Recombination Among Three Mitochondrial Genes in Yeast (Saccharomyces cerevisiae)

Eight crosses involving three different mitochondrial genes were made in Saccharomyces cerevisiae. All three genes were linked, but an unambiguous linkage map could not be constructed.     

Endotoxin and Arachidonic Acid Metabolites in Portal,Hepatic and Arterial Blood of Cattle with Acute Ruminai Acidosis

Ruminai acidosis was induced experimentally with 70 g barley / kg body weight in 2 rumen fistulated cows with chronic indwelling catheters in the portal vein, in a hepatic vein and the carotid artery. The cows were followed for 24 and 20h after the overfeeding and evaluated clinically and by clinical chemistry. The 2 cows exerted different responses to the treatment. Both cows showed signs of severe ruminai acidosis. Both cows had endotoxin in portal and hepatic vein blood, but only 1 of the cows convincingly developed a systemic endotoxaemia. A pre-hepatic release of the stable prostacyclin and thromboxane metabolites, 6-ketoprostaglandin Flα and thromboxane B2 was demonstrated in this cow. The results of the present study show that endotoxin and arachidonic acid metabolites of pre-hepatic origin may be factors involved in the pathogenesis of ruminai acidosis, and that investigation of the factors affecting translocation of ruminai endotoxin and subsequent clearing in the liver, will be of importance.     

On Intermediate Products of Induced Autolysis of Intracellular Proteins of the Saccharomyces cerevisiae Yeast (in Russian)

It was discovered that in the process of baker's yeast autolysis the accumulation of intermediate peptides takes place. The separation and isolation of autolysate peptide fractions as obtained in various times from the beginning of the process, were accomplished. The qualitative characteristic of molecular-weight variation of this fractions dependant on the time was also determined. The intermediate peptides were shown to be hydrolysed additionally with the help of their own immobilized proteases.     

De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae)

