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.     

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.     

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.     

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.     

Biotechnology of natural and winery-associated strains of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

A new body of evidence challenges the original consolidated theory of Pasteur on the natural (vineyard) origin of wine strains of Saccharomyces cerevisiae and instead indicates a local, winery-restricted life cycle. The findings open novel biotechnological perspectives for obtaining autochthonous selected starters for the wine industry. A local, individual, and specific fermenting yeast flora, mass selected year after year through many generations of S. cerevisiae in grape must, is present on the surfaces of every winery. These yeast strains are endowed with exceptional enological properties and capable of producing an assortment of volatile compounds apparently contributing to the specific bouquet of locally produced wines.     

Acrolein toxicity involves oxidative stress caused by glutathione depletion in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Exposure of yeast cells to allyl alcohol results in intracellular production of acrolein. The toxicity of so formed acrolein involves oxidative stress, as (1) strains deficient in antioxidant defense are hypersensitive to allyl alcohol, (2) exposure to allyl alcohol increases the level of thiobarbituric-acid-reactive substances and decreases glutathione level in the cells, (3) hypoxic and anoxic atmosphere and antioxidants protect against allyl alcohol toxicity, and (4) allyl alcohol causes activation of Yap1p. No increased formation of reactive oxygen species was detected in cells exposed to allyl alcohol, so oxidative stress is due to depletion of cellular thiols and thus alteration in the redox state of yeast cells.     

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.     

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.     

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     

The effect of growth medium on the antioxidant defense of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We compared the oxidation of dihydrorhodamine 123, glutathione contents and activities of superoxide dismutase (SOD) and catalase for three wild-type strains of Saccharomyces cerevisiae grown on media with different carbon sources. The rate of oxidation of dihydrorhodamine 123 was much higher in respiring cells grown on ethanol or glycerol media than in fermenting cells grown on glucose medium. The total SOD activity was highest on glycerol medium and lowest on ethanol medium, while the catalase activity was highest on glycerol medium. The sequence of glutathione content values was: glucose > ethanol > glycerol.     

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.     

An evaluation of evolutionary theories based on genomic structures in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Encephalitozoon cuniculi</Emphasis>

Codon usage patterns in 16 chromosomes coincided with each other in Saccharomyces cerevisiae, and the same result was obtained from Encephalitozoon cuniculi consisting of 11 chromosomes, although each chromosome function differs. In addition, preferential codon usage in the regenerated coding systems for Leu and Lys differed between Saccharomyces cerevisiae and Encephalitozoon cuniculi. These results cannot be explained by Darwins natural selection theory or by the neutral theory proposed against Darwins. Furthermore, the codon usage patterns were examined in both prokaryotes and eukaryotes. The use of G or C at the third codon position was much lower than T or A in Ureaplasma urealyticum, whereas inversely the use of G or C at the third codon position was much higher than T or A in Mycobacterium tuberculosis. Additionally, Candida albicans and Plasmodium falciparum also showed a very low usage of G or C at the third codon position. It is a difficult leap to speculate that the inverse codon usage change occurred over the genome during biological evolution. Thus, the present results strongly suggest that organisms were derived from different origins, indicating that the origin of life was plural, based on genomic structures.     

The patterns of utilization and accumulation of polyphosphates in the cytosol of the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under inactivation of exopolyphosphatase genes <Emphasis Type="Italic">PPX1</Emphasis> and <Emphasis Type="Italic">PPN1</Emphasis>

The effect of inactivation of the PPX1 and PPN1 genes encoding the yeast exopolyphosphatases on the activities of these enzymes and polyphosphate content in the cytosol of Saccharomyces cerevisiae was studied under P_i deficit and P_i excess in the cultivation medium. Under P_i deficit, exopolyphosphatase activity in strain CRN (with inactivated PPN1 gene) and in the parent strain CRY increased 3- and 1.5-fold, respectively. In the strain CRX (with inactivated PPX1 gene), exopolyphosphatase activity did not change under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by an ～1.7-fold increase of exopolyphosphatase activities in the cytosol preparations of strains CRY, CRX, and CRN. In the cytosol of the double mutant, exopolyphosphatase activity was practically absent under all of the above cultivation conditions. The content of polyphosphates in the cytosol preparations of all strains under study substantially decreased under P_i deficit. Transfer from P_i-deficient to P_i-rich medium was accompanied by polyphosphate over-accumulation only in the cytosol preparations of stains CRX and CNX, where their levels increased ～1.3 and 3.5-fold, respectively. No over-accumulation was observed in the parent strain CRY and in the PPN1-deficient strain CRN. These data suggest that the exopolyphosphatases encoded by the PPX1 and PPN1 genes are not involved in polyphosphate synthesis.     

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.     

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.