Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.     

New applications for Schizosaccharomyces pombe in the alcoholic fermentation of red wines

The fermentation of grape must using non‐Saccharomyces yeasts with particular metabolic and biochemical properties is of growing interest. In the present work, red grape must was fermented using four strains of Schizosaccharomyces pombe (935, 936, 938 and 2139), Saccharomyces cerevisiae 7VA and Saccharomyces uvarum S6U, and comparisons were made over the fermentation period in terms of must sugar (glucose + fructose), malic acid, acetic acid, ammonia, primary amino nitrogen, lactic acid, urea (a possible fermentation activator or precursor of other metabolites) and pyruvic acid (a molecule affecting vitisin formation and therefore colour stability) concentration. The colour intensity of the fermenting musts was also recorded. The Schizosaccharomyces strains consumed less primary amino nitrogen and produced less urea and more pyruvic acid than other Saccharomyces species. Further, three of the four Schizosaccharomyces strains completed the breakdown of malic acid by day 4 of fermentation. The main negative effect of the use of Schizosaccharomyces was strong acetic acid production. The Schizosaccharomyces strains that produced most pyruvic acid (938 and 936) were associated with better ‘wine’ colour than the remaining yeasts. The studied Schizosaccharomyces could therefore be of oenological interest.     

生物发酵法制丁二酸生产工艺的研究与应用

丁二酸是一种重要的化工原料,广泛用于食品、医药和化工等行业,市场前景广阔。微生物发酵法生产丁二酸具有高效、环保、可持续利用的特点,是一种新型的绿色化工生产工艺。本文介绍了微生物发酵法制备丁二酸的生产工艺并对其应用前景进行了展望。     

Transport of malic acid in the yeast Schizosaccharomyces pombe: evidence for a proton-dicarboxylate symport.

The transport system for malic acid present in Schizosaccharomyces pombe cells, growing in batch culture on several carbon sources, has been studied. It was found that the dicarboxylic acid carrier of S. pombe is a proton-dicarboxylate symporter that allows uphill transport and accumulation as a function of delta pH with the following kinetic parameters at pH 5.0: Vmax = 0.1 nmol of total malic acid s-1 mg (dry weight) of cells-1 and Km = 1.0 mM total malic acid. Malic acid uptake (pH 5.0) was accompanied by disappearance of extracellular protons, the uptake rates of which followed Michaelis-Menten kinetics as a function of the acid concentration. The Km values calculated as the concentrations either of anions or of undissociated acid, at various extracellular pH values, pointed to the monoanionic form as the transported species. Furthermore, accumulated free acid suffered rapid efflux after the addition of the protonophore carbonyl cyanid m-chlorophenyl hydrazone. These results suggested that the transport system was a dicarboxylate-proton symporter. Growth of cells in a medium with glucose (up to 14%, w/v) and malic acid (1.5%, w/v) also resulted in proton-dicarboxylate activity, suggesting that the system, besides being constitutive, was still active at high glucose concentrations. The following dicarboxylic acids acted as competitive inhibitors of malic acid transport at pH 5.0: D-malic acid, succinic acid, fumaric acid, oxaloacetic acid, alpha-ketoglutaric acid, maleic acid and malonic acid. In addition, all of these dicarboxylic acids induced proton movements that followed Michaelis-Menten kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a fed-batch culture process for enhanced production of recombinant human antithrombin by Chinese hamster ovary cells

Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, primarily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an increased glutamate concentration on cell metabolism and rAT production were investigated. When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maximum lactate concentration decreased with decreasing pH. The highest production level of rAT was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glucose consumption rate (q(Glc)) with increasing culture time was observed in all the above-mentioned experiments, while the glucose concentration was maintained above 0.7 g l(-1). In addition, a decrease in the specific rAT production rate (q(rAT)) was observed after the depletion of lactate in the late cultivation stage. Taken together, these results suggest that the reduced availability of cellular energy caused by the decrease in q(Glc) and depletion of lactate led to the decrease in q(rAT). This decrease in q(rAT) was partially prevented by increasing the residual glutamate concentration from 1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The optimized fed-batch culture process yielded 1.0 g l(-1) rAT at 287 h of cultivation.     

Quantitative analysis of human ras localization and function in the fission yeast Schizosaccharomyces pombe

Ras signalling is central to fundamental and diverse cellular processes. In higher eukaryotes ras signalling is highly complex, involving multiple isoforms, regulatory proteins and effectors. As a consequence, the study of ras activity in mammalian systems presents a number of technical challenges. The model organism Schizosaccharomyces pombe has previously proved a key system for the study of human signalling components and provides an ideal model for the study of ras, as it contains just one ras protein (Ras1p), which is non‐essential and controls a number of downstream processes. Here we present data demonstrating the quantitative analysis of three distinct Ras1‐related signalling outputs, utilizing the three most abundant human ras isoforms, H‐Ras, N‐Ras and K‐Ras4B, in Sz. pombe. Further, we have characterized the localization of these three human ras isoforms in Sz. pombe, utilizing quantitative image analysis techniques. These data indicate that all three human ras isoforms are functional in fission yeast, displaying differing localization patterns which correlate strongly with function in the regulation of pheromone response and cell shape. These data demonstrate that such yeast strains could provide powerful tools for the investigation of ras biology, and potentially in the development of cancer therapies. Copyright © 2013 John Wiley & Sons, Ltd.     

