Survival of Saccharomyces cerevisiae and Sarcina lutea at Refrigerator Temperatures

Assay organisms Saccharomyces cerevisiae and Sarcina lutea, when suspended in a weak buffer and stored at refrigerator temperatures, are stable for 1 year or more.     

Survival of inoculated Saccharomyces cerevisiae strain on wine grapes during two vintages

AIMS: To investigate the influence of a specific ecological niche, the wine grape, on the survival and development of Saccharomyces cerevisiae. METHODS AND RESULTS: A strain with a rare phenotype was sprayed onto the grape surfaces and monitored through two vintages using a specific indicative medium and analysing the internal transcribed spacer regions in the 5.8S rDNA. During the ripening process, there was a progressive colonization of the surface of the undamaged and damaged grapes by epiphytic yeasts, up to the time of harvest. The damaged wine grapes showed a much greater epiphytic yeast population. However, the inoculated S. cerevisiae strain showed a scarce persistence on both undamaged and damaged wine grapes, and the damaged grapes did not appear to improve the grape surface colonization of this strain. CONCLUSIONS: Results indicated that wine grape is not a favourable ecological niche for the development and colonization of S. cerevisiae species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this work are further evidence that S. cerevisiae is not specifically associated with natural environments such as damaged and undamaged wine grapes.     

Saccharomyces cerevisiae

Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23–46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae.     

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H₂O₂), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H₂O₂ in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.Download video file.^{(79M, mov)}     

Factors Affecting the Survival of Auxotrophs and Prototrophs of Saccharomyces cerevisiae in Mixed Populations

Moat, Albert G. (Hahnemann Medical College, Philadelphia, Pa.), Isabel J. Barnes, and Eleanor H. McCurley. Factors affecting the survival of auxotrophs and prototrophs of Saccharomyces cerevisiae in mixed populations. J. Bacteriol. 92:297-301. 1966.-The conditions under which the number of yeast prototrophs, as well as respiration-deficient mutants, could be materially decreased, while allowing the survival of auxotrophic mutants in recoverable numbers, have been investigated in detail. Neither the use of carbohydrates other than glucose to prevent development of respiration-deficient mutants, nor treatment with acriflavine to render all surviving wild types respiration-deficient, provided a selective advantage for the auxotrophs. Increased concentrations of the antifungal agents amphotericin B or endomycin, while reducing the number of respiration-deficient mutants, did not significantly increase the final mutant-wild type ratio. A more soluble form of amphotericin B (Fungizone), when used under carefully defined physiological conditions, produced a significant reduction in the number of surviving prototrophs relative to the surviving auxotrophs, without development of respiration-deficient mutants.     

The Role of Trehalose and Glycogen in the Survival of Aging Saccharomyces cerevisiae Cells

The role of the storage carbohydrates trehalose and glycogen in the survival of aging Saccharomyces cerevisiae cells was studied. Culture aging for one week did not reduce cell viability. During this period, the cells accumulated the storage carbohydrates and showed increased activity of the glycolytic enzymes hexokinase and phosphofructokinase. However, further aging led to a drastic drop in cell viability and to a decrease in the cellular content of trehalose and glycogen and in the activity of hexokinase and phosphofructokinase. The possible reasons for these changes are discussed.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.     

Survival and antioxidant defence of the yeast Saccharomyces cerevisiae during starvation and oxidative stress

The role of catalase in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide under starvation was investigated. It was shown that under conditions used in this study 0.5 mM H2O2 did not change the number of viable cells in the wild strain YPH250, but this parameter was decreased by 15% in the acatalsaemic strain YWT1. Cells treatment with 0.5 mM H2O2 for 30 min did not modify the levels of carbonyl proteins in the parental strain, but caused its 1.4-fold increase in the defective strain. The observed 1.5-fold activation of catalase in the wild strain cells in response to H2O2-stress suggests that under starvation conditions catalase can be involved in the yeast cell protection, particularly they can prevent oxidative modification of some antioxidant and associated enzymes.     

Suspended Animation Extends Survival Limits of Caenorhabditis elegans and Saccharomyces cerevisiae at Low Temperature

The orderly progression through the cell division cycle is of paramount importance to all organisms, as improper progression through the cycle could result in defects with grave consequences. Previously, our lab has shown that model eukaryotes such as Saccharomyces cerevisiae, Caenorhabditis elegans, and Danio rerio all retain high viability after prolonged arrest in a state of anoxia-induced suspended animation, implying that in such a state, progression through the cell division cycle is reversibly arrested in an orderly manner. Here, we show that S. cerevisiae (both wild-type and several cold-sensitive strains) and C. elegans embryos exhibit a dramatic decrease in viability that is associated with dysregulation of the cell cycle when exposed to low temperatures. Further, we find that when the yeast or worms are first transitioned into a state of anoxia-induced suspended animation before cold exposure, the associated cold-induced viability defects are largely abrogated. We present evidence that by imposing an anoxia-induced reversible arrest of the cell cycle, the cells are prevented from engaging in aberrant cell cycle events in the cold, thus allowing the organisms to avoid the lethality that would have occurred in a cold, oxygenated environment.     

