Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.     

酿酒酵母ADH3基因的敲除

设计含有与酿酒酵母(Saccharomyces cerevisiae)编码乙醇脱氢酶Ⅲ的ADH3基因ORF两侧序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除组件。转化酿酒酵母YS3(Saccharomyces cerevisiae),并将质粒pSH65转入阳性克隆子。半乳糖诱导表达Cre酶切除Kanr基因,在YPD培养基中连续传代培养丢失pSH65质粒,在原ORF处留下一个loxP位点,获得ADH3单倍体缺陷型菌株。利用同样的方法再次敲除双倍体的另一个等位基因。最终获得ADH3双倍体基因缺陷型突变株YS3-ADH3。     

A new transformation system of Saccharomyces cerevisiae with blasticidin S deaminase gene

A new method for transformation of Saccharomyces cerevisiae that allows selection was developed. As the frequency of spontaneous blasticidin S resistant mutants from diploid type yeast strain (X-2180AB) was 5.2×10^–6, which was a thousandfold less than that from haploid type yeast strain (X-2180B), it was considered that the mechanism of spontaneous blasticidin S resistant mutations was related to recessive gene. Industrial yeasts, which were diploid, were transformed with blasticidin S deaminase gene from Aspergillus terreus to blasticidin S resistance. Expression of blasticidin S deaminase gene allowed selection of transformants from industrial yeasts.     

Production of Extracellular lipases by Saccharomyces cerevisiae

Seven strains of Saccharomyces cerevisiae all produced lipase when grown in shake flask culture. The best strain, DSM 1848, produced 4.0U of lipase in the medium containing olive oil and yeast extract. Production of the lipase was growth-associated.     

Reaction kinetics of d-glucose fermentation by Saccharomyces cerevisiae

Data obtained on the conversion of d-glucose to alcohol using Saccharomyces cerevisiae in batch culture has been analysed kinetically. The effects of different kinetic parameters, e.g. rates of ethanol and biomass formation, rate of d-glucose utilization and variation of pH have been studied. Analysis of data was made on the basis of Michaelis-Menten, Leudeking-Piret and simple kinetics. Unsteady rate behaviour in the lag phase was observed and explained.     

PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

Improvement of the transformation efficiency of Sacchaaromyces cerevisiae by altering carbon sources in pre-culture

We show here that the transformation efficiency of Saccharomyces cerevisiae is improved by altering carbon sources in media for pre-culturing cells prior to the transformation reactions. The transformation efficiency was increased up to sixfold by combination with existing transformation protocols. This method is widely applicable for yeast research since efficient transformation can be performed easily without changing any of the other procedures in the transformation.     

The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates

Abstract Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases. The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains. However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol₂₄P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors. It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants. Moreover, a block in the ergosterol branch of the metabolic pathway ( erg 9 ) causes an increase in the de novo synthesis of dolichyl phosphate.     

Characterization of natural hybrids of Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum

Nine yeast strains were isolated from spontaneous fermentations in the Alsace area of France, during the 1997, 1998 and 1999 grape harvests. Strains were characterized by pulsed-field gel electrophoresis, PCR-restriction fragment length polymorphism (RFLP) of the MET2 gene, delta-PCR, and microsatellite patterns. Karyotypes and MET2 fragments of the nine strains corresponded to mixed chromosomal bands and restriction patterns for both Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum. They also responded positively to amplification with microsatellite primers specific to both species and were demonstrated to be diploid. However, meiosis led to absolute nonviability of their spores on complete medium. All the results demonstrated that the nine yeast strains isolated were S. cerevisiaexS. bayanus var. uvarum diploid hybrids. Moreover, microsatellite DNA analysis identified strains isolated in the same cellar as potential parents belonging to S. bayanus var. uvarum and S. cerevisiae.     

Activation of Class I histone deacetylases contributes to mitochondrial dysfunction in cardiomyocytes with altered complex activities

Histone deacetylases (HDACs) play vital roles in the pathophysiology of heart failure, which is associated with mitochondrial dysfunction. Tumor necrosis factor-α (TNF-α) contributes to the genesis of heart failure and impairs mitochondria. This study evaluated the role of HDACs in TNF-α-induced mitochondrial dysfunction and investigated their therapeutic potential and underlying mechanisms. We measured mitochondrial oxygen consumption rate (OCR) and ATP production using Seahorse XF24 extracellular flux analyzer and bioluminescent assay in control and TNF-α (10 ng/ml, 24 h)-treated HL-1 cells with or without HDAC inhibition. TNF-α increased Class I and II (but not Class IIa) HDAC activities (assessed by Luminescent) with enhanced expressions of Class I (HDAC1, HDAC2, HDAC3, and HDAC8) but not Class IIb HDAC (HDAC6 and HDAC10) proteins in HL-1 cells. TNF-α induced mitochondrial dysfunction with impaired basal, ATP-linked, and maximal respiration, decreased cellular ATP synthesis, and increased mitochondrial superoxide production (measured by MitoSOX red fluorescence), which were rescued by inhibiting HDACs with MPT0E014 (1 μM, a Class I and IIb inhibitor), or MS-275 (1 μM, a Class I inhibitor). MPT0E014 reduced TNF-α-decreased complex I and II enzyme (but not III or IV) activities (by enzyme activity microplate assays). Our results suggest that Class I HDAC actions contribute to TNF-α-induced mitochondrial dysfunction in cardiomyocytes with altered complex I and II enzyme regulation. HDAC inhibition improves dysfunctional mitochondrial bioenergetics with attenuation of TNF-α-induced oxidative stress, suggesting the therapeutic potential of HDAC inhibition in cardiac dysfunction.     

