Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana ^r -6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana ^r -6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.     

Trypanosomes lacking uracil-DNA glycosylase are hypersensitive to antifolates and present a mutator phenotype

Cells contain low amounts of uracil in DNA which can be the result of dUTP misincorporation during replication or cytosine deamination. Elimination of uracil in the base excision repair pathway yields an abasic site, which is potentially mutagenic unless repaired. The Trypanosoma brucei genome presents a single uracil-DNA glycosylase responsible for removal of uracil from DNA. Here we establish that no excision activity is detected on U:G, U:A pairs or single-strand uracil-containing DNA in uracil-DNA glycosylase null mutant cell extracts, indicating the absence of back-up uracil excision activities. While procyclic forms can survive with moderate amounts of uracil in DNA, an analysis of the mutation rate and spectra in mutant cells revealed a hypermutator phenotype where the predominant events were GC to AT transitions and insertions. Defective elimination of uracil via the base excision repair pathway gives rise to hypersensitivity to antifolates and oxidative stress and an increased number of DNA strand breaks, suggesting the activation of alternative DNA repair pathways. Finally, we show that uracil-DNA glycosylase defective cells exhibit reduced infectivity in vivo demonstrating that efficient uracil elimination is important for survival within the mammalian host.     

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.     

Hydrogen peroxide mediates the radiation-induced mutator phenotype in mammalian cells

Chronic oxidative stress has been associated with genomic instability following exposure to ionizing radiation. However, results showing direct causal linkages between specific ROS (reactive oxygen species) and the ionizing radiation-induced mutator phenotype are lacking. The present study demonstrates that ionizing radiation-induced genomically unstable cells (characterized by chromosomal instability and an increase in mutation and gene amplification frequencies) show a 3-fold increase in steady-state levels of hydrogen peroxide, but not superoxide. Furthermore, stable clones isolated from parallel studies showed significant increases in catalase and GPx (glutathione peroxidase) activity. Treatment of unstable cells with PEG-CAT (polyethylene glycol-conjugated catalase) reduced the mutation frequency and mutation rate in a dose-dependent fashion. In addition, inhibiting catalase activity in the stable clones using AT (3-aminotriazole) increased mutation frequency and rate. These results clearly demonstrate the causal relationship between chronic oxidative stress mediated by hydrogen peroxide and the mutator phenotype that persists for many generations following exposure of mammalian cells to ionizing radiation.     

Fission yeast Schizosaccharomyces pombe as a new system for the investigation of corticosterone methyloxidase deficiency-causing mutations

The aldosterone synthase, CYP11B2, catalyses the conversion of 11-deoxycorticosterone to aldosterone, a process that requires three steps: a hydroxylation at position 11β to form corticosterone, another one at position 18 to produce 18-hydroxycorticosterone, and, finally, an oxidation at position 18 to form aldosterone. Aldosterone synthase deficiency usually finds its expression in infancy as a life-threatening electrolyte imbalance, caused by mutations in the CYP11B2 gene. Therefore, in depth studies of mutations and their enzymatic activities will provide information for the diagnosis and management of hypoaldosteronism caused by CYP11B2 deficiencies. Here, we report the development of a fast and cheap whole-cell technology for the enzymatic characterisation of CYP11B2 mutations. The principle of the new system is the heterologous expression of the mutants of CYP11B2 in fission yeast (Schizosaccharomyces pombe) followed by steroid bioconversion assays for the enzymatic characterisation of the investigated mutants. The new system was validated and 10 known mutations of CYP11B2 have been investigated, two of them for the first time concerning their effect on the CYP11B2 three-step reaction. The results of the fission yeast system were in good agreement with the cell culture results presenting this new system as an alternative non radioactive method that can be applied for the enzymatic characterisation of CYP11B2 mutations.     

Apoptosis and lipoapoptosis in the fission yeast Schizosaccharomyces pombe

Yeasts being simple eukaryotes are established genetic systems that are often employed to solve important biological questions. Recently, it has become evident that certain cell death programs exist in these unicellular organisms. For example, it has been shown recently that strains of the fission yeast Schizosaccharomyces pombe deficient in triacylglycerol synthesis undergo cell death with prominent apoptotic markers. This minireview is intended to discuss key developments that have rendered fission yeast useful both as a tool and as a model for apoptosis and lipoapoptosis research. It is attempted to delineate a putative signaling pathway leading to the execution of lipoapoptosis in the fission yeast. Although in its infancy, apoptosis research in the fission yeast promises exciting breakthroughs in the near future.     

