Studies on plasmid stability,cell metabolism and superoxide dismutase production by Pgk− strains of Saccharomyces cerevisiae

Summary A double mutant sod1/pgk1 strain of Saccharomyces cerevisiae has been constructed in order to investigate the effects of different environmental conditions on yeast physiology, plasmid stability, and superoxide dismutase (SOD) production. Strains were transformed with yeast episomal plasmids (YEp) containing both PGK1 and SOD1 genes and were grown on fermentable carbon sources and under vigorous aeration. Under these conditions, the presence of the PGK1 gene was made essential for growth and both genes were efficiently expressed. However, plasmid-borne PGK1 was found not to increase the stability of YEp vectors in batch cultures of Pgk^– cells. Paradoxically, plasmid stability increased during the respiratory phase of growth. An investigation of the metabolism of Pgk^– cells demonstrated that these glycolytic pathway mutants do not appreciably metabolize glycerol. Thus Pgk⁺, plasmid-containg, cells have a selective advantage during the respiratory phase of batch growth since they can utilize both glycerol and ethanol. Correspondence to: S. G. Oliver     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

Insertion of transposon Tn9 into the spinal (Escherichia coli-Saccharomyces cerevisiae) plasmids and the expression of the prokaryotic gene of chloramphenicol resistance in yeast cells

Transposon Tn9 carrying camr gene which controls resistance to chloramphenicol has been introduced in vivo (in cells of Escherichia coli) into two chimeric shuttle plasmids pYF91 and YEp13. These plasmids consist of the different parts of the E. coli plasmid pBR322, the yeast 2mkm DNA plasmid and the yeast LEU2 structural gene. The plasmidis able to autonomously replicate in both yeast and bacterial cells. A recipient yeast strain carrying cams and leu2 markers was constructed to study the functional expression of the prokaryotic camr gene in eukaryotic yeast cells. The chimeric plasmids pYF91::Tn9 and YEp13::Tn9 were introduced into the yeast and bacterial recipient strains by transformation. The camr LEU2 yeast transformants were isolated. They were genetically unstable when grown on non-selective medium and they simultaneously lost camr and LEU2 markers with a frequency of 10 to 30%. The E. coli transformants were genetically stable under nonselective conditions and they maintain all plasmid markers. The chimeric plasmid pYF91::Tn9 was isolated from the yeast transformants and reintroduced into the cams leuB bacterial strain by transformation. The camr LEUB transformants were obtained. All these data confirm the possibility of the expression of the prokaryotic camr gene in yeast cells and present evidence for introduction of transposon Tn9 into chimeric plasmids.     

The Surface Display of the Alginate Lyase on the Cells of Yarrowia lipolytica for Hydrolysis of Alginate

The alginate lyase structural gene (AlyVI gene) was amplified from plasmid pET24-ALYVI carrying the alginate lyase gene from the marine bacterium Vibrio sp. QY101 which is a pathogen of Laminaria sp. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying the alginate lyase could form clear zone on the plate containing sodium alginate, indicating that they had high alginate lyase activity. The cells displaying alginate lyase can be used to hydrolyze poly-β-d-mannuronate (M) and poly-α-l-guluronate (G) and sodium alginate to produce different lengths of oligosaccharides (more than pentasaccharides). This is the first report that the yeast cells displaying alginate lyase were used to produce different lengths of oligosaccharides from alginate.     

Physiological role of d-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of d-amino acids

Saccharomyces cerevisiae is sensitive to d-amino acids: those corresponding to almost all proteinous l-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that d-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of d-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to d-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to d-amino acids than the wild type. We further confirmed that, upon cultivation with d-phenylalanine, N-acetyl-d-phenylalanine was accumulated in the culture but not in the wild type and hpa3Δ cells overproducing DNT cells. Thus, d-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.     

Co-production of S-adenosyl-l-methionine and glutathione from spent brewer’s yeast cells

Both S-adenosyl-l-methionine (AdoMet) and glutathione (GSH) are important small molecules with pharmaceutical importance. The co-production of AdoMet and GSH using abundant spent brewer’s yeast cells from the beer industry and with l-methionine supplement was successfully realized. Experimental data showed that improvement of GSH productivity was accompanied by AdoMet accumulation. AdoMet productivity of 40–45 mg g⁻¹ (DCW) was successfully achieved and an additional 13–18 mg g⁻¹ (DCW) GSH was synthesized in spent brewer’s yeast cells.     

