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.     

一种新型酿酒酵母附加型分泌表达载体的构建

用化学法合成克鲁维酵母的菊粉酶基因的信号肽序列(INU),将其嵌入酵母附加型表达质粒pYES2,得到一套新型的分泌表达载体pYES2I,pYES2Ⅱ,pYES2Ⅲ。然后用PCR方法分别扩增大肠杆菌的天冬酰胺酶基因(ASN)和短芽孢杆菌α乙酰乳酸脱羧酶(ALDC)基因,连接到INU下游,得到重组质粒pASN和pALDC。分别将这两个重组质粒转化酿酒酵母菌株INVScⅠ中表达,胞内和胞外的酶活分析表明ASN和ALDC基因都能在酿酒酵母中分泌表达,表明菊粉酶信号肽序列能很好地将酿酒酵母中的重组蛋白分泌到胞外。稳定性分析表明,重组酵母菌株在没有选择压力的条件下连续接种培养100h,未发现重组质粒的不稳定性。     

Regulation of the yeast trehalose–synthase complex by cyclic AMP-dependent phosphorylation

Background

Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control.

Methods

To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry.

Results

Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg^2 + in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant.

Conclusion

Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1.

General significance

A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.     

TPS1 Terminator Increases mRNA and Protein Yield in a Saccharomyces cerevisiae Expression System

Both terminators and promoters regulate gene expression. In Saccharomyces cerevisiae, the TPS1 terminator (TPS1t), coupled to a gene encoding a fluorescent protein, produced more transgenic mRNA and protein than did similar constructs containing other terminators, such as CYC1t, TDH3t, and PGK1t. This suggests that TPS1t can be used as a general terminator in the development of metabolically engineered yeast in high-yield systems.     

Detection of inactive precursors of beta-glucanases in Saccharomyces cerevisiae

Accumulation and secretion of beta-glucanases have been studied in vivo by using a thermosensitive secretory mutant of Saccharomyces cerevisiae blocked at the endoplasmic reticulum level (sec 18-1). When incubated at the restrictive temperature no accumulation of active glucanases was observed. Following a shift to permissive conditions in the presence of cycloheximide a rise in the internal activity took place. The increase in total glucanase activity was partially due to the activation of an exo-glucanase that hydrolyzes PNPG. It is concluded that glucanases are synthesized in inactive precursor forms and are converted to the active forms in their secretory pathway.     

A genetically isolated population of Saccharomyces cerevisiae in Malaysia

A divergent population of Saccharomyces cerevisiae has been identified in Malaysia by molecular and genetic analysis. It has also demonstrated that the yeast S. bayanus may be found in South America. The origin of S. cerevisiae is discussed.     

基因重组酵母在葡萄酒酿造中的应用前景

基因重组技术是菌种改良的重要手段。就基因重组酵母在葡萄酒酸度调解、增香、抗氧化及果胶分解方面的潜在应用价值及近年来相关研究成果进行了概述。     

Construction of a recombinant autolytic wine yeast strain overexpressing the csc1‐1 allele

During the aging step of sparkling wines and wines aged on lees, yeast cells kept in contact with the wine finally die and undergo autolysis, releasing cellular compounds with a positive effect on the wine quality. In view of the interest of autolysis for wine properties, biotechnologists have tried to improve autolytic yield during winemaking. In this work we used genetic engineering techniques to construct an autolytic industrial strain by expressing the csc1‐1 allele from the RDN1 locus. The expression of this mutant allele, that causes a “constitutive in autophagy phenotype,” resulted in accelerated autolysis of the recombinant strain. Although autophagic phenotype due to csc1‐1 expression has been reported to require the mutant allele in multicopy, autolytic acceleration was achieved by expressing only one or two copies of the gene under the control of the constitutive promotor pTDH3. The acceleration of autolysis together with the unaltered fermentative capacity, strongly supported the overexpression of csc1‐1 allele as a strategy to obtain wines with aged‐like properties in a shortened time. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

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。     

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.     

Strategies for the Genetic Manipulation of Saccharomyces cerevisiae

Abstract

The budding yeast Saccharomyces cerevisiae is now widely used as a model organism in the study of gene structure, function, and regulation in addition to its more traditional use as a workhorse of the brewing and baking industries. In this article the plethora of methods available for manipulating the genome of S. cerevisiae are reviewed. This will include a discussion of methods for manipulating individual genes and whole chromosomes, and will address both classic genetic and recombinant DNA-based methods. Furthermore, a critical evaluation of the various genetic strategies for genetically manipulating this simple eukaryote will be included, highlighting the requirements of both the new and the more traditional biotechnology industries.     

Molecular engineering of Rhizopus oryzae lipase using a combinatorial protein library constructed on the yeast cell surface

We constructed a combinatorial yeast library through cell-surface display of the pro- and mature region of lipase from Rhizopus oryzae (ProROL) and obtained clones retaining lipase activity in fluorescent plate assay. The initial reaction rates of hydrolysis and methanolysis could be measured directly as whole-cell biocatalyst without complex treatments such as concentration, purification, and immobilization. The selected clones showed wide-ranging variation of reaction specificity. The K138R mutant showed a 1.3-fold shift of reaction specificity toward methanolysis compared to the wild type, while the V-95D, I53V, P-96S/F196Y, and Q128H/Q197L mutants showed shifts toward hydrolysis of 1.6–5.9-fold. Predictions of the mutants’ three-dimensional structure suggested that the hydrogen-bond distance between threonine 83 and aspartic acid 92 may influence reaction specificity, which shifted toward hydrolysis in mutants where this distance was shorter than in the wild type, but toward methanolysis where it was longer. The positions of amino acid residues (aa) 53, 138 and 196 in ProROL are considered the sites that influence hydrogen-bond distance and change reaction specificity. Construction of a surface-displayed combinatorial library in yeast cells is thus a powerful tool in accelerating the combinatorial approach to enzyme engineering and novel whole-cell biocatalyst development.