An edeine resistant mRNA-dependent protein synthesis system from a Saccharomyces cerevisiae mutant

A cell-free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2 microM edeine, while a similar system from wild-type strain was completely inhibited by the drug. The mutant ribosomes showed an affinity for [125I]edeine comparable to the wild-type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine-resistant capacity of the mutant.     

Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Ethanol production by Saccharomyces cerevisiae in biofilm reactors

Biofilms are natural forms of cell immobilization in which microorganisms attach to solid supports. At ISU, we have developed plastic composite-supports (PCS) (agricultural material (soybean hulls or oat hulls), complex nutrients, and polypropylene) which stimulate biofilm formation and which supply nutrients to the attached microorganisms. Various PCS blends were initially evaluated in repeated-batch culture-tube fermentation with Saccharomyces cerevisiae (ATCC 24859) in low organic nitrogen medium. The selected PCS (40% soybean hull, 5% soybean flour, 5% yeast extract-salt and 50% polypropylene) was then used in continuous and repeated-batch fermentation in various media containing lowered nitrogen content with selected PCS. During continuous fermentation, S. cerevisiae demonstrated two to 10 times higher ethanol production in PCS bioreactors than polypropylene-alone support (PPS) control. S. cerevisiae produced 30 g L⁻¹ ethanol on PCS with ammonium sulfate medium in repeated batch fermentation, whereas PPS-control produced 5 g L⁻¹ ethanol. Overall, increased productivity in low cost medium can be achieved beyond conventional fermentations using this novel bioreactor design. Received 20 May 1997/ Accepted in revised form 29 August 1997     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Purification and Characterization of an Endo-Polygalacturonase from a Mutant of Saccharomyces cerevisiae

An extracellular endo-polygalacturonase (PGase) produced by a mutant of Saccharomyces cerevisiae was isolated. The enzyme was regarded, immunologically, as a PGase belonging to the Kluyveromyces marxianus group. The enzyme had properties similar to the PGase from K. marxianus in heat and pH stability, and N-terminal amino acid sequence. However, the enzyme showed different properties in optimum pH and temperature, molecular weight, and reactivity in antiserum against PGase from K. marxianus, indicating that the enzyme has a different molecular structure from the PGase from K. marxianus.     

酿酒酵母产油脂条件的优化

微生物细胞通常仅含2%3%油脂,但少数微生物含油脂率却可达70%以上,所以高含油脂量使微生物油脂实际开发成为可能。目前用于生产多不饱和脂肪酸的微生物主要为藻类和真菌。尽管微生物油脂是当前的研究热点,已经引起广大研究者的重视,但目前国内外研究大都集中在含油脂量在干重20%以上的微生物,如浅白色隐性酵母、粘红酵母等,而对于酿酒酵母来说,则很少见到研究其产油脂的相关报道。     

rDNA介导的酿酒酵母稳定表达载体的构建

将一段含有质粒ColE1复制起始区（ori）和氨苄青霉素抗性基因（bla）的DNA片段插入到酿酒酵母rDNA片段中部的EcoR I或Hpa I位点之间,在pYES2载体的基础上构建了两个rDNA介导的酿酒酵母稳定表达载体pHBM367E或pHBM367H。将黑曲霉糖化酶基因导入载体pHBM367H,获得表达载体pHBM166。为生物安全性的考虑,pHBM166经Hpa I酶切去掉2.2kb的ColE1 ori和bla片段后转化酿酒酵母Y33菌株,获得不含任何抗生素抗性基因的工程菌。随后,对糖化酶基因在酿酒酵母中的表达、稳定性进行了分析,结果显示,rDNA介导的糖化酶基因在酿酒酵母中的表达呈现不同的剂量效应,挑选高表达的菌株传80代之后仍能保持其产糖化酶的稳定性。     

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

Abstract Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae , chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L^-1 glutathione with glutathione content and yield of 4.3% and 25.6 mg_glutathione/g_glucose, respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L^-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.     

