Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

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

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

A gene encoding Achlya bisexualis β-amylase and its expression in Saccharomyces cerevisiae

Part of a -amylase genomic DNA sequence from the oomycete, Achlya bisexualis was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotide primers derived from the conserved regions of other known -amylase sequences. The 5- and 3-regions of the -amylase gene were amplified by genome walking method. The Ach. bisexualis -amylase gene consisted of a 1338bp open reading frame, encoding a protein of 446 amino acids with a molecular weight of 49 381Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 67% similarity to the -amylase of Saprolegnia ferax, followed by 40% similarity to that ofArabidopsis thaliana. The -amylase gene was expressed in Saccharomyces cerevisiae placing it under the control of the alcohol dehydrogenase gene (ADC1) promoter.     

Cloning and expression in Saccharomyces cerevisiae of an endo-β-glucanase gene from a thermophilic cellulolytic anaerobe

Summary The gene encoding heat-stable -glucanase from a thermophilic cellulolytic anaerobe was recloned in Saccharomyces cerevisiae. Yeast transformant expressed the heat-stable endo--glucanase and produced a level of enzyme activity similar to the Escherichia coli transformant.This work was supported in part by the Biomass Conversion Project of the Ministry of Agriculture, Forestry and Fisheries, Japan     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.     

δ-Aminolevulinic acid transport in Saccharomyces cerevisiae

• 1.1. This work represents the first approach to characterize the transport system of haem pathway precursors, such as δ-aminolevulinic acid (ALA), in two strains of Saccharomyces cerevisiae, a wild type, D27, and a HEM R⁺ mutant.
• 2.2. ALA transport occurs unidirectionally by a sole active system with an apparent K_M of 0.10 mM, at the optimum pH of 5.0. ALA uptake is influenced by both the carbon and nitrogen source; this suggests a rather complex regulation mechanism.
• 3.3. This transport is not mediated by the general amino acid permease (GAP).
• 4.4. ALA uptake is strongly inhibited by compounds harboring a methyl-amine terminus suggesting that this group is essential for ALA transport; however, the electric environment of the carboxylic group may be also important for the interaction between ALA and its transporter active site.
• 5.5. We have found differences in ALA transport which would indicate a different regulation mechanism for this system in both strain cells.

     

Fungal β-glucosidase expression in Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae strains expressing β-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best β-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB?+?Lac12]. The recombinant extracellular β-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75?°C. The S.?cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more β-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8?g?l(-1) after 24?h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB?+?Lac12] exhibited limited growth.     

Authentication and identification of Saccharomyces cerevisiae‘flor’ yeast races involved in sherry ageing

Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate 'flor' and non-'flor' S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that 'flor' yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.     

Expression of Bacillus sp. β-xylosidase gene (xylB) in Saccharomyces cerevisiae

-Xylosidase gene (xylB) from Bacillus sp. was amplified and inserted between GAL10 promoter and GAL7 terminator. For the secretory production of xylB in Saccharomyces cerevisiae, in-frame fusion of the exoinulinase signal sequence (INU1s) of Kluyveromyces marxianus to the upstream of xylB was conducted. When a transformant of S. cerevisiae harboring the resulting plasmid was grown on galactose-containing medium, most of -xylosidase activity was localized in the periplasmic space of yeast and a maximum total activity reached about 2.9 unit ml^–1 at 42 h cultivation. The recombinant -xylosidase was produced as an active dimer form.     

Is Saccharomyces cerevisiae apoptotic cell death associated with gene transfer?

Programmed cell death in unicellular organisms is difficult to account for in evolutionary terms. In the budding yeast, Saccharomyces cerevisiae, existence of several morphological and biochemical features of apoptosis has been described, and genes responsible for execution of the death program have been identified. It is here suggested that apoptosis of yeast cells could provide direct benefit to the genes of the dying cells, by facilitating DNA transfer to surrounding cells. The biochemical details of yeast apoptotic death are considered in light of a gene transfer hypothesis.     

Does crossover interference count in Saccharomyces cerevisiae?

