Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae

1993 ◽  
Vol 237-237 (1-2) ◽  
pp. 17-25 ◽  
Author(s):  
Bruno André ◽  
Claudine Hein ◽  
Marcelle Grenson ◽  
Jean-Claude Jauniaux
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transport Protein ◽  
Cloning And Expression ◽  
Specific Transport ◽  
Gene Coding

scholarly journals Engineering of Saccharomyces cerevisiae for the accumulation of high amounts of triacylglycerol

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon Arhar ◽  
Gabriela Gogg-Fassolter ◽  
Mojca Ogrizović ◽  
Klavdija Pačnik ◽  
Katharina Schwaiger ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Lipid Level ◽  
Pharmaceutical Products ◽  
Acetyl Coa Carboxylase ◽  
Oleaginous Yeasts ◽  
Regulatory Subunits ◽  
Gene Coding ◽  
Tag Accumulation ◽  
Competing Pathways

Abstract Background Fatty acid-based substances play an important role in many products, from food supplements to pharmaceutical products and biofuels. The production of fatty acids, mainly in their esterified form as triacylglycerol (TAG), has been intensively studied in oleaginous yeasts, whereas much less effort has been invested into non-oleaginous species. In the present work, we engineered the model yeast Saccharomyces cerevisiae, which is commonly regarded as non-oleaginous, for the storage of high amounts of TAG, comparable to the contents achieved in oleaginous yeasts. Results We investigated the effects of several mutations with regard to increased TAG accumulation and identified six of them as important for this phenotype: a point mutation in the acetyl-CoA carboxylase Acc1p, overexpression of the diacylglycerol acyltransferase Dga1p, deletions of genes coding for enzymes involved in the competing pathways glycogen and steryl ester synthesis and TAG hydrolysis, and a deletion of CKB1, the gene coding for one of the regulatory subunits of casein kinase 2. With the combination of these mutations in a S. cerevisiae strain with a relatively high neutral lipid level already in the non-engineered state, we achieved a TAG content of 65% in the dry biomass. High TAG levels were not only obtained under conditions that favor lipid accumulation, but also in defined standard carbon-limited media. Conclusions Baker's yeast, which is usually regarded as inefficient in the storage of TAG, can be converted into a highly oleaginous strain that could be useful in processes aiming at the synthesis of fatty acid-based products. This work emphasizes the importance of strain selection in combination with metabolic engineering to obtain high product levels.

Cloning and expression of ?-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

1990 ◽  
Vol 12 (7) ◽  
pp. 499-504 ◽  
Author(s):  
Byong H. Lee ◽  
Normand Robert ◽  
Chantal Jacques ◽  
Line Ricard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Streptococcus Thermophilus ◽  
Cloning And Expression

Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum

1984 ◽  
Vol 4 (11) ◽  
pp. 2273-2278
Author(s):  
B C Dowds ◽  
W F Loomis
Keyword(s):  
Dictyostelium Discoideum ◽  
Cdna Clone ◽  
Single Gene ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
Spore Coat ◽  
Coat Proteins ◽  
Developmentally Regulated ◽  
Gene Coding ◽  
Spore Coat Proteins

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

Histone H1 expressed in Saccharomyces cerevisiae binds to chromatin and affects survival, growth, transcription, and plasmid stability but does not change nucleosomal spacing

1994 ◽  
Vol 14 (4) ◽  
pp. 2822-2835
Author(s):  
C Linder ◽  
F Thoma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasmid Stability ◽  
Apparent Molecular Weight ◽  
Histone H1 ◽  
Expression Levels ◽  
Inhibition Of Growth ◽  
Gene Coding ◽  
Gal1 Promoter ◽  
Core Histones ◽  
Low Levels

Histone H1 is proposed to serve a structural role in nucleosomes and chromatin fibers, to affect the spacing of nucleosomes, and to act as a general repressor of transcription. To test these hypotheses, a gene coding for a sea urchin histone H1 was expressed from the inducible GAL1 promoter in Saccharomyces cerevisiae by use of a YEp vector for high expression levels (strain YCL7) and a centromere vector for low expression levels (strain YCL1). The H1 protein was identified by its inducibility in galactose, its apparent molecular weight, and its solubility in 5% perchloric acid. When YCL7 was shifted from glucose to galactose for more than 40 h to achieve maximal levels of H1, H1 could be copurified in approximately stoichiometric amounts with core histones of Nonidet P-40-washed nuclei and with soluble chromatin fractionated on sucrose gradients. While S. cerevisiae tolerated the expression of low levels of H1 in YCL1 without an obvious phenotype, the expression of high levels of H1 correlated with greatly reduced survival, inhibition of growth, and increased plasmid loss but no obvious change in the nucleosomal repeat length. After an initial induction, RNA levels for GAL1 and H1 were drastically reduced, suggesting that H1 acts by the repression of galactose-induced genes. Similar effects, but to a lower extent, were observed when the C-terminal tail of H1 was expressed.

Cloning and expression of the gene coding for FtsH protease from Mycobacterium tuberculosis H37Rv

Gene ◽  
1998 ◽  
Vol 214 (1-2) ◽  
pp. 7-11 ◽  
Author(s):  
Gopalakrishnapillai Anilkumar ◽  
Mehbubhussein M Chauhan ◽  
Parthasarathi Ajitkumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tuberculosis H37rv ◽  
Cloning And Expression ◽  
Mycobacterium Tuberculosis H37rv ◽  
Gene Coding ◽  
Ftsh Protease

scholarly journals Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
María-Efigenia Álvarez-Cao ◽  
María-Esperanza Cerdán ◽  
María-Isabel González-Siso ◽  
Manuel Becerra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Food Industry ◽  
Culture Conditions ◽  
Raffinose Family Oligosaccharides ◽  
Recombinant Production ◽  
Gene Coding ◽  
Productive Process ◽  
Hydrolysis Of ◽  
Degree Of Purification

Abstract Background α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

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