Browsing by Subject "Saccharomyces cerevisiae"

Sort by: Order: Results:

Now showing items 1-12 of 12
  • Lohva, Henri (Helsingfors universitet, 2016)
    Saccharomyces cerevisiae is a popular organism in the production of biofuels, chemicals and pharmaceuticals. This is thanks to a good understanding of its metabolism, GRAS status and the ease of modification. Traditionally its genetic modification has been based on the use of selectable markers. Modifying multi gene pathways has required a sequential process consisting of multiple single gene disruptions together with marker recycling. Additionally, many industrial S. cerevisiae strains are polyploid and lack the same tools for their modification as laboratory strains. In this study we sought to develop CRISPR/Cas9 based genetic engineering method for the modification of industrial S. cerevisiae strains. The CRISPR/Cas9 system is based on the adaptive immunity system of bacteria. It makes use of the Cas9 endonuclease which produces double stranded DNA brake to any location determined by a gRNA molecule. This causes the activation of DNA repair mechanisms which can be utilized to for the genomic integration of a template DNA. This makes transformation events much more likely and thus enables producing multiple modifications at once and removes the need for the of use selectable markers. In our approach Cas9 and gRNA were transformed into the cell in a plasmid together with a separate template DNA molecule. We used this method to remove lyp1, ura3 and can1 genes from diploid and polyploid industrial S. cerevisiae strains multiple genes at a time. Simultaneously we evaluated the effect of the NHEJ repair mechanism on CRISPR/Cas9 by repeating the tests with a deletion strain missing the ku70 gene required by NHEJ. Finally the method was used for the metabolic engineering by integrating the five gene violacein metabolic pathway into two loci in a single transformation event. This study demonstrated the CRISPR/Cas9 method is well suited for the modification of industrial S. cerevisiae strains and is capable of modifying up to three loci at a time in a polyploid yeast strain.
  • Peltonen, Kaisa (Helsingin yliopisto, 2018)
    New alternative feedstocks are needed for biofuel production to fulfil the growing demand in the coming years. The industry is moving away from second-generation biofuels, produced from food and feed crops, to using waste streams from industrial processes. An abundant, cheap and attractive waste stream for processing in Europe is the pectin-rich pulp from sugar beet processing and fruit juice industry. Sugar beet pulp is particularly rich in D-galacturonic acid and arabinose, but neither are naturally used by the yeast Saccharomyces cerevisiae, which would be an interesting candidate for the microbial fermentation of the biomass. S. cerevisiae is one of the most used organisms in the industrial biotechnology, and methods for the genetic engineering of the organism are highly developed. To overcome the natural limitations of the yeast for D-galacturonic acid fermentation, the metabolic pathways present in other organisms could be integrated in the yeast genome. Two bacterial and one fungal pathway are known to convert D-galacturonic acid into metabolites of the yeast glycolytic and ethanol fermentation pathways, and are thus considered promising for engineering in yeast. A major engineering challenge in integrating the fungal pathway in yeast is the redox imbalance caused by the two NADPH-specific reducing enzymes. The aim of this thesis was to review the potential of different D-galacturonic acid pathways for yeast fermentation. S. cerevisiae is a well-characterised organism for heterologous protein expression, but at times functional expression of foreign proteins is not achieved. One approach to study the pathways was to clone and express enzymes of the bacterial isomerase and dehydratase pathways in S. cerevisiae, and to test their activity in culture lysates. In addition, to overcome the redox imbalance in the eukaryotic pathway, two approaches were used to obtain an NADH-spesific D-galacturonic acid reductase. First, a mutant library of the Trichoderma reesei gar1 reductase was designed with the structure-guided cofactor specificity reversal tool CSR-SALAD. An automated high-throughput screening method for expression in Escherichia coli was developed, and the library was screened for enzymatic activity. The second approach was to try to identify the sequence for the characterised NADH-utilising reductase from the single-cell algae Euglena gracilis. A cDNA library of the algae was made and screened with PCR and in vivo methods. The reductase uxaB of the isomerase pathway and dehydrogenase kduD of the dehydratase pathway were functionally expressed in S. cerevisiae, with specific activities of 1.1 µmol min-1 mg-1 and 0.22 µmol min-1 mg-1 , respectively. The enzymes dehydratase uxaA and isomerase kduI did not exhibit activity in activity assays. The galurD of the dehydratase pathway was expressed in E. coli, and the purified enzyme was successfully used to convert D-galacturonate to 5-keto-4-deoxy galacturonate. The approaches to change the cofactor specificity of the NADPH-specific reductase of the eukaryotic pathway did not lead to a discovery of a NADH-specific enzyme. More research is needed for engineering active enzymes for S. cerevisiae expression and constructing a fully functional D-galacturonic acid pathway for feasible D-galacturonic acid fermentation.
