Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae

BACKGROUND: Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. RESULTS: In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. CONCLUSION: Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

BACKGROUND: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. RESULTS: We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. CONCLUSION: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production

Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

<p>Abstract</p> <p>Background</p> <p>Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric <it>n</it>-butanol. <it>Saccharomyces cerevisiae </it>was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials.</p> <p>Results</p> <p>The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes <it>ILV2, ILV3</it>, and <it>ILV5 </it>in valine metabolism in anaerobic fermentation of glucose in mineral medium in <it>S. cerevisiae</it>. Isobutanol yield was further improved by twofold by the additional overexpression of <it>BAT2</it>, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of <it>ILV6</it>, encoding the regulatory subunit of Ilv2, in the <it>ILV2 ILV3 ILV5 </it>overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the <it>ILV2 ILV3 ILV5 </it>overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively.</p> <p>Conclusions</p> <p>Overexpression of genes <it>ILV2, ILV3, ILV5</it>, and <it>BAT2 </it>in valine metabolism led to an increase in isobutanol production in <it>S. cerevisiae</it>. Additional overexpression of <it>ILV6 </it>in the <it>ILV2 ILV3 ILV5 </it>overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of <it>ILV2, ILV3</it>, and <it>ILV5 </it>on isobutanol production. Aerobic cultivations of the <it>ILV2 ILV3 ILV5 </it>overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the reference strain, but not for the <it>ILV2 ILV3 ILV5 </it>overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after <it>ILV2, ILV3</it>, and <it>ILV5 </it>have been overexpressed, which most probably includes the valine inhibition to Ilv2.</p

Role of cultivation media in the development of yeast strains for large scale industrial use

The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized

Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering

<p>Abstract</p> <p>Background</p> <p>Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by <it>Saccharomyces cerevisiae </it>requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.</p> <p>Results</p> <p>Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial <it>Saccharomyces cerevisiae </it>strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.</p> <p>Conclusion</p> <p>To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant <it>S. cerevisiae </it>strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.</p

Carbonic anhydrase IX in oligodendroglial brain tumors

Background Carbonic anhydrase IX is a hypoxia-induced enzyme that has many biologically important functions, including its role in cell adhesion and invasion. Methods This study was set out to investigate the role of CA IX in a series of 86 oligodendroglial brain tumors (71 primary and 15 recurrent; 48 pure oligodendrogliomas and 40 mixed oligoastrocytomas). Results 80% of the tumors showed CA IX expression by immunohistochemistry. Tumors with moderate or strong CA IX expression had decreased level of cell proliferation compared to weak or no CA IX expression (median 2.9 vs. 5.8, p = 0.015). CA IX correlated with two antioxidative enzymes, manganese superoxide dismutase (MnSOD) and regulatory gammaglutamylcysteine synthetase (GLCL-R): CA IX expression was significantly higher in MnSOD-positive tumors (p = 0.008) and decreased in GLCL-R-positive tumors (p = 0.044). In Cox multivariate analysis CA IX expression, patient age and histological component (pure oligodendroglioma vs. mixed oligoastrocytoma) showed independent prognostic values (p = 0.009, p = 0.003 and p = 0.022, respectively), CA IX positivity predicting poorer outcome. Conclusion CA IX was proved to be an independent prognostic indicator in oligodendroglial brain tumors, and it also correlates reversely with cell proliferation. It may have a role in the biology of oligodendrogliomas, and most interestingly, as it is mainly expressed in tumor tissue, CA IX could serve as a target molecule for anticancer treatments.BioMed Central Open acces