Construction and characterization of a series of vectors for Schizosaccharomyces pombe

A set of vectors was created to allow cloning and expression studies in Schizosaccharomyces pombe. These vectors had a uniform backbone with an efficient Sz. pombe ARS, ARS3002, but different selectable markers--his3+, leu1+, ade6+ and ura4+. The vectors functioned efficiently as autonomously replicating plasmids that could also be converted into integrating vectors. The ura4+-containing vector was used to construct a Sz. pombe genomic library.     

Characterization of a Schizosaccharomyces pombe mutant deficient in UDP-galactose transport activity.

In fission yeast, Schizosaccharomyces pombe, the carbohydrate components of the cell wall consist of galactomannan, unlike in Saccharomyces cerevisiae. We previously found that the disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, led to the complete defect of cell surface galactosylation in Sz. pombe. The Deltagms1 strain is therefore useful for the analysis of physiological properties of galactose residues in Sz. pombe. The deletion strain of gms1+ was viable; however, itshowed an aberrant cell morphology and increased sensitivities to digestion with beta-glucanase and to various drugs, such as hygromycin B, sodium orthovanadate and Calcofluor white. A reduction of galactomannan layers of the cell wall in the Deltagms1 strain was observed by scanning and transmission electron microscopic analyses. The addition of osmotic stabilizer suppressed the morphologic defect of the Deltagms1 cells, while other phenotypes were weakly suppressed. The Deltagms1 (h90) strain was incapable of sexual conjugation during nutritional starvation. These results suggest that the cell surface galactosylation is required not only for non-sexual flocculation but also for sexual conjugation in Sz. pombe.     

Acetate-glycerol cometabolism: cultivating Schizosaccharomyces pombe on a non-fermentable carbon source in a defined minimal medium

The growth of the fission yeast Schizosaccharomyces pombe on glucose and glycerol was monitored on-line in shake flasks and microtiter plates. The Edinburgh Minimal Medium 2 was improved by doubling its concentrations, improving its buffer and increasing its sulphur and iron concentrations additionally. By growing S. pombe on mixed carbon sources, it was shown that glycerol and glucose complement one another. Several tests were performed to establish the cultivation of S. pombe with non-fermentable glycerol as the main carbon source in minimal medium. Interestingly, a synergistic effect of glycerol and acetate was discovered which can significantly improve the growth of the fission yeast on glycerol. S. pombe showed optimal respiration activity, growth, and product formation by co-utilizing 20g/L glycerol and 2.5g/L sodium acetate.     

Production of active lipase by Rhizopus oryzae from sugarcane bagasse: solid state fermentation in a tray bioreactor

This study deals with production of lipase in solid state fermentation by Rhizopus oryzae from sugarcane bagasse. A tray bioreactor was designed for the extracellular enzyme production. Daily, lipase production was evaluated at several incubation temperatures. Furthermore, the influence of temperature and humidity of the cabinet, depth of solid bed, particle size, initial moisture content and supplementary substrate (olive oil) as carbon source was investigated. The obtained results showed that bioreactor temperature of 45 °C, humidity of 80%, solid bed depth of 0.5 cm, particle size in the range of 0.335–1 mm, substrate initial moisture content of 80% for the top tray and 70% for the middle tray and supplementary substrate of 8% (v/w) olive oil led to maximum lipase production. Under optimum fermentation conditions after 72‐h incubation, maximum lipase activities for the top, middle and bottom trays were 215.16, 199.36 and 52.64 U gds^?1, respectively.     

Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe

To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4 Delta as well as ypt7 Delta, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4-64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3-GFP were inhibited in the end4 mutant. Deletion of end4 resulted in pleiotropic phenotypes consistent with F-actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects. Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant. These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post-internalization step after the intersection of the endocytic and the vacuolar protein-sorting pathways in fission yeast.     