  • Omoruyi, I.M.; Pohjanvirta, R. (2018)
    Mounting evidence of the effects of endocrine-disrupting chemicals (EDCs) in humans has led to assaying a vast array of food items (processed or packaged) as possible sources of human exposure to estrogens. In this study, we investigated the current situation in this respect of different food supplements and beer brands. Eleven food supplements and 24 beer brands were obtained from Helsinki, Finland. Sample preparation was carried out by established methods while estrogenic activities were assessed by a yeast bioluminescent assay, using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc). All the food supplements as well as 81% of the beer samples tested were found to be estrogenic, with estradiol equivalent concentrations of food supplements and beer brands ranging from 7.5 to 11.5 µg/ml and from below detection limits to 43.6 ng/ml, respectively. The estrogenic activities detected in beer samples were not dependent on the beer's alcoholic content, the country of production, or the size of the production brewery. The results of our study imply that both food supplements and beers can be a significant source of human exposure to estrogens. Therefore, further studies and regular surveillance are warranted. © 2018, © 2018 Taylor & Francis Group, LLC.
  • Viitasalo, Liisa; Iltanen, Sari; Huhtala, Heini; Saavalainen, Päivi; Kaukinen, Katri; Lindfors, Katri; Kurppa, Kalle (2020)
    Risk of celiac disease (CD) is increased in relatives of CD patients due to genetic and possible environmental factors. We recently reported increased seropositivity to anti-Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (anti-I2) and Bacteroides caccae TonB-linked outer membrane protein (anti-OmpW) antibodies in CD. We hypothesized these markers also to be overrepresented in relatives. Seropositivity and levels of ASCA, anti-I2 and anti-OmpW were compared between 463 first-degree relatives, 58 untreated and 55 treated CD patients, and 80 controls. CD-associated human leukocyte antigen (HLA)-haplotypes and transglutaminase (tTGab) and endomysium (EmA) antibodies were determined. One or more of the microbial antibodies was present in 75% of relatives, 97% of untreated and 87% of treated CD patients and 44% of the controls. The relatives had higher median ASCA IgA (9.13 vs. 4.50 U/mL, p <0.001), ASCA IgG (8.91 vs. 5.75 U/mL, p <0.001) and anti-I2 (absorbance 0.74 vs. 0.32, p <0.001) levels than controls. There was a weak, positive correlation between tTGab and ASCA (r = 0.31, p <0.001). Seropositivity was not significantly associated with HLA. To conclude, seropositivity to microbial markers was more common and ASCA and anti-I2 levels higher in relatives of CD patients than controls. These findings were not associated with HLA, suggesting the role of other genetic and environmental factors.
