Analysis of alpha-factor secretion signals by fusing with acid phosphatase of yeast

In yeast, Saccharomyces cerevisiae, the PHO5 gene encodes the repressible acid phosphatase (APase) whose activity can be easily monitored by either the staining of colonies or by colorimetric assay. Therefore, gene fusions to PHO5 provide a convenient system for structural and functional analysis of yeast genes. We have constructed fusions of the PHO5 gene with a MF alpha 1 gene of yeast to delineate the secretion signal(s) in the alpha-factor leader peptide. Gene fusion between MF alpha 1 and PHO5 codes for a hybrid protein in which the alpha-factor leader peptide of 89 amino acids (aa) directed the export of APase, a periplasmic protein, into the medium. Since the hybrid gene is transcribed from the alpha-factor promoter, expression of the APase activity from these hybrid genes showed cell type-specific regulation. Further analyses of another MF alpha 1-PHO5 fusion showed that only the first 22 aa of the 89-aa alpha-factor leader peptide contained sufficient information for the secretion of APase into the medium. This shows that, in addition to the analysis of gene regulation, PHO5 fusions can be used to study signals involved in the proper localization of proteins.     

The effect of initial bioenergetic state on the cryostability of yeast cells

The cryostability of Saccharomyces cerevisiae cells decreased when they were cultivated under anaerobic conditions in a liquid growth medium YEPD as compared to the culture grown under aerobic conditions. The effect of cultivation conditions on the different cryostability of S. cerevisiae cells is discussed. The initial state of their bioenergetics was shown to influence the cryostability of yeast cells.     

酵母系统表达人表皮生长因子研究进展

人源表皮生长因子是一种从人体内分离出的蛋白质,具有刺激细胞生长的作用,临床用途广泛.但由于天然来源有限,化学合成成本高,利用基因工程技术生产人表皮生长因子具有很好的前景.酵母系统作为一种典型的真核表达系统,在生产异源蛋白时能进行翻译后修饰,如糖基化、形成二硫键等,并能有效地将重组蛋白分泌到细胞外,有益于下游分离提取,在...     

Factors influencing excretive production of human epidermal growth factor (hEGF) with recombinant Escherichia coli K12 system

Human epidermal growth factor (hEGF) was expressed and excreted into culture medium in an excretory recombinant Escherichia coli system. Conditions for the production of hEGF in this system were investigated. Results showed that the slight reduction of promoter strength improved the stability of plasmid and the production of hEGF in this recombinant system. One favorable MMBL medium for hEGF production was formulated by evaluating the effects of medium components, ampicilin addition and pH. hEGF production was affected obviously by culture conditions, especially fermentation temperature. High temperature (32 °C) was very beneficial for culture process by increasing productivity and reducing the quantity of isopropyl-#-d-thiogalactopyranoside (IPTG) for sufficient induction. High cell density and hEGF productivity could be accomplished concomitantly by inducing the culture at middle or late log phase of cell growth. In comparison with the batch process, fed-batch cultivation could improve plasmid stability from ca. 72% to ca. 83%, and increased hEGF productivity by 24.4%. Under all these circumstances, almost all expressed hEGF (⃋%) was fully excreted into the culture medium.     

Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry

AIMS: To study the effects of assimilable nitrogen concentration on growth profile and on fermentation kinetics of Saccharomyces cerevisiae. METHODS AND RESULTS: Saccharomyces cerevisiae was grown in batch in a defined medium with glucose (200 g l(-1)) as the only carbon and energy source, and nitrogen supplied as ammonium sulphate or phosphate forms under different concentrations. The initial nitrogen concentration in the media had no effect on specific growth rates of the yeast strain PYCC 4072. However, fermentation rate and the time required for completion of the alcoholic fermentation were strongly dependent on nitrogen availability. At the stationary phase, the addition of ammonium was effective in increasing cell population, fermentation rate and ethanol. CONCLUSIONS: The yeast strain required a minimum of 267 mg N l(-1) to attain complete dryness of media, within the time considered for the experiments. Lower levels were enough to support growth, although leading to sluggish or stuck fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to elucidate the role of nitrogen on growth and fermentation performance of wine yeast. This information might be useful to the wine industry where excessive addition of nitrogen to prevent sluggish or stuck fermentation might have a negative impact on wine stability and quality.     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

人工合成表皮生长因子基因在甲基营养型酵母中的高效表达

选用酵母菌偏爱密码子人工合成了编码５１个氨基酸的人表皮生长因子（ｈＥＧＦ）基因．将合成基因与编码酵母α因子前导肽８５个氨基酸的ＤＮＡ片段融合后克隆到醇氧化酶基因启动子下游,并构建出多拷贝表达载体．此载体转化甲基营养型酵母株ＧＳ１１５后筛选出整合型ＭｕｔＳＨｉｓ＋基因型菌株．高密度培养及诱导表达后该株可分泌具完好生物活性和正确物理化学性质的人表皮生长因子,产量达１００ｍｇ／Ｌ,经３次柱层析纯度达９５％以上,为观察其生物学作用打下了良好基础     

Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae.