We previously proposed a "counting model" for meiotic crossover interference, in which double-strand breaks occur independently and a fixed number of noncrossovers occur between neighboring crossovers. Whereas in some organisms (group I) this simple model alone describes the crossover distribution, in other organisms (group II) an additional assumption--that some crossovers lack interference--improves the fit. Other differences exist between the groups: Group II needs double-strand breaks and some repair functions to achieve synapsis, while repair in group I generally occurs after synapsis is achieved; group II, but not group I, has recombination proteins Dmc1, Mnd1, and Hop2. Here we report experiments in msh4 mutants that are designed to test predictions of the revised model in a group II organism. Further, we interpret these experiments, the above-mentioned differences between group I and II meiosis, and other data to yield the following proposal: Group II organisms use the repair of leptotene breaks to promote synapsis by generating double-Holliday-junction intermediates that lock homologs together (pairing pathway). The possible crossover or noncrossover resolution products of these structures lack interference. In contrast, for both group I and group II, repair during pachytene (disjunction pathway) is associated with interference and generates only two resolution types, whose structures suggest that the Holliday junctions of the repair intermediates are unligated. A crossover arises when such an intermediate is stabilized by a protein that prevents its default resolution to a noncrossover. The protein-binding pattern required for interference depends on clustering of sites that have received, or are normally about to receive, meiotic double-strand breaks.     

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode

Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.     

Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae

A recombinant plasmid, pGMF, containing a gamma-glutamylcysteine synthetase gene (GSH-I) from Saccharomyces cerevisiae, was constructed with a copper-resistance gene as the selection marker and was introduced into S. cerevisiae YSF-31. The glutathione content of the recombinant strain was 1.5-fold (13.1 mg g dry cells(-1)) of that in the host strain.     

Introduction of a feed back resistant α-isopropylmalate synthase gene of Saccharomyces cerevisiae into sake yeast

A mutant LEU4 gene (LEU4^fbr-2), responsible for both the overproduction of iso-amyl alcohol in yeast and the phenotype of yeast resistant to 5,5,5-trifluoro-dl-leucine (TFL), was isolated from a TFL-resistant mutant of Saccharomyces cerevisiae F-7. The single copy number of LEU4^fbr-2 complemented the leucine auxotrophy of S. cerevisiae HB190 (a, leu4, leu5), and also transformed it to TFL-resistant. Leucine-insensitive α-isopropylmalate synthase activity was detected in the crude extract of the Leu⁺ transformant. Also sake yeast Kyokai no. 7 (K-7) was transformed by the LEU4^fbr-2 gene to TFL-resistant. The resulting transformants produced 3∼30-fold higher levels of iso-amyl alcohol (approx. 50∼475 ppm) in shaking cultures, while in static cultures the increase in productivity was only 2.5-fold compared with that of recipient strain K-7. The isolated LEU4^fbr-2 gene may be useful as a positive selectable marker for the transformation of industrial yeast.     

N -glycosylation is involved in the sensitivity of Saccharomyces cerevisiae to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895

Saccharomyces cerevisiae rhk mutants were previously shown to have a phenotype that is resistant to HM-1 killer toxin secreted from Hansenula mrakii IFO 0895. The RHK1/ALG3 gene encodes a mannosyltransferase that is involved in the synthesis of an oligosaccharide in protein N-glycosylation. Previously, this gene was cloned and shown to complement the rhk1 mutation. In this study, the RHK2 gene, which complements the rhk2 mutation, was cloned. The RHK2 gene was found to be identical to the essential gene STT3, which encodes a subunit of the oligosaccharyltransferase complex. This complex transfers the core oligosaccharide to proteins. The rhk2 mutants showed supersensitivity to several drugs (Calcofluor White, caffeine and FK506), suggesting that these strains have cell-wall defects. Activity staining of invertase in an acrylamide gel indicated that it was underglycosylated. These results suggest that one or more mannoproteins are involved in the cytocidal process of HM-1. Received: 13 July 1998 / Received last revision: 18 September 1998 / Accepted: 2 October 1998