<Emphasis Type="Italic">MPK1</Emphasis> gene is required for filamentous growth induced by isoamyl alcohol in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from the alcoholic fermentation

The aim of this study was to evaluate the MPK1 (SLT2) gene deletion upon filamentous growth induced by isoamyl alcohol (IAA) in two haploid industrial strains of Saccharomyces cerevisiae using oligonucleotides especially designed for a laboratory S. cerevisiae strain. The gene deletion was performed by replacing part of the open reading frames from the target gene with the KanMX gene. The recombinant strains were selected by their resistance to G418, and after deletion confirmation by polymerase chain reaction, they were cultivated in a yeast extract peptone dextrose medium + 0.5% IAA to evaluate the filamentous growth in comparison to wild strains. Mpk1 derivatives were obtained for both industrial yeasts showing the feasibility of the oligonucleotides especially designed for a laboratory strain (Σ1278b) by Martinez-Anaya et al. (In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint. J Cell Sci 116:3423–3431, 2003). The filamentation rate in these derivatives was significantly lower for both strains, as induced by IAA. This drastic reduction in the filamentation ability in the deleted strains suggests that the gene MPK1 is required for IAA-induced filamentation response. The growth curves of wild and derivative strains did not differ substantially. It is not known yet whether the switch to filamentous growth affects the fermentative characteristics of the yeast or other physiological traits. A genetically modified strain for nonfilamentous growth would be useful for these studies, and the gene MPK1 could be a target gene. The feasibility of designed oligonucleotides for this deletion in industrial yeast strains is shown.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

Anaerobic glycerol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains under hyperosmotic stress

Glycerol formation is vital for reoxidation of nicotinamide adenine dinucleotide (reduced form; NADH) under anaerobic conditions and for the hyperosmotic stress response in the yeast Saccharomyces cerevisiae. However, relatively few studies have been made on hyperosmotic stress under anaerobic conditions. To study the combined effect of salt stress and anaerobic conditions, industrial and laboratory strains of S. cerevisiae were grown anaerobically on glucose in batch-cultures containing 40 g/l NaCl. The time needed for complete glucose conversion increased considerably, and the specific growth rates decreased by 80–90% when the cells were subjected to the hyperosmotic conditions. This was accompanied by an increased yield of glycerol and other by-products and reduced biomass yield in all strains. The slowest fermenting strain doubled its glycerol yield (from 0.072 to 0.148 g/g glucose) and a nearly fivefold increase in acetate formation was seen. In more tolerant strains, a lower increase was seen in the glycerol and in the acetate, succinate and pyruvate yields. Additionally, the NADH-producing pathway from acetaldehyde to acetate was analysed by overexpressing the stress-induced gene ALD3. However, this had no or very marginal effect on the acetate and glycerol yields. In the control experiments, the production of NADH from known sources well matched the glycerol formation. This was not the case for the salt stress experiments in which the production of NADH from known sources was insufficient to explain the formed glycerol.     

Domain engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg ^–1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.     

Availability of substratum enhances ethanol production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Novel additives that act as substratum for attachment of the yeast cells, increased ethanol production in Saccharomyces cerevisiae. The addition of 2 g rice husk, straw, wood shavings, plastic pieces or silica gel to 100 ml medium enhanced ethanol production by 30–40 (v/v). Six distillery strains showed an average enhancement of 34 from 4.1 (v/v) in control to 5.5 (v/v) on addition of rice husk. The cell wall bound glycogen increased by 40–50 mg g ^–1 dry yeast while intracellular glycogen decreased by 10–12 mg g^–1 dry yeast in cells grown in presence of substratum     

A database of microsatellite genotypes for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A system for genotyping Saccharomyces cerevisiae is described based on a multiplex of ten microsatellite loci and the MAT locus. A database of genotypes has been developed for 246 yeast strains, including a large set of commercial wine yeasts, as well as 35 sequenced natural isolates currently being sequenced. The latter allow us, for the first time, to make direct comparisons of the relationship between DNA sequence data and microsatellite-based genotypes. The genotyping system provides a rapid and valuable system for strain identification as well as studying population genetics of S. cerevisiae.     

<Emphasis Type="Italic">N</Emphasis>-Acetyltransferase Mpr1 confers ethanol tolerance on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by reducing reactive oxygen species

N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H₂O₂, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H₂O₂ or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H₂O₂. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.     

Novel methods of genome shuffling in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Genome shuffling can improve complex phenotypes; however, there are several obstacles towards its broader applicability due to increased complexity of eukaryotic cells. Here, we describe novel, efficient and reliable methods for genome shuffling to increase ethanol production of Saccharomyces cerevisiae. Using yeast sexual and asexual reproduction by itself, mutant diploid cells were shuffled through highly efficient sporulation and adequate cross among the haploid cells, followed by selection on the special plates. The selected strain obtained after three round genome shuffling not only distinctly improved the resistance to ethanol, but also, increased ethanol yield by up to 13% compared with the control.     

In situ monitoring by quantitative Raman spectroscopy of alcoholic fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under high pressure

We monitored alcoholic fermentation in Saccharomyces cerevisiae as a function of high hydrostatic pressure. Ethanol production from 0.15 M glucose was measured by Raman spectroscopy in situ in a diamond-anvil cell. At 10 MPa, fermentation proceeds three times faster than at ambient pressure and the fermentation yield is enhanced by 5% after 24 h. Above 20 MPa, the reaction kinetics slows down with increasing pressure. The pressure above which no more ethanol is produced is calculated to be 87 ± 7 MPa. These results indicate that the activity of one or several enzymes of the glycolytic pathway is enhanced at low pressure up to 10 MPa. At higher pressures, they become progressively repressed, and they are completely inhibited above 87 MPa. Although fermentation was predicted to stop at ca. 50 MPa, due to the loss of activity of phosphofructokinase, the present study demonstrates that there is still an activity of ca. 30% of that measured at ambient pressure at 65 MPa. This study also validates the use of Raman spectroscopy for monitoring the metabolism of living microorganisms.     

Assessing the viability of a clumpy <Emphasis Type="Italic">mnn9</Emphasis> strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> used in the manufacture of recombinant pharmaceutical proteins

Demonstration of the viability of cryopreserved cell bank used to make a biopharmaceutical product is an important indicator of the ability to consistently manufacture over a long period of time, and is mandated in regulatory guidances. A mnn9 strain of Saccharomyces cerevisiae, chosen for its inability to hypermannosylate vaccine antigens, has a clumpy growth tendency due to the inactivation of the gene MNN9 (wild-type), complicating the interpretation of conventional viability measurements useful for single cells. Therefore, two growth-based measurements as well as staining by a membrane-impermeable dye were examined for their ability to reflect changes in viability of a clumpy mnn9 (defective) strain. The cell clumps proved to be stable to mixing, and variability of agar-plate-based viable counts (VC) of undisrupted suspensions of this clumpy mnn9 strain was consistent with variability observed for cell banks of a non-clumpy MNN9 strain. Both the VC and the growth times in an oxygen-sensing broth-based microplate assay corresponded well with shake-flask growth times for a set of stressed and unstressed samples, although the correlation was highest between the two broth-based systems. Counts of trypan-blue-stained cells within clumps also increased with time of stress, suggesting that this method could be adapted as a simple index of viability as well.     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as “enzyme bags” and incubated at 30°C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 μg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-β-d-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26°C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.     

Functional expression of amine oxidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> (AO-I) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.