLN1 gene, potentially encoding a histidine kinase receptor, is crucial for oxidative stress adaptation when the fungus grows as budding yeast and during the early steps of pseudohyphal development. In the current study, we characterized the SHO1 gene of this opportunistic fungus. Complete loss of SHO1 function causes profound defects in pseudohyphal differentiation, especially in high osmolarity and oxidative stress conditions, suggesting a crucial role of SHO1 in the pseudohyphae morphogenetic transitions. Moreover, when grown as budding yeast, the sho1Δ mutant revealed a sensitivity to compounds that interfere with the cell wall assembly, pointing to a potential role of Sho1p in cell wall biogenesis. However, the sho1Δ mutant does not display evident cell‐wall architecture modifications, such as aggregation phenotypes. Although not hypersusceptible to antifungals of clinical relevance, the sho1Δ mutants are susceptible to the filamentous fungi‐specific antifungals dicarboximides and phenylpyrroles. Finally, our findings highlight some significant phenotypic differences when the C. lusitaniae sho1Δ mutant is compared with the corresponding mutants described in Saccharomyces cerevisiae, Candida albicans and Aspergillus fumigatus. The GeneBank Accession No. for C. lusitaniae SHO1 gene is EU797514. Copyright © 2008 John Wiley & Sons, Ltd.     

精酿啤酒酿造中存在的乙醇胁迫问题及对策

精酿啤酒麦汁浓度高、酒精度高、浓烈香郁,越来越受到啤酒爱好者的青睐,是我国啤酒酿造的新兴重要领域。但是较高浓度的乙醇对酵母的胁迫危害已经成为制约精酿啤酒高酒精度发酵的瓶颈。该文阐述了国内外精酿啤酒发展现状,指出了精酿啤酒的乙醇胁迫问题。结合酿酒酵母乙醇耐受机理研究进展从乙醇耐受菌株选育,发酵过程中增加海藻糖浓度、提高麦角甾醇含量、添加氮源4个方面探讨了解决精酿啤酒乙醇胁迫问题的对策,为精酿啤酒的高酒精度酿造提供了思路和方法。     

酿酒酵母SOD1、SOD2基因缺失对胁迫耐受性的影响

以sod1Δ、sod2Δ、sod1Δsod2Δ酿酒酵母基因缺失菌株为遗传材料,采用休止细胞梯度生长法,分析SOD1和SOD2基因缺失对高温、乙醇毒性、高渗透压、高盐、乙酸毒性及营养饥饿胁迫条件耐受性的影响。结果显示,与野生型菌株相比,sod1Δ菌株对高温、高渗透压和乙酸胁迫的耐受性降低;sod2Δ菌株耐受性无明显变化;sod1Δsod2Δ双缺失菌株对高温、乙醇毒性、乙酸毒性、高渗透压和高盐的耐受性均下降,表明酵母超氧化物歧化酶基因与多种胁迫耐受性密切相关。