Production of S-adenosyl-l-methionine by Saccharomyces sake

In a study of the optimization of the culture conditions for the intracellular accumulation of S-adenosyl-l-methionine (AdoMet) by Saccharomyces sake Kyokai No. 6, a medium containing 10% sucrose, 1.8% urea, 1% yeast extract and 0.75–1.5% l-methionine as the main components was found to be suitable for the production. Addition of CaCl₂, biotin and glycylglycine to the medium increased the accumulation of AdoMet. Under optimal conditions in a 10-liter fermenter, the yeast produced 10.8 g L⁻¹ AdoMet (260 mg per g dry cells).In order to increase the recovery yield and purity of AdoMet, a new and efficient isolation procedure involving freezing and thawing of cells for the extraction of AdoMet and chromatographies on Amberlite IRA-45, XAD-2 and IRC-50 was developed.     

Construction of a threonine-hyperproducing strain of Serratia marcescens by amplifying the phosphoenolpyruvate carboxylase gene

Summary The phosphoenolpyruvate carboxylase gene (ppc) of Escherichia coli K-12 was cloned on the multi-copy plasmid pLG339. Plasmid pST101, which carried a 4.3-kb SalI fragment, was introduced into Serratia marcescens T-1165, which carried the seven regulatory mutations for three aspartokinases and two homoserine dehydrogenases. Strain T-1165[pST101] produced phosphoenolpyruvate carboxylase at a rate 26 times higher than the control strain T-1165[pLG339]. While T-1165[pST101] produced 63 mg/ml l-threonine in a medium containing sucrose and urea, whereas T-1165 only produced 52 mg/ml.     

Nuclear suppression of a mitochondrial RNA splice defect: nucleotide sequence and disruption of the MRS3 gene

Summary A mitochondrial RNA splice defect in the first intron of the COB gene (bI1) can be suppressed by a dominant nuclear mutation SUP-101. Starting with a gene bank of yeast nuclear DNA from a SUP-101 suppressor strain cloned in the YEp13 plasmid, we have isolated a recombinant plasmid which exerts a suppressor activity similar to the SUP-101 allele. The N3(2) insert of this plasmid contains an open reading frame (ORF) of 1014 bp which is transcribed to a 12 S RNA. Deletion of the 5 end of this ORF and its upstream sequences abolishes the suppressor activity. The N3(2) insert thus carries a functional gene (called MRS3) which can suppress a mitochondrial splice defect. The chromosomal equivalent of the cloned gene has been mapped to chromosome 10. Disruption of this chromosomal gene has no phenotypic effect on wild-type cells.     

Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii

Abstract An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii , which is resitant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 μm origin and URA3 derived from Saccharomyces cerevisiae . The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene ( PLB1 ) in cells of disruption mutant ( plbI : : URA3 ) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed ∼ 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 Δ mutant showed increased survival when cells of plb1 Δ mutant and wild-type strain were incubated in water at 30 °C. Cells of PLB1 -over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.     

Regulation of the gene encoding glutathione synthetase from the fission yeast

The fission yeast cells that contained the cloned glutathione synthetase (GS) gene showed 1.4-fold higher glutathione (GSH) content and 1.9-fold higher GS activity than the cells without the cloned GS gene. Interestingly, gamma-glutamylcysteine synthetase activity increased 2.1-fold in the S. pombe cells that contained the cloned GS gene. The S. pombe cells that harbored the multicopy-number plasmid pRGS49 (containing the cloned GS gene) showed a higher level of survival on solid media with cadmium chloride (1 mM) or mercuric chloride (10 microM) than the cells that harbored the YEp357R vector. The 506 bp upstream sequence from the translational initiation point and N-terminal 8 amino acid-coding region were fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate the fusion plasmid pUGS39. Synthesis of beta-galactosidase from the fusion plasmid pUGS39 was significantly enhanced by cadmium chloride and NO-generating S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SN). It was also induced by L-buthionine-(S,R)-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase (GCS). We also found that the expression of the S. pombe GS gene is regulated by the Atf1-Spc1-Wis1 signal pathway.     