Vanillin is one of the world''s most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world''s vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker''s yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.In 2007, the global market for flavor and fragrance compounds was an impressive $20 billion, with an annual growth of 11 to 12%. The isolation and naming of vanillin (3-methoxy-4-hydroxybenzaldehyde) as the main component of vanilla flavor in 1859 (8), and the ensuing chemical synthesis in 1874 (41), in many ways marked the true birth of this industry, and this compound remains the global leader in aroma compounds. The original source of vanillin is the seed pod of the vanilla orchid (Vanilla planifolia), which was grown by the Aztecs in Mexico and brought to Europe by the Spaniards in 1520. Production of natural vanillin from the vanilla pod is a laborious and slow process, which requires hand pollination of the flowers and a 1- to 6-month curing process of the harvested green vanilla pods (37). Production of 1 kg of vanillin requires approximately 500 kg of vanilla pods, corresponding to the pollination of approximately 40,000 flowers. Today, only about 0.25% (40 tons out of 16,000) of vanillin sold annually originates from vanilla pods, while most of the remainder is synthesized chemically from lignin or fossil hydrocarbons, in particular guaiacol. Synthetically produced vanillin is sold for approximately $15 per kg, compared to prices of $1,200 to $4,000 per kg for natural vanillin (46).An attractive alternative is bioconversion or de novo biosynthesis of vanillin; for example, vanillin produced by microbial conversion of the plant constituent ferulic acid is marketed at $700 per kilogram under the trade name Rhovanil Natural (produced by Rhodia Organics). Ferulic acid and eugenol are the most attractive plant secondary metabolites amenable for bioconversion into vanillin, since they can be produced at relatively low costs: around $5 per kilogram (37). For the bioconversion of eugenol or ferulic acid into vanillin, several microbial species have been tested, including gram-negative bacteria of the Pseudomonas genus, actinomycetes of the genera Amycolatopsis and Streptomyces, and the basidiomycete fungus Pycnoporus cinnabarinus (19, 23, 25, 27, 31, 34, 35, 36, 45, 48). In experiments where the vanillin produced was absorbed on resins, Streptomyces cultures afforded very high vanillin yields (up to 19.2 g/liter) and conversion rates as high as 55% were obtained (15). Genes for the responsible enzymes from some of these organisms were isolated and expressed in Escherichia coli, and up to 2.9 g/liter of vanillin were obtained by conversion of eugenol or ferulic acid (1, 3, 32, 49).Compared to bioconversion, de novo biosynthesis of vanillin from a primary metabolite like glucose is much more attractive, since glucose costs less than $0.30/kilogram (42). One route for microbial production of vanillin from glucose was devised by Frost and coworker Li (6, 20), combining de novo biosynthesis of vanillic acid in E. coli with enzymatic in vitro conversion of vanillic acid to vanillin. 3-Dehydroshikimic acid is an intermediate in the shikimate pathway for biosynthesis of aromatic amino acids, and the recombinant E. coli was engineered to dehydrate this compound to form protocatechuic acid (3,4-dihydroxybenzoic acid) and methylate this to form vanillic acid. The vanillic acid was subsequently converted into vanillin in vitro using carboxylic acid reductase isolated from Neurospora crassa. The main products of the in vivo step were protocatechuic acid, vanillic acid, and isovanillic acid in an approximate ratio of 9:4:1, indicating a bottleneck at the methylation reaction and nonspecificity of the OMT (O-methyltransferase) enzyme for the meta-hydroxyl group of protocatechuic acid. Serious drawbacks of this scheme are the lack of an in vivo step for the enzymatic reduction of vanillic acid, demanding the addition of isolated carboxylic acid reductase and costly cofactors such as ATP, NADPH, and Mg²⁺, and the generation of isovanillin as a contaminating side product.In this study, we have genetically engineered single-recombination microorganisms to synthesize vanillin from glucose, according to the metabolic route depicted in Fig. ​Fig.1.1. To avoid the synthesis of isovanillin as an undesired side product, a large array of OMTs was screened for the desired high substrate specificity, and an appropriate enzyme was identified. A synthetic version of an aromatic carboxylic acid reductase (ACAR) gene, optimized for yeast codon usage, was introduced to achieve the reduction step. The vanillin pathway was introduced into both Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, and significant levels of vanillin production were obtained in both organisms. Vanillin β-d-glucoside is the form in which vanillin accumulates and is stored in the fresh pod of the vanilla orchid (Vanilla planifolia). During the “curing” process of the pod, β-glucosidases are liberated and facilitate a partial conversion of the vanillin β-d-glucoside into vanillin. Upon consumption or application, the conversion of vanillin β-d-glucoside into free vanillin by enzymes in the saliva or in the skin microflora can provide for a slow-release effect that prolongs and augments the sensory event, as is the case for other flavor glycosides investigated, such as menthol glucoside (14, 16). In addition to the increased value of vanillin β-d-glucoside as an aroma or flavor compound, production of the glucoside in yeast may offer several advantages. Vanillin β-d-glucoside is more water soluble than vanillin, but most importantly, compounds such as vanillin in high concentrations are toxic to many living cells (4). It has been shown that glucosides of toxic compounds are less toxic to yeasts (24). We found this to be the case with vanillin and S. cerevisiae yeast as well. Thus, to facilitate storage and accumulation of higher vanillin yields, we introduced a step for vanillin glucosylation in S. pombe.Open in a separate windowFIG. 1.Biosynthetic scheme for de novo biosynthesis of vanillin in Schizosaccharomyces pombe and outline of the different vanillin catabolites and metabolic side products observed in different yeast strains and constructs. Gray arrows, primary metabolic reactions in yeast; black arrows, enzyme reactions introduced by metabolic engineering; diagonally striped arrows, undesired inherent yeast metabolic reactions.     

Heat Shock–Induced Changes in the Respiration of the Yeast Saccharomyces cerevisiae

The incubation of Saccharomyces cerevisiaeat elevated temperature (45°C) stimulated the respiration of yeast cells and decreased their survival rate. The respiration-deficient mutant of this yeast was found to be more tolerant to the elevated temperature than the wild-type strain. At the same time, the cultivation of the wild-type strain in an ethanol-containing medium enhanced the respiration, catalase activity, and thermotolerance of yeast cells, as compared with their growth in a glucose-containing medium. It is suggested that the enhanced respiration of yeast cells at 45°C leads to an intense accumulation of reactive oxygen species, which may be one of the reasons for the heat shock–induced cell death.     

Detection of Active Yeast Cells (Saccharomyces cerevisiae) in Frozen Dough Sections

A new method based on fluorescence microscopy was developed to detect active yeast cells in cryosections of wheat dough. The sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and counterstained with Evans blue. The active yeast cells in the sections appeared brilliant yellow and were readily distinguished from the red dough matrix. The dead cells allowed penetration of the Evans blue through the cell membrane, which interfered with the DAPI staining and caused the dead cells to blend into the red environment. The number of active yeast cells in fermenting dough sections containing different proportions of living and dead yeast cells correlated well with the gas-forming capability of the yeast in the dough but not with the results of the conventional plate count method. The new method allows the study of yeast activity not only during the different stages of frozen dough processing but also during the fermentation of doughs.     

Ascospore Formation in the Yeast Saccharomyces cerevisiae

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Atg9 Trafficking in Yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and non-selective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element.5 Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.

Addendum to:

Atg9 Sorting from Mitochondria is Impaired in Early Secretion and VFT Complex Mutants in Saccharomyces cerevisiae

F. Reggiori and D.J. Klionsky

J Cell Sci 2006: 119:2903-11     

MAP Kinase Pathways in the Yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.     