Expression and secretion of a prokaryotic protein streptokinase without glycosylation and degradation in Schizosaccharomyces pombe

Streptokinase (SK) is an important thrombolytic protein that is secreted by pathogenic strains of Streptococcus. Expression of streptokinase has been so far attempted in Pichia pastoris, Escherichia coli and Bacillus subtilis and shown to yield protein that was either highly glycosylated or degraded. Since the fission yeast, Schizosaccharomyces pombe, shares several molecular characteristics with higher eukaryotes, we decided to express the streptokinase gene in this yeast. A chimeric gene comprising the signal sequence of the Plus pheromone of Sz. pombe fused in-frame with the mature streptokinase from Streptococcus sp. was constructed and inserted into the expression vector containing the thiamine-regulated promoter. We obtained a high level of expression of streptokinase comparable to that in E. coli and P. pastoris, with 50-100% processing of the signal sequence and secretion of the mature streptokinase into the periplasmic fraction. The mature enzyme co-migrates with the authentic mature SK in SDS gels, lacks any major modification and is functional. Importantly, a higher level of expression under stationary phase conditions and improved extractability of the mature and undegraded streptokinase was achieved in a novel mutant of Sz. pombe defective for a potent extracellular protease activity. We suggest that the unique vector/strain system developed here could be advantageous for large-scale production of prokaryotic proteins without significant modification or degradation in Sz. pombe.     

New insights into galactose metabolism by Schizosaccharomyces pombe: isolation and characterization of a galactose-assimilating mutant

The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase δ variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7+), SPBPB2B2.12c (gal10+) and SPBPB2B2.13 (gal1+), homologous to Saccharomyces cerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7Δ strain, carrying an integrated ura4+ marker at the gal7+ locus, grew on 5-fluoroorotic acid (5-FOA)-containing medium. In contrast, the FG2-8 gal7Δ strain could not grow on 5-FOA medium. In addition, expression of gal7+, SPBPB2B2.13, gal10+ and gal1+ genes increased in the wild-type strain when carried on a vector, and these transformants grew on galactose medium. We suggest that gal7+, gal10+, and gal1+ are localized close to a chromosomal terminal repressed by gene silencing in S. pombe. In contrast, gene silencing was defective in the FG2-8 strain making galactose assimilation possible.     

脂肪酸乙酯的制备与纯化

介绍了合成脂肪酸乙酯的油脂乙醇解法和脂肪酸与乙醇酯化法的制备过程和优缺点,并分析了脂肪酸乙酯纯化方法及现状。     

发酵法生产乳酸的提取与精制研究进展

乳酸发酵液的分离提取技术,直接影响产品的质量和收率。根据国内外有关研究报导,详述了提取与精制乳酸的各种新技术:溶剂萃取法、液膜法、离子交换法、电渗析法、吸附法、双水相萃取法、双蒸馏法,并指出了各种工艺的优缺点。     

Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis

In a systematic approach to study genes that are related to nucleocytoplasmic trafficking in the fission yeast Schizosaccharomyces pombe, the open reading frames (ORFs) of 26 putative nucleoporins and transport factors were deleted. Here we report the initial characterization of these deletion mutants. Of the 26 putative genes deleted, 14 were found to be essential for viability. Null mutations of essential genes resulted in failure to either complete one round or to sustain cell division. Four of the 14 essential genes, SPBC582.11c, SPBC17G9.04c, SPBC3B9.16c and SPCC162.08c, encode putative nucleoporins and a myosin-like protein with homologues NUP84, NUP85, NUP120 and MLP1, respectively, that are not required for viability in Saccharomyces cerevisiae, suggesting that their gene products perform critical functions in Sz. pombe. On the basis of combined drug sensitivity assays and genetic analysis we have identified five non-essential null mutants that were hypersensitive to the microtubule depolymerizing drug thiabendazole (TBZ) and exhibited a cut phenotype upon TBZ treatment, suggesting possible involvement in microtubule function. Three of the corresponding ORFs, SPCC18B5.07c, nup40 and SPAC1805.04, encode putative nucleoporins with low similarity to the S. cerevisiae nucleoporins NUP2p, NUP53p and NUP133p, respectively. Further genetic analysis revealed that one of the nucleoporin genes, nup40, and another gene, SPCC1322.06, encoding a putative importin-beta/Cse1p superfamily protein may have a spindle checkpoint function.     

Physiological and biochemical analysis of cytochrome P-450 in the yeast Schizosaccharomyces pombe

The yeast Schizosacchromyces pombe has been shown to contain a microsomal cytochrome P-450 (cyt. P-450) inducible under conditions of glucose repression. Under these conditions the enzyme has maximal expression of 0.43 nmol g^?1 wet wt at the end of the exponential phase of growth. Substrate and inhibitor affinity was examined using studies of spectral changes on binding and revealed a type II spectrum with ketoconazole (K_s = 23 μM ) and a type I spectrum with benzo(a)pyrene (K_s = 77 μM ). A K_m of 112 μM was found in the aryl hydrocarbon hydroxylas assay. These properties show broad comparability with the cyt. P-450 of Saccharomyces cerevisiae.