CUP1为筛选标记的酿酒酵母整合型多基因表达载体的构建

为了赋予工业酿酒酵母对淀粉和纤维素的降解活性,提高酿酒酵母对粗木薯粉进行酒精发酵时的酒精产率;另一方面,为了解决工业酿酒酵母不适于使用营养缺陷型筛选标记对转化子进行筛选的问题,以及避免引入抗药性标记基因带来的安全性问题,构建了以抗铜蛋白基因CUP1为筛选标记的酿酒酵母整合型多基因表达载体.以载体pYES2-PMF-rDNA为基础,以新的筛选标记基因CUP1替换原有的尿嘧啶Ura-基因,得到载体pYES2M.再顺序插入葡聚糖内切酶基因eg3、葡萄糖淀糖酶基因gal和β-葡萄糖苷酶基因bgl1,构建得到以CUP1为筛选标记的酵母整合型三价表达载体pYES2M-eg3-ga1 -bgl1,其中每个基因都具有独立而完整的表达盒,包括启动子、信号肽和终止子,从而实现多基因单表达载体一次转化.     

The immunosuppressive drug leflunomide affects mating-pheromone response and sporulation by different mechanisms in Saccharomyces cerevisiae

Leflunomide (LFM) is a novel anti-inflammatory and immunosuppressive drug, and inhibits the growth of cytokine-stimulated lymphoid cells in vitro. The effect of LFM on haploid and diploid cells of Saccharomyces cerevisiae was investigated to elucidate the molecular mechanism of action of the drug. Using a halo assay, LFM was shown to enhance the cell cycle arrest of haploid cells induced by mating pheromone alpha-factor. LFM also inhibited sporulation of diploid cells completely. S. cerevisiae genes which were cloned to suppress the anti-proliferative effect when present in increased copy number were introduced and examined for their activity to suppress the effect of LFM. Out of them, MLF4/SSH4, was found to suppress the sporulation-inhibitory effect of LFM. However, MLF4 failed to suppress the enhancing effect of LFM on pheromone response. Thus, LFM is suggested to act on haploid and diploid cells by different mechanisms.     

压力对酵母菌及其海藻糖生成的影响

酵母菌海藻糖是其在培养条件发生“恶劣”变化时生成的一种应激代谢产物。当压力在0．5．1．0MPa时,酵母菌海藻糖含量为6．5mg／g,较对照提高27％。确定压力提高酵母海藻糖的最适条件为：采用复合培养基,菌体前培养时间20h,压力1．0MPa、加压温度34℃,pH6．0,升降压速度为0．10MPa/min,加压培养3h,此时,酵母海藻糖含量达到11mg／g。     

Genetic evidence of a new flocculation suppressor gene in Saccharomyces cerevisiae

Abstract The flocculation character in strain IM1-8b of Saccharomyces cerevisiae is controlled by a single and dominant gene shown to be allelic to FLO1 . Such a gene has been both mitotically and meiotically mapped on the right arm of chromosome I at 4.7 cM from PHO11 . The phenotype was suppressed by a single gene of wide distribution among non-flocculent strains (proposed as fsu3 ) that, however, was unable to suppress other FLO1 genes in other flocculent strains.     

酿酒酵母细胞衰老机理的研究进展

酿酒酵母的细胞衰老研究作为生命科学领域的前沿课题,对解析高等真核生物衰老的分子机制具有重要意义。迄今为止,在酵母中已经确立的衰老模式有两种,即复制型衰老和时序型衰老。细胞衰老的影响因子较多,涉及到很多过程,所以研究起来非常复杂。综述了两种细胞衰老机制的研究进展。     

Cloning and Nucleotide Sequence of the Alcohol Acetyltransferase II Gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7

The ATF2 gene, which encodes alcohol acetyltransferase II (AATase II), was cloned from Saccharomyces cerevisiae Kyokai No. 7 (sake yeast). The ATF2 gene coded for a protein of 535 amino acid residues with a calculated molecular mass of 61,909 daltons. The deduced amino acid sequences of the ATF2 showed 36.9% similarity with that of ATF1, which encodes AATase I. The hydrophobicity profiles for the Atf2 protein and Atf1 protein were similar. A transformant carrying multiple copies of the ATF2 gene had 2.5-fold greater AATase activity than the control, and this activity was not significantly inhibited by linoleic acid. A Southern analysis of the yeast genomes in which the ATF2 gene was used as a probe showed that S. cerevisiae and brewery lager yeast have one ATF2 gene, while S. bayanus has no similar gene.     

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.