啤酒酵母乙醇脱氢酶Ⅰ(ADHI)的遗传变异

在啤酒酵母(Saccharomyces cerevisiae)中观察到存在一种新的ADHI:ADHIF,电泳迁移率明显快于文献报道的ADHI:ADHIS。这种ADHIF在10％葡萄糖的培养条件下,以及在呼吸缺陷型菌株进行无氧呼吸时都能够出现。ADHIF性状的遗传分析表明,它受1个与结构基因ADC-1S(编码ADHIS)等位的基因ADC-1F控制。     

啤酒酵母菌对无机硒的有机转化

利用啤酒酵母菌对无机硒(亚硒酸钠)进行有机转化。通过在培养基中加入不同浓度的无机硒溶液和不同时间加入无机硒溶液,于28℃、220 r/min摇床条件下培养5 d,离心得菌细胞,测定前样品预处理:破碎菌细胞,显微镜下计数,计算破碎率,破碎后的菌体装入透析袋于蒸馏水中透析除去无机硒。准确测定无机硒,用浓硫酸-高氯酸的消化体系消化样品后,紫外分光光度法于335 nm处测量吸光度,在标准曲线上查出硒含量,计算无机硒的转化率。啤酒酵母菌的最佳加硒时间为24 h,亚硒酸钠浓度大于12μg/mL对啤酒酵母菌转化无机硒有明显抑制作用,啤酒酵母菌对无机硒的摄入率约为62%,转化率约为53%;超生波细胞粉碎仪破碎细胞的破碎率为55%左右。结果表明,啤酒酵母菌可以转化无机硒。     

Vesicular Transport of Extracellular Acid Phosphatases in Yeast Saccharomyces cerevisiae

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.     

Acetate and Bicarbonate in the Correction of Uraemic Acidosis

Uraemic acidosis was readily corrected by the infusion of sodium acetate solution, which gave comparable results to equivalent amounts of sodium bicarbonate infusion. Preliminary data suggest that this was true even in severe liver disease.     

Random Phenotypic Variation of Yeast (Saccharomyces cerevisiae) Single-Gene Knockouts Fits a Double Pareto-Lognormal Distribution

Background

Distributed robustness is thought to influence the buffering of random phenotypic variation through the scale-free topology of gene regulatory, metabolic, and protein-protein interaction networks. If this hypothesis is true, then the phenotypic response to the perturbation of particular nodes in such a network should be proportional to the number of links those nodes make with neighboring nodes. This suggests a probability distribution approximating an inverse power-law of random phenotypic variation. Zero phenotypic variation, however, is impossible, because random molecular and cellular processes are essential to normal development. Consequently, a more realistic distribution should have a y-intercept close to zero in the lower tail, a mode greater than zero, and a long (fat) upper tail. The double Pareto-lognormal (DPLN) distribution is an ideal candidate distribution. It consists of a mixture of a lognormal body and upper and lower power-law tails.

Objective and Methods

If our assumptions are true, the DPLN distribution should provide a better fit to random phenotypic variation in a large series of single-gene knockout lines than other skewed or symmetrical distributions. We fit a large published data set of single-gene knockout lines in Saccharomyces cerevisiae to seven different probability distributions: DPLN, right Pareto-lognormal (RPLN), left Pareto-lognormal (LPLN), normal, lognormal, exponential, and Pareto. The best model was judged by the Akaike Information Criterion (AIC).

Results

Phenotypic variation among gene knockouts in S. cerevisiae fits a double Pareto-lognormal (DPLN) distribution better than any of the alternative distributions, including the right Pareto-lognormal and lognormal distributions.

Conclusions and Significance

A DPLN distribution is consistent with the hypothesis that developmental stability is mediated, in part, by distributed robustness, the resilience of gene regulatory, metabolic, and protein-protein interaction networks. Alternatively, multiplicative cell growth, and the mixing of lognormal distributions having different variances, may generate a DPLN distribution.     

The Yeast Protein Database (YPD): a curated proteome database for Saccharomyces cerevisiae.

The Yeast Protein Database (YPD) is a curated database for the proteome of Saccharomyces cerevisiae . It consists of approximately 6000 Yeast Protein Reports, one for each of the known or predicted yeast proteins. Each Yeast Protein Report is a one-page presentation of protein properties, annotation lines that summarize findings from the literature, and references. In the past year, the number of annotation lines has grown from 25 000 to approximately 35 000, and the number of articles curated has grown from approximately 3500 to >5000. Recently, new data types have been included in YPD: protein-protein interactions, genetic interactions, and regulators of gene expression. Finally, a new layer of information, the YPD Protein Minireviews, has recently been introduced. The Yeast Protein Database can be found on the Web at http://www.proteome.com/YPDhome. html