Kinetics of invertase synthesis by Saccharomyces cerevisiae in synthetic medium

Present investigation deals with optimization of appropriate substrate concentration and incubation temperature both for growth of Saccharomyces cerevisiae and invertase production. Submerged fermentation technique was employed in the present study. The maximal production of invertase during the course of study was achieved after 48 h of incubation using initial sucrose concentration, 15.0 g l-1. The sugar consumption and dry cell mass were also examined. Data was subjected to kinetic analysis and on the basis of kinetic parameters such as Yp/x (amount of enzyme produced mg-1 cell mass), Yp/s (amount of enzyme produced mg-1 sugar consumed), Yx/s (mg cells mg-1 substrate consumed), Ys/x (mg sugar consumed mg-1 cell mass produced), qp (Amount of enzyme produced mg-1 sugar consumed h-1), qs (mg mg-1 cells h-1), qx (mg cells mg-1 sugar consumed h-1), µ (mg cells produced h-1), it was found that temperature had a direct influence both on substrate consumption and synthesis of enzyme. Similarly, higher concentrations of sucrose in fermentation medium induced catabolite repression of yeast invertase

PENGARUH VARIASI MASSA RAGI SACCHAROMYCES CEREVISIAE DAN LAMA FERMENTASI TERHADAP DENSITAS DAN RENDEMEN BIOETANOL ALANG-ALANG (Imperata Cy-lindrica)

Ketersediaan energi fosil dalam penggunaan bahan bakar semakin menipis dan tingkat residu yang dihasilkan semakin menigkat, sehingga diperlukan sumber energi alternatif untuk menunjang kebutuhan energi salah satunya bioetanol. Alang- alang (Imperata cylindrica) memiliki kandungan selulosa sebanyak 40,22% dapat digunakan sebagai bahan dasar bioetanol dengan bantuan Saccharomyces cerevisiae pengubah glukosa menjadi etanol. Tujuan penelitian ini untuk menganalisis karakteristik bioetanol berdasarkan densitas dan persen rendemen bioetanol dengan variasi massa ragi roti Saccharomyces cerevisiae dan waktu fermentasi. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL), dengan 4 perlakuan dan 3 kali ulangan. Jumlah takaran larutan untuk setiap perlakuan adalah 100 ml. Nilai densitas tertinggi pada perlakuan 4 massa   roti Saccharomyces cerevisiae 15 gram dengan lama fermentasi 6 hari sebesar 0.9041 g/ml, nilai densitas terendah pada perlakuan 1 massa ragi roti Saccharomyces cerevisiae 10 gram dan lama fermentasi 3 hari sebesar 0,9340 g/ml. Persen rendemen tertinggi diperoleh dari perlakuan 4 massa ragi roti Saccharomyces cerevisiae 15 gram dan lama fermentasi 6 hari sebanyak 23,11% dan persen rendemen terendah pada perlakuan 1 dengan massa ragi roti Saccharomyces cerevisiae 10 gram dan lama fermentasi 3 hari sebesar 17,90%. Hasil penelitian menunjukkan ada pengaruh variasi konsentrasi  Saccharomyces cerevisiae dan lama fermentasi terhadap nilai densitas dan persen rendemen

The geographic distribution of saccharomyces cerevisiae isolates within three Italian neighboring winemaking regions reveals strong differences in yeast abundance, genetic diversity and industrial strain dissemination

In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore, the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over 3 years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota

KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes

The KNR4 gene, originally isolated by complementation of a K9 killer-toxinresistant mutant displaying reduced levels of both 1,3-b-glucan and 1,3-bglucan synthase activity, was recloned from a YCp50 genomic library as a suppressor of Saccharomyces cerevisiae calcofluor-white-hypersensitive (cwh) mutants. In these mutants, which were characterized by increased chitin levels, the suppressor effect of KNR4 resulted, for some of them, in a lowering of polymer content to close to wild-type level, with no effect on the contents of b-glucan and mannan. In all cases, this effect was accompanied by a strong reduction in mRNA levels corresponding to CHS1, CHS2 and CHS3, encoding chitin synthases, without affecting expression of FKS1 and RHO1, two genes encoding the catalytic subunit and a regulatory component of 1,3-b-glucan synthase, respectively. Overexpression of KNR4 also inhibited expression of CHS genes in wild-type strains and in two other cwh mutants, whose sensitivity to calcofluor white was not suppressed by this gene. The physiological relevance of the KNR4 transcriptional effect was addressed in two different ways. In a wild-type strain exposed to a-factor, overexpression of this gene inhibited CHS1 induction and delayed shmoo formation, two events which are triggered in response to the pheromone, whereas it did not affect bud formation and cell growth in a chs1 chs2 double mutant. A chimeric protein made by fusing green fluorescent protein to the C terminus of Knr4p which fully complemented a knr4D mutation was found to localize in patches at presumptive bud sites in unbudded cells and at the incipient bud site during bud emergence. Taken together, these results demonstrate that KNR4 has a regulatory role in chitin deposition and in cell wall assembly. A mechanism by which this gene affects expression of CHS genes is proposed