酿酒酵母代谢工程技术

自20世纪90年代初期诞生以来,代谢工程历经了30年的快速发展。作为代谢工程的首选底盘细胞之一,酿酒酵母细胞工厂已被广泛应用于大量大宗化学品和新型高附加值生物活性物质的生物制造,在能源、医药和环境等领域取得了巨大的突破。近年来,合成生物学、生物信息学以及机器学习等相关技术也极大地促进了代谢工程的技术发展和应用。文中回顾了近30年来酿酒酵母代谢工程重要的技术发展,首先总结了经典代谢工程的常用方法和策略,以及在此基础上发展而来的系统代谢工程和合成生物学驱动的代谢工程技术。最后结合最新技术发展趋势,展望了未来酿酒酵母代谢工程发展的新方向。     

构建酿酒酵母工程菌合成香紫苏醇

香紫苏醇是一种来源于植物的双环二萜醇,常用于香味成分且具有重要生物学活性。为实现香紫苏醇的微生物生产,以酿酒酵母为宿主,表达焦磷酸赖百当烯二醇酯合酶和香紫苏醇合酶,构建香紫苏醇的人工生物合成途径。发现过表达前体代谢关键酶、蛋白质融合增强底物通道效应及去除异源蛋白信号肽等,有利于香紫苏醇合成。在摇瓶培养条件下,组合优化得到的工程菌株S6的香紫苏醇产量达到8.96 mg/L。研究结果对其他萜类化合物的异源生物合成具有参考价值。     

Engineering Saccharomyces cerevisiae for isoprenol production

Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.     

Membranes of Saccharomyces cerevisiae

A crude small particle pellet, obtained from postmitochondrial supernatant fractions of Saccharomyces cerevisiae, contains about half the ergosterol and phospholipid of crude cell homogenates. Most of the phospholipid of this pellet is in a “heavy” fraction which, with the aid of electron microscopy, shows membranous elements in addition to discrete particles. The “heavy” fraction, upon treatment with deoxycholate, can be freed of membranes, or, with ribonuclease treatment, ribosomes can be removed, leaving relatively clean membranes. The “heavy” fraction resembles the microsomes of animal cells, but contains considerably less lipids, including phospholipids, thus suggesting a less well-developed intracellular membrane system.     

Procedure for mutagenizing spores of Saccharomyces cerevisiae

A procedure for inducing mutants of a homothallic strain of Saccharomyces cerevisiae is described. The essential parts of the procedure are long incubation in Glusulase, which preferentially kills vegetative cells instead of spores, and treatment in 9% ethyl methanesulfonate, which also preferentially kills vegetative cells instead of spores. Consequently, the viable population is virtually 100% spores.     

A method for enucleation of Saccharomyces cerevisiae

Abstract We have analysed the response of the acidophilic chemolithotroph Thiobacillus ferrooxidans to phosphate starvation. Cultivation of the bacteria in the absence of added phosphate induced a remarkable filamentation of the cells. Polyacrylamide gel electrophoresis revealed several proteins whose levels increased upon phosphate limitation, as well as some polypeptides that were exclusively synthesized under this growth limitation. One of the proteins whose level increased by the lack of phosphate was apparently an acid phosphatase with a pH optimum of about 3.8, and a molecular mass of 26 kDa, which was located in the periplasm. The N-terminal sequence of a 26 kDa protein derepressed by starvation, which may correspond to the T. ferrooxidans phosphatase, showed 30% and 35% identity with the known sequence of Lysobacter enzymogenes and Escherichia coli alkaline phosphatases, respectively.     

Survival of Saccharomyces cerevisiae Y5 during starvation in the presence of osmotic supports.

The viability of starved or dying Saccharomyces cerevisiae Y5 cells was extended for up to 1,500 h by adding osmotic supports (0.6 M sorbitol or mannitol) to the starvation media. Replacement of these polyols with 0.3 M KCl, 0.5 M MgSO4, or 0.2 M MgCl2 postponed the death of cells incubated in N-free medium, but accelerated it in distilled water.