  • Fatal, Netta (University of Helsinki, 1997)
  • Viitasalo, Liisa; Kurppa, Kalle; Ashorn, Merja; Saavalainen, Päivi; Huhtala, Heini; Ashorn, Sara; Mäki, Markku; Ilus, Tuire; Kaukinen, Katri; Iltanen, Sari (2018)
    Background and AimsIn nonresponsive celiac disease (NRCD), the symptoms and duodenal damage persist despite a gluten-free diet. Celiac disease patients with persistent symptoms are found to have a dysbiotic microbiota. We thus hypothesized that increased seroreactivity to the serum gluten-sensitive microbial antibodies Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (I2), and Bacteroides caccae TonB-linked outer membrane protein (OmpW) is associated with NRCD.MethodsASCA, I2 and OmpW were measured in 20 seronegative CD patients with persistent villous damage despite strict dietary treatment (NRCD group). Fifty-eight responsive patients served as CD controls (55 on gluten-free treatment) and 80 blood donors as non-CD controls.ResultsAt least one microbial marker was positive in 80% of NRCD patients, in 97% of untreated CD and 87% of treated CD patients, and in 44% of controls. NRCD patients had the highest frequency of ASCA positivity (65% vs 52, 20, and 0%, respectively) and also significantly higher ASCA IgA (median 14.5 U/ml) and IgG (32.5 U/ml) titers than treated CD patients (7.0 U/ml, 13.0 U/ml) and non-CD controls (4.5 U/ml, 5.8 U/ml). The frequencies of I2 and OmpW were lower in NRCD than in untreated CD (65% and 45% vs 86% and 59%, respectively), and I2 titers were higher in NRCD (median absorbance 0.76) and untreated (1.0) and treated (0.83) CD than controls (0.32). OmpW was elevated in untreated (1.1) and treated (0.94) CD patients compared with controls (0.79).ConclusionsSeropositivity and high titers of ASCA are associated with NRCD and might serve as an additional follow-up tool in CD.
  • Aro-Kärkkäinen, Niina (Helsingin yliopisto, 2021)
    Orgaanisilla hapoilla on useita käyttötarkoituksia muun muassa lääke-, elintarvike- ja polymeeriteollisuudessa. Tässä työssä perehdyttiin L-arabonaatin biotekniseen tuottamiseen L- arabinoosista, jota esiintyy runsaasti biomassan hemiselluloosassa toisen pentoosisokerin, D- ksyloosin, ohella. Neljä potentiaalista dehydrogenaasientsyymiä, jotka tuottavat L-arabinoosista L-arabonaattia katalysoimalla hapetusreaktiota, identifioitiin kirjallisuuden ja bioinformatiikan avulla. L-arabonaatin tuottoa testattiin ilmentämällä näitä geenejä Saccharomyces cerevisiae -hiivassa, jota kasvatettiin L-arabinoosia sisältävällä kasvatusalustalla. Näistä entsyymeistä tehokkain, joka oli peräisin juurinystyrästä eristetystä Rhitzobium leguminosarum bv. trifolii -bakteerista, puhdistettiin ja karakterisoitiin. Entsyymi nimettiin L-arabinoosi/D-galaktoosi 1dehydrogenaasiksi (EC 1.1.1.-), Rl AraDH. Rl AraDH kuuluu Gfo/Idh/MocA -proteiiniperheeseen ja suosii kofaktorina NADP+:a verrattuna NAD+:iin. Rl AraDH entsyymillä on suurin katalyyttinen aktiivisuus (kcat/Km) käytettäessä substraattina L-arabinoosia, D-galaktoosia ja D-fukoosia. Rl AraDH:n rakennetutkimukset, jotka suoritettiin käyttäen ydinmagneettista resonanssia (NMR) ja proteiinimallinnusta, osoittivat entsyymin suosivan substraattina L-arabinoosin α-pyranoosimuotoa. Entsyymin hapetusreaktion stabiili tuote havaittiin olevan L-arabino-1,4-laktoni, joka voi myös aueta hitaasti neutraalissa pH:ssa lineaariseksi L-arabonaatiksi. Entsyymin pH optimi on 9, mutta hiivan kasvatukseen sopivan puskurin käyttö pH:ssa 6.8 osoitti, että hyvää katalyyttista aktiivisuutta voitiin odottaa myös in vivo -olosuhteissa. Rl AraDH:n ilmentäminen S. cerevisiae -hiivassa yhdessä L-arabinoosin sisäänottoa soluun lisäävän galaktoosipermeaasin (Gal2) kanssa johti 18 g/l L-arabonaatin tuottoon nopeudella 248 mg/l/h, saantoprosentin ollessa 86 %. Näin ollen uuden työssä karakterisoidun Rl AraDH:n osoitettiin olevan käyttökelpoinen ja tehokas entsyymi tuotettaessa L-arabonaattia S. cerevisiae -hiivassa. L- arabonaatin tuottaminen L-arabinoosista on osa biojalostamoiden kehittämisprosessia, jonka tavoitteena lignoselluloosaa sisältävästä biomassasta on tuottaa uusia käyttökelpoisia yhdisteitä.