Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts

The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol-grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH-gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH-gradient is strongly related to the cell's integrity.     

Production of metabolites by methylotrophic yeasts

Recent advances in biotechnology of methanol-utilizing yeasts are briefly summarized. The emphasis is given to production of some fine and commercial chemicals such as formaldehyde, formate, hydrogen peroxide, dihydroxyacetone, ATP, FAD as well as proteins, specifically alcohol oxidase. The advantages of mutants and recombinants derived from methylotrophic yeasts for efficient production of various useful materials are demonstrated.     

Emerging applications of the methylotrophic yeasts

Abstract The use of methylotrophic yeasts for the production of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the production of heterologous recombinant proteins by Pichia pastoris that is emerging as the most significant application of the methylotrophic yeasts.     

Emerging applications of the methylotrophic yeasts

The use of methylotrophic yeasts for the production of single-cell-protein (SCP), alcohol oxidase and fine chemicals has been proposed. Fermentation technology developed for the growth of these yeasts on methanol at high cell densities has been commercialized. However, it is the production of heterologous recombinant proteins by Pichia pastoris that is emerging as the most significant application of the methylotrophic yeasts.     

A medium for detecting sulphite-binding yeasts in meat products

The loss of free sulphite in meat products, and thereby preservative potential, is associated with an increase in bound sulphite resulting in part from acetaldehyde production by yeasts. A medium containing Schiff's reagent was devised to detect sulphite-binding yeasts. Yeast colonies that caused red colouration were recorded as acetaldehyde producers. Initially acetaldehyde-producing strains (as enumerated on the detection medium) represented 60% of the yeast flora of preserved minced lamb but, as the free sulphite level diminished, non-sulphite-binding yeasts increased in number.     

甲醇酵母代谢工程研究进展

甲醇酵母由于独特优点被认为是绿色生物制造的潜在宿主.特别是其天然甲醇利用性能有望建立甲醇生物转化路线,拓展生物炼制底物,具有重要经济价值和环保意义.文中综述了代谢工程改造甲醇酵母合成蛋白质和化学品的最新研究进展,并比较了其与模式生物酿酒酵母作为细胞工厂的优缺点.随后,分析了甲醇酵母代谢工程改造面临的挑战,并展望了潜在解...     

甲醇酵母表达系统研究进展

甲醇酵母是一种新的基因表达系统,有着许多突出的优点。多种重组蛋白已在甲醇酵母获得成功表达.该文综述几种甲醇酵母表达系统的特点和现状。     

Trehalase activation in yeasts is mediated by an internal acidification

It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCl depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide m-chlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential.     

非常规酵母天然产物合成

以解脂耶氏酵母(Yarrowia lipolytica)、巴斯德毕赤酵母(Pichia pastoris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、圆红冬孢酵母(Rhodosporidium toruloides)、多形汉逊酵母(Hansenula polymorpha)为代表的非常规酵母凭借较广的底物利用谱、较强的环境耐受性等优势,已成功实现多种天然产物的高效生产。随着合成生物学及基因编辑技术的发展,针对非常规酵母代谢工程改造的工具和策略也逐渐丰富。本文介绍了几类常见的非常规酵母的生理特性、工具开发及应用现状,并总结归纳了天然产物合成优化中常用的代谢工程策略;最后讨论了现阶段非常规酵母作为天然产物合成细胞工厂的优势和不足,并对后续研究和发展趋势进行了展望。     

Effect of formaldehyde on the level of cytochrome c in methylotrophic yeasts

The effect of formaldehyde on the cytochrome system of the methylotrophic yeasts Candida tropicalis, Candida boidinii, Candida methylica, Hansenula capsulata, Hansenula polymorpha, Pichia pastoris and nonmethylotrophic yeasts Saccharomyces cerevisiae was studied. In the investigated concentration range of 0.58 – 2.5 mol · 1^?1, formaldehyde decreases the level of the reduced cytochrome c in the above-mentioned microorganisms. The kinetics of the change in the reduced cytochrome c content has two phases: a phase of slow decrease and a phase of complete disappearance of the reduced cytochrome c in the cells. The duration of the first phase shortens with increasing concentrations of formaldehyde.     

Redox balances in the metabolism of sugars by yeasts

Abstract The central role of the redox couples NAD⁺/NADH and NADP⁺/NADPH in the metabolism of sugars by yeasts is discussed in relation to energy metabolism and product formation. Besides their physical compartmentation in cytosol and mitochondria, the two coenzyme systems are separated by chemical compartmentation as a consequence of the absence of transhydrogenase activity. This has considerable consequences for the redox balances of both coenzyme systems and hence for sugar metabolism in yeasts.
As examples, the competition between respiration and fermentation of glucose, the Crabtree effect, the Custers effect, adaptation to anaerobiosis, the activities of the hexose monophosphate pathway, and the fermentation of xylose in yeast are discussed.     

Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera

Methylotrophic yeasts, named after their ability to grow on methanol as the sole carbon source, have raised large interest as recombinant protein factories. In this review, we explain the basic mechanisms underlying this interest and describe the minimal requirements to transform the two genera recognized as methylotrophic, Pichia and Candida, into a powerful protein production tool. We present a comparison between this group of yeasts and the conventional yeasts used as expression system in view of productivity, level of secretion and quality of post-translational modifications. Selected examples of recombinant protein produced by methylotrophic yeast are also included.     

The significance of peroxisomes in methanol metabolism in methylotrophic yeast

The capacity to use methanol as sole source of carbon and energy is restricted to relatively few yeast species. This may be related to the low efficiency of methanol metabolism in yeast, relative to that of prokaryotes. This contribution describes the details of methanol metabolism in yeast and focuses on the significance of compartmentalization of this metabolic pathway in peroxisomes.     

Degradation of PCBs by white rot fungi,methylotrophic and hydrocarbon utilizing yeasts and bacteria

Summary Of the white rot fungi tested, two strains alone and other three in combination with specific yeasts or bacteria degraded PCBs in polluted soil within interval 20–30%. When all organisms were applied together PCB degradation achieved was about 50%.     

Fermentation and aerobic metabolism of cellodextrins by yeasts

The fermentation and aerobic metabolism of cellodextrins by 14 yeast species or strains was monitored. When grown aerobically, Candida wickerhamii, C. guilliermondii, and C. molischiana metabolized cellodextrins of degree of polymerization 3 to 6. C. wickerhamii and C. molischiana also fermented these substrates, while C. guilliermondii fermented only cellodextrins of degree of polymerization less than or equal to 3. Debaryomyces polymorphus, Pichia guilliermondii, Clavispora lusitaniae, and one of two strains of Kluyveromyces lactis metabolized glucose, cellobiose, and cellotriose when grown aerobically. These yeasts also fermented these substrates, except for K. lactis, which fermented only glucose and cellobiose. The remaining species/strains tested, K. lactis, Brettano-myces claussenii, B. anomalus, K. dobzhanskii, Rhodotorula minuta, and Dekkera intermedia, both fermented and aerobically metabolized glucose and cellobiose. Crude enzyme preparations from all 14 yeast species or strains were tested for ability to hydrolyze cellotriose and cellotretose. Most of the yeasts produced an enzyme(s) capable of hydrolyzing cellotriose. However, with two exceptions, R. minuta and P. guilliermondii, only the yeasts that metabolized cellodextrins of degree of polymerization greater than 3 produced an enzyme(s) that hydrolyzed cellotretose.