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.     

Effects of spermine on the detection of induced forward mutation at the Can1 locus in yeast: evidence for selection against canavanine-resistant mutants.

The effect of exogenous spermine tetrahydrochloride (0.5 mg/ml) on hydrazine- and nitrous acid-induced forward mutation to canavanine resistance (CAN1 leads to can1, normal to defective arginine permease) was examined in stationary-phase haploid Saccharomyces cerevisiae. Post-treatment cell division (specifically DNA replication) is required for hydrazine mutagenesis at this locus, whereas nitrous acid mutagenesis exhibits, in addition, a significant post-treatment-independent component. Spermine addition only during mutagenic treatments in buffer did not affect mutagen cytotoxicity, but did result in a slight yet consistent decrease in induced mutation frequencies. Addition of spermine to the yeast extract--peptone--dextrose (YEPD) post-treatment growth medium resulted in dramatic reductions of induced mutation frequencies, which could be alleviated by pregrowth in spermine-containing YEPD. Such a medium was found to cause an apparent temporary growth inhibition for almost 40 h, after which the growth rate of the culture increased rapidly. Cultures "recovering" from spermine inhibition were no longer inhibitable by spermine in fresh medium, suggesting an outgrowth of spontaneous and/or induced spermine-resistant derivatives. Genetic analysis of one isolate revealed a single dominant nuclear gene conferring resistance by some means other than defective spermine uptake. Growth of this mutant was only slightly inhibited by spermine (20% increase in doubling time), while mutation expression remained high. Results of competitive growth experiments indicated that spermine-containing YEPD exerted a selection pressure against canavanine-resistant cells, while YEPD by itself did not. The mechanism for this selection is not presently understood. With respect to replication-dependent induced mutation at CAN1, our initial observation of a strong apparent antimutagenic action of spermine was found to be best explained by this specific selection against can1 mutants. This underscores the need for caution in the interpretation of experiments designed to study physiological modification of mutagenic potential.     

Reduced proteolysis of secreted gelatin and Yps1-mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae alpha-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the alpha-factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the alpha-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.     

Production of recombinant human granulocyte-colony-stimulating factor in high cell density yeast cultures

The recombinant human granulocyte-colony-stimulating factor (rhG-CSF) was synthesized in a fusion protein using a GAL1-10 UAS in recombinant Saccharomyces cerevisiae and the intracellular KEX2 cleavage led excretion of mature rhG-CSF into the extracellular culture broth. The recombinant yeast growth in fed-batch cultures was controlled by precise computer-aided medium feed. The optimal C/N ratio in preinduction (glucose/Casamino acids) and post-induction (galactose/yeast extract) feed media was determined at 3 and 2, respectively. The final rhG-CSF and cell concentration was more than 60 mg/L and 70 g/L, respectively, with around 90% plasmid stability and negligible ethanol accumulation. Comparing the cell growth between the hG-CSF + and hG-CSF - recombinant strains shows that the cloned gene product does not hamper the host cell growth.     

Enhanced production of human epidermal growth factor under control of the <Emphasis Type="Italic">pho</Emphasis>A promoter by acetate-tolerant <Emphasis Type="Italic">Escherichia coli</Emphasis> DB15 in a chemically defined medium

The phoA expression system is an efficient one and is successfully used in foreign gene expression. In a previous study, it was found that pH during the expression phase had a significant effect on extracellular hEGF production under control of the phoA promoter by Escherichia coli DA19, an acetate-tolerant strain of E. coli DH5α, in a chemically defined medium, but the level of hEGF production was only 75.5 mg/L. E. coli DB15 is another acetate tolerant mutant of DH5α. In the present study, production of hEGF under control of the phoA promoter by DB15 was further investigated. When transition from the growth phase, where phosphate was abundant, to the expression phase where phosphate was limited, was performed based on cell density, the extracellular hEGF reached 165 mg/L, twice that when transition was based on dissolved oxygen. Furthermore, adding 0.22 g/L of CaCl₂ during the growth phase, further increased hEGF production to 228 mg/L, which is 3-fold the level produced by DA19 (pAET-8) cultured in the same medium.     

Purification of recombinant human epidermal growth factor secreted from the methylotrophic yeast Hansenula polymorpha.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae-derived prepro alpha-factor leader in the methylotrophic yeast Hansenula polymorpha. The recombinant hEGF(1-53), when secreted by H. polymorpha, rapidly cleaved to hEGF(1-52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1-52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase ysc(alpha) was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.     