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.     

Induction of sexual co-flocculation of heterothallic fission yeast (Schizosaccharomyces pombe) cells by mating pheromones

Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10 min. By contrast, comparable flocculation requires over 1 h if untreated control cells are mixed with obverse mating-type cells. The agglutinin of P cells is more inducible than that of M cells. These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea. These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability). Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.     

Macromolecular changes and commitment to sporulation in the fission yeast Schizosaccharomyces pombe

Homothallic cultures of Schizosaccharomyces pombe in stationaryphase may be induced to flocculate by aeration. Flocculationis followed by copulation, conjugation, zygote formation, meiosisand sporulation. This developmental sequence was monitored forrespiratory activity, changes in protein, RNA and DNA, cataboliterepression, and commitment to sporulation. Respiratory activity,apparently a prerequisite to induction, increased 5-fold priorto maximum flocculation and remained at that level up to theend of the sequence. Protein and RNA content increased priorto conjugation but gradually decreased shortly thereafter. Around of premeiotic DNA synthesis occurred after copulation,presumably during conjugation. The developmental sequence wasrepressible by glucose. Cyclic AMP at low concentrations stimulatedsporulation somewhat, but the stimulatory effect was not sufficientto offset repression due to glucose. Commitment to sporulationwas determined by adding glucose at various times during thedevelopmental sequence and then observing refractoriness ofthe events to catabolite repression. Cells not committed wererepressed by glucose and reverted to mitotic cell cycles. Committedcells proceeded to sporulate in the presence of exogenous glucose.Commitment to sporulation appears to occur soon after premeioticDNA synthesis. ¹NRCC No.: 18664. (Received February 22, 1980; )     

Regularities and irregularities in the cell cycle of the fission yeast,Schizosaccharomyces pombe (a review)

In an exponentially growing wild-type fission yeast culture a size control mechanism ensures that mitosis is executed only if the cells have reached a critical size. However, there is some scattering both in cell length at birth (BL) and in cycle time (CT). By computational simulations we show here that this scattering cannot be explained solely by asymmetric cell division, therefore we assume that nuclear division is a stochastic, asymmetric process as well. We introduce an appropriate stochastic variable into a mathematical model and prove that this assumption is suitable to describe the CT vs. BL graph in a wild-type fission yeast population. In a double mutant of fission yeast (namely wee1-50 cdc25 delta) this CT vs. BL plot is even more curious: cycle time splits into three different values resulting in three clusters in this coordinate system. We show here that it is possible to describe these quantized cycles by choosing the appropriate values of the key parameters of mitotic entry and exit and even more the clustered behavior may be simulated by applying a further stochastic parameter.     

RNA interference and heterochromatin in the fission yeast Schizosaccharomyces pombe

Fission yeast is a useful model for RNA interference because it has single-copy genes for components of the RNAi pathway such as argonaute, dicer and RNA-dependent RNA polymerase (RdRP). Functions for RNAi revealed in S. pombe, such as heterochromatic silencing and chromosome segregation, are likely to be ancient because they are shared with some other eukaryotes. The underlying mechanisms are being rapidly unraveled.     

Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe

Genetically engineered microorganisms are being increasingly used for the industrial production of complicated chemical compounds such as steroids; however, there have been few reports on the use of the fission yeast Schizosaccharomyces pombe for this purpose. We previously have demonstrated that this yeast is a unique host for recombinant expression of human CYP11B2 (aldosterone synthase), and here we report the functional production of human CYP11B1 (steroid 11beta-hydroxylase) in S. pombe using our new integration vector pCAD1. In the human adrenal, the mitochondrial cytochrome P450 enzyme CYP11B1 catalyses the conversion of 11-deoxycortisol to cortisol, a key reaction in cortisol biosynthesis that in addition is of fundamental interest for the technical synthesis of glucocorticoids. We observed that the endogenous mitochondrial electron transport system detected previously by us is capable of supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. Under optimised cultivation conditions the transformed yeasts show in vivo the inducible ability to efficiently and reliably convert deoxycortisol to cortisol at an average rate of 201 microM d(-1) over a period of 72h, the highest value published to date for this biotransformation.