Cloning and mapping of the sporulation gene, spoT7, in Saccharomyces cerevisiae

Summary In order to isolate a DNA fragment able to complement a sporulation-deficient mutation in Saccharomyces cerevisiae, a simple screening procedure was devised which was based on the difference in osmotic sensitivity between protoplasts and spores. A plasmid (pHT7) containing a 13 kb DNA insert that complemented the spoT7 mutation was isolated from a yeast genomic library prepared in the vector YEp13. Gene spoT7 was linked to rna1 at 1.2 cM and to mak27 at 7.2 cM on the right arm of chromosome XIII. Mapping of the cloned gene following integration into the chromosome showed that the cloned gene was allelic to spoT7 and that a part of the RNA1 gene was also cloned into the same fragment. Gene spoT7 was localized on a 5 kb DNA fragment by further subcloning.Abbreviations kb kilobase pairs - cM centiMorgans     

Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.     

A set of Hansenula polymorpha integrative vectors to construct lacZ fusions

A set of YEp Saccharomyces cerevisiae-based, integrative Hansenula polymorpha plasmids was constructed to express lacZ gene under yeast gene promoters. The HpLEU2 and HpURA3 genes were used both as markers and to target the integration of plasmids into the corresponding H. polymorpha genome locus. The frequency of transformation reached with these plasmids linearised either in HpLEU2 or HpURA3 was around 100 transformants per microgram of plasmid DNA; in all transformants checked by Southern blotting the plasmid was integrated into the genome locus corresponding to the gene plasmid marker. PCR showed that about 50% of the transformants contained more than one plasmid copy per genome. Experiments carried out using the developed plasmids to determine the strength of the gene promoters involved in nitrate assimilation in H. polymorpha revealed that, in the presence of nitrate, the nitrate reductase gene promoter (YNR1) was the strongest, followed by nitrite reductase (YNI1) and nitrate transporter (YNT1). Received: 5 February 1999 / Received revision: 31 May 1999 / Accepted: 4 June 1999     

Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast

Summary Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS-polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

Movement of Shuttle Plasmids from Escherichia coli into Yeasts Other Than Saccharomyces cerevisiae Using Trans-kingdom Conjugation

Transfer of bacteria/yeast shuttle plasmids from Escherichia coli into the yeast species Kluyveromyces lactis, Pichia angusta (Hansenula polymorpha), and Pachysolen tannophilus has been accomplished, presumably through inter-kingdom conjugal transfer. Plasmid pEK2 was transferred into a K. lactis mutant to complement trp auxotrophy, while plasmid YEp13 was mobilized into and complemented P. angusta and P. tannophilus Leu^- auxotrophs. Plasmid DNA in the recipient strains was detected by transformation of E. coli with crude yeast cell extracts. Freely replicating plasmids without detectable alterations as well as plasmids with rearrangements were recovered from yeast transconjugants.     

Comparative studies on S-adenosyl-l -methionine binding sites of protein N-methyltransferases, using 8-azido-S-adenosyl-l -methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase

The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.     

Overexpression of bacterioferritin comigratory protein (Bcp) enhance viability and reduced glutathione level in the fission yeast under stress

The structural gene encoding bacterioferritin comigratory protein (Bcp) was amplified using PCR from the genomic DNA of Schizosaccharomyces pombe, and transferred into the shuttle vector pRS316 to generate the recombinant plasmid pBCPlO. The bcp ⁺ mRNA level in the pBCPlO-containing yeast cells was significantly higher than that in the control yeast cells, indicating that the cloned gene is functioning. The S. pombe cells harboring the plasmid pBCPIO exhibited higher survival on the solid minimal media with hydrogen peroxide, tert-BOOH or cadmium than the control yeast cells. They also exhibited enhanced cellular viability in the liquid media containing the stressful agents. The increased viabilities of the fission yeast cells harboring the plasmid pBCP10 were also obtained with 0.4% glucose or 0.4% sucrose as a sole carbon source, and nitrogen starvation, compared with those of the control yeast cells. The total glutathione (GSH) content and total GSH/GSSG ratio were significantly higher in the yeast cells harboring the plasmid pBCP10 than in the control yeast cells. In brief, the S. pombe Bcp plays a protective role in the defensive response to oxidative stress possibly via up-regulation of total and reduced glutathione levels.