Quantitative study of interactions between Saccharomyces cerevisiae and Oenococcus oeni 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, SO2 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

The influence of microgravity on invasive growth in Saccharomyces cerevisiae

This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Sigma 1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Sigma 1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Sigma 1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium

Phosphatidylinositol (4,5)-bisphosphate turnover by INP51 regulates the cell wall integrity pathway in "Saccharomyces cerevisiae"

Signal transduction pathways are important for the cell to transduce external or internal stimuli where second messengers play an important role as mediators of the stimuli. One important group of second messengers are the phosphoinositide family present in organisms ranging from yeast to mammals. The dephosphorylation and phosphorylation cycle of the phosphatidylinositol species are thought to be important in signaling for recruitment or activation of proteins involved in vesicular transport and/or to control the organization of the actin cytoskeleton. In mammals, phosphatidylinositol (4,5)bisphosphate (PI(4,5)P2) signaling is essential and regulated by various kinases and phosphatases. In the model organism Saccharomyces cerevisiae PI(4,5)P2 signaling is also essential but the regulation remains unclear. My dissertation focuses on the regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae. The organization of the actin cytoskeleton in Saccharomyces cerevisiae is regulated by different proteins such as calmodulin, CMD1, and here I present data that CMD1 plays a role in the regulation of the only phosphatidylinositol 4-phosphate 5-kinase, MSS4, in Saccharomyces cerevisiae. CMD1 regulates MSS4 activity through an unknown mechanism and thereby controls the organization of the actin cytoskeleton. MSS4 and CMD1 do not physically interact but MSS4 seems to be part of a large molecular weight complex as shown by gel filtration chromatography. This complex could contain regulators of the MSS4 activity. The complex is not caused by dimerization of MSS4 since MSS4 does not interact with itself. Two pathways, the cell wall integrity pathway and TORC2 (target of rapamycin complex 2) signaling cascade are important for the organization of the actin cytoskeleton. Loss of TOR2 function results in a growth defect that can be suppressed by MSS4 overexpression. To further characterize the link between MSS4 and the TORC2 signaling pathway and the cell wall integrity pathway we looked for targets of PI(4,5)P2. The TORC2 pathway and the cell wall integrity pathway signal to the GEF ROM2, an activator of the small GTPase RHO1. In our study we identified ROM2 as a target of PI(4,5)P2 signaling. We observed that the ROM2 localization changes in an mss4 conditional mutant. This suggests that the proper localization needs PI(4,5)P2. This could be mediated by the putative PI(4,5)P2 binding pleckstrin homology (PH) domain of ROM2. To better understand the regulation of PI(4,5)P2 levels in Saccharomyces cerevisiae we focused on one of the PI(4,5)P2 5-phosphatases, INP51. Here we present evidence that INP51 is a new negative regulator of the cell wall integrity pathway as well as the TORC2 pathway. INP51 probably regulates these two pathways by the turnover of PI(4,5)P2 thereby inactivating the effector/s. The deletion of INP51 does not result in any phenotype, but when combined with mutations of the cell wall integrity pathway we observe synthetic interaction. INP51 together with the GTPase activating protein (GAP) SAC7, responsible for the negative regulation of RHO1, negatively regulates the cell wall integrity pathway during vegetative growth. One of the targets of cell wall integrity pathway, the cell wall component chitin, which is normally deposited at the bud end, bud neck and forms bud scars, is delocalized in the mother cell in the sac7 inp51 double deletion mutant. In addition, another downstream component of the cell wall integrity pathway, the MAP kinase MPK1, has increased phosphorylation and protein level in the sac7 inp51 double deletion mutant. This suggests that INP51 is important for the negative regulation of the cell wall integrity pathway. Furthermore, we show evidence that INP51 forms a complex with TAX4 or IRS4, with two EH-domain containing proteins, that positively regulates the activity of INP51 and in this manner negatively regulate the cell wall integrity pathway. The EH-domain is known to bind the NPF-motif. This motif is present in INP51 and is important for INP51 interaction with TAX4 or IRS4. The EH-NPF interaction is a conserved mechanism to build up protein networks. The interaction between an EH-domain containing protein and a PI(4,5)P2 5-phosphatase is conserved. This is demonstrated by the epidermal growth factor substrate EPS15 (EH) interaction with the PI(4,5)P2 5-phosphatase synaptojanin the mammalian orthologue of the Saccharomyces cerevisiae INP proteins. In summary, INP51 together with TAX4 and IRS4, forms complexes important for regulation of PI(4,5)P2 levels. The complexes are linked to the TORC2 signaling pathway and the cell wall integrity pathway, specifically regulating MPK1 activation and chitin biosynthesis. The work presented in this dissertation facilitates the development of a model of the complex regulation of PI(4,5)P2 signaling in Saccharomyces cerevisiae