补料方式对酵母菌生产谷胱甘肽的影响

比较了酵母菌发酵生产谷胱甘肽（GSH）的几种补料分批培养方式。实验发现补料可以明显地促进酵母菌的生长和谷胱甘肽的合成,同时还发现不同的补料方式对发酵液中的菌体浓度和GSH浓度有不同的影响。采用指数流加方式可获得极高的菌体浓度,但菌体中的GSH浓度较低;而采用恒－pH补料分批培养既可以达到较高菌体浓度,菌体中又含有较高的GSH含量,因此,其总的GSH产量最高,可达到977.8mg/L。     

Enhancement of production of cloned glucoamylase under conditions of low aeration from recombinant yeast using a SUC2 promoter

The effect of aeration rate on the production of cloned glucoamylase in a recombinant yeast was investigated. This system consisted of Saccharomyces cerevisiae transformed with the 2 μ-based plasmid YEpSUCSTA which contains the SUC2 promoter, the STA signal sequence, and the STA structural gene. In contrast to typical yeast expression reports, high production of cloned glucoamylase was achieved at low aeration level (0·3 vvm). The recombinant yeast grown at 0·3 vvm aeration produced more glucoamylase (0·94 units/ml) than when grown at 0·0 vvm, 0·6 vvm, or 0·9 vvm (9·4, 1·4, and 3·1 times more, respectively). A high dissolved oxygen level early in the cultivation was important for cell growth and a low dissolved oxygen level during the production stage was important for glucoamylase production. In large scale processes for the production of recombinant proteins, the maintenance of aeration and dissolved oxygen at high levels is difficult and expensive. In this work, we have evaluated the coordination of oxygen level with growth and protein production and developed optimal conditions. Since a low aeration rate was optimal, our results demonstrate that the method described at the laboratory scale should be successfully applied at an industrial scale.     

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.     

一个酿酒酵母呼吸突变株在高糖胁迫下的生理特性研究

MF11a为甘蔗糖蜜乙醇发酵野生型高产菌株MF1002的呼吸突变体,对糖分的利用能力显著高于MF1002。本文研究了这两菌株应激高糖胁迫的生理特性变化。结果表明,高糖培养条件下,MF11a菌株的生长和乙醇发酵受抑制的程度均明显低于MF1002,培养基的葡萄糖浓度为30%和40%时,其最大菌体密度、最高出芽率和乙醇浓度等已显著高于MF1002,表明MF11a较MF1002具有更强的高糖耐受能力。在30%葡萄糖的胁迫培养条件下,两菌株胞内的总超氧化物歧化酶(SOD)活力、过氧化氢酶活力、过氧化物酶活力,及它们细胞质和线粒体的ATP酶活力均显著上升,说明这五种酶均参与了两菌株的高糖胁迫反应。其中,MF11a的胞内过氧化氢酶活性、过氧化物酶活力、细胞质ATP酶活力在高糖胁迫下的上升幅度显著高于MF1002,表明这三种酶活力可能与MF11a菌株的高糖耐受能力有关,可作为该菌株进一步改造的指导指标。     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究,我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因,并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3力标记基因,在不舍尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株,该菌株在低K 培养基中导入拟南芥K 转运体基因AtKuP1可恢复正常生长.     

酿酒酵母TRK1和TRK2钾吸收缺陷型菌株的构建

为更好的进行钾素营养有关基因表达调控和功能性研究, 我们采用同源重组法通过重叠引物扩增分别将URA3和HIS3基因替代酿酒酵母的TRK1和TRK2基因, 并以酿酒酵母的尿嘧啶合成酶URA3基因和组氨酸合成酶HIS3为标记基因, 在不含尿嘧啶和组氨酸的基本培养基筛选转化子获得了钾离子转运蛋白TRK1和TRK2基因缺失的酿酒酵母钾素营养缺陷型菌株, 该菌株在低K+培养基中导入拟南芥K+转运体基因AtKuP1可恢复正常生长。     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.