Alpha-factor-directed synthesis of Bacillus stearothermophilus alpha-amylase in Saccharomyces cerevisiae

Promoter and leader sequence of Bacillus stearothermophilus alpha-amylase gene were removed and the gene was joined in-frame to sequences encoding the leader region of Saccharomyces cerevisiae mating pheromone alpha-factor on plasmid p69A (a hybrid of pBR322 and S. cerevisiae 2-microns plasmid). S. cerevisiae cells were transformed with plasmids containing the hybrid genes, obtaining yeast transformants which exhibit a significant extra-cellular amylolytic activity in solid medium, but not in liquid medium. Levels of alpha-amylase activity in solid medium were found to depend on the mode of fusion of the alpha-amylase gene to the alpha-factor leader region.     

Effect of plasmid size and medium on growth kinetics and plasmid copy number in Saccharomyces cerevisiae

Saccharomyces cerevisiae cultures were selected to determine the effects of plasmid size on host growth kinetics and plasmid copy number. A complete synthetic medium was verified and the effects of yeast nitrogen base without amino acids medium and leucine selection were established for the strain. The dependence of copy number on the plasmid size, medium, and oxygen availability was also measured. This study was part of a comprehensive effort to elucidate the behavior of recombinant yeast.     

Automated spectrophotometric assay for cell division regulation in yeast

A spectrophotometric assay is presented for monitoring the regulation of cell division by the polypeptide alpha-factor in cultures of living cells of Saccharomyces cerevisiae yeast. This assay is simple, automated, and may have wider application in the study of other eucaryotic cells that do not require anchorage for cell growth. The kinetics of absorbance change were monitored continuously over time in yeast cell cultures that were mixed and aerated in cuvettes fitted with top-loading propeller stirrers. The absorbance doubling time. TD(Abs), was identical to the cell number doubling time in the absence of cell division arrest by alpha-factor. alpha-Factor lengthened the TD(Abs) during division arrest. At pH 5.8, 10(5) 381G cells/ml, the Khalf-maximal was 250 +/- 50 nM alpha-factor for the TD(Abs) increase during arrest, with a maximum increase of five-fold. After a period of time the TD(Abs) abruptly shortened. This is defined as the spectrophotometric recovery time (RTspec) and was compared to the time of recovery that is due to the reinitiation of cell division monitored by bud emergence (RTBE). RTBE occurred 40 +/- 5 min prior to RTspec when recovery was spontaneous or was artificially induced by the removal of alpha-factor (pH 5.8, 381G). The difference between RTBE and RTspec was independent of alpha-factor concentration between 0.05 and 1 microM and cell concentration between 1 and greater than or equal to 25 x 10(5) cells/ml (pH 5.8, 381G) but was both pH and cell strain dependent. At pH 5.8 and 2.7 the recovery from arrest occurred by inactivation of alpha-factor. The TD(Abs) increase during arrest appears to be due to an alpha-factor-induced inhibition of net cell mass increase, an effect that has not been reported previously. Evidence is presented that this process is also correlated with the formation of cell projections.     

Production of human epidermal growth factor by an ampicillin resistant recombinant Escherichia coli strain

Summary The effect of medium composition and initial glucose concentration on production of hEGF by recombinant E. coli cells was investigated. Optimum hEGF production was observed in a yeast extract/acid hydrolysed casein/salts media containing an initial glucose concentration of 10 g.l^-1. A maximum hEGF titer of 250 mg.l^-1 was obtained in this medium after 32 h in laboratory fermenters with pH, temperature, agitation and aeration set at 6.8, 30°C, 500 rpm and 2 vvm, respectively.     

Fed-batch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures ofEscherichia coliYK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression, was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically defined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of 0.25 gg⁻¹h⁻¹, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.     

Effect of Putative Deoxyribonucleic Acid Inhibitors on Macromolecular Synthesis in Saccharomyces cerevisiae

The effects of inhibitors of bacterial deoxyribonucleic acid (DNA) synthesis upon logarithmically growing cultures of Saccharomyces cerevisiae were investigated. Cell division, ribonucleic acid (RNA) synthesis, and DNA synthesis were measured after addition of nalidixic acid, fluorodeoxyuridine, or phenethyl alcohol to cultures of yeast growing in defined and complex media. Both nalidixic acid and fluorodeoxyuridine had only temporary effects on nucleic acid synthesis in cultures growing in defined medium, and little or no observable effect on cultures growing in complex medium. Neither compound inhibited colony formation on complex solid medium, although growth was slow on defined solid medium. Phenethyl alcohol caused complete inhibition of DNA synthesis, RNA synthesis, and cell division in cultures growing in defined medium. In cultures growing in complex medium, RNA synthesis and cell division were inhibited to a lesser extent. A slight increase in DNA was observed in the presence of the inhibitor.