Maahanmuuttajapoikien ja -miesten seksuaaliterveyden edistäminen

Tiivistelmä Maahanmuuttajien määrä on kasvanut merkittävästi Suomessa tämän vuosituhannen aikana. Maahanmuuttajilla on todettu olevan enemmän seksuaaliterveyteen liittyviä ongelmia ja vähemmän seksuaaliterveydestä tietoa kuin kantaväestöllä. Maahanmuuttaja-asiakkaan ammatillinen, laadukas kohtaaminen sosiaali- ja terveyshuollossa edistää pitkällä aikavälillä asiakkaan sopeutumista ja kotoutumista suomalaiseen yhteiskuntaan. Opinnäytetyön aiheena oli maahanmuuttajapoikien ja -miesten seksuaaliterveyden edistäminen. Opinnäytetyön tarkoituksena oli edistää maahanmuuttajapoikien ja -miesten seksuaaliterveyttä. Opinnäytetyön tavoitteena on kehittää sosiaali- ja terveysalan henkilöstön valmiuksia maahan-muuttajapoikien ja -miesten seksuaaliterveyden edistämisessä. Opinnäytetyössä tuotettiin ope-tusmateriaalia sosiaali- ja terveysalan ammattilaisille sekä opiskelijoille maahanmuuttajapoikien ja -miesten seksuaaliterveyden edistämisestä. Tämä opinnäytetyö tehtiin yhteistyössä Terveyden ja hyvinvoinninlaitoksen (THL) rahoittaman maahanmuuttajien seksuaaliterveyttä ja turvataitoja edistävän MAUSTE – hankkeen kanssa. MAUSTE- hanke oli Savonia -ammattikorkeakoulun ja Jyväskylän ammattikorkeakoulun yhteistyössä toteuttama kaksivuotinen hanke. Opinnäytetyö toteutettiin kuvailevanana kirjallisuuskatsauksena. Opetusmateriaalin teoriaosuus koottiin kirjallisuuskatsauksella keräämällä tietoa eri lähteistä. Opinnäytetyön tulokseksi saatiin, että sosiaali- ja terveysalan ammattilaiset kokevat tarvitsevansa enemmän tietoa maahanmuuttaja poikien ja miesten seksuaaliterveydestä ja sen edistämisestä. Tulevaisuudessa maahanmuuttaja poikien ja miesten seksuaaliterveyden edistämiseen voidaan panostaa antamalla lisää tietoa sekä kouluttamalla ammattilaisia enemmän.Abstract The number of immigrants has increased significantly in Finland during this millennium. Immigrants have been found to have more sexual health problems and less knowledge than other people in the population. The professional, high-quality encounter of an immigrant client in social and health care promotes long-term customer adaptation and integration into Finnish society. The theme of the thesis was to promote sexual health for immigrant boys and men. The purpose of the thesis was to promote the immigrant's sexual and physical health for boys and men. The aim of the Bachelor's thesis is to develop the skills of the social and health personnel in the promotion of the sexual health for boys and men. The aim was to produce teaching materials for social and health professionals and for immigrants to promote the sexual health of boys and men. This thesis was made in cooperation with the Health and Welfare Institute (THL) funded by the MAUSTE project promoting sexual health and safety skills for immigrants. The MAUSTE project was a two-year project carried out by the Savonia Polytechnic and the Jyväskylä Polytechnic. The descriptive literature review was used as the method of the thesis. The theoretical part of the teaching material was compiled by literature review by collecting information from different sources. Studies show that social and health professionals feel that they need more information on immigrant and sexual health and promotion of boys and men. In the future, it can be invested in promoting sexual health for immigrant boys and men by providing more information and training more profes-sionals

Engineering xylose and arabinose metabolism in recombinant Saccharomyces cerevisiae

Utilization of all sugars in lignocellulose hydrolysates is a prerequisite for economically feasible bioethanol production. The yeast commonly used for industrial ethanol production, Saccharomyces cerevisiae, is naturally unable to utilize pentose sugars xylose and arabinose, which constitute a large fraction of many lignocellulosic materials. Xylose utilization by S. cerevisiae can be achieved by heterologous expression of a xylose utilization pathway, consisting either of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK), or alternatively, of xylose isomerase (XI) and XK. Xylitol formed by XR is a major by-product in xylose fermentation when using the XR-XDH pathway. In this thesis, high-level expression of both XR and XDH was shown to decrease xylitol formation. The influence of other genetic modifications was also evaluated. It was shown that the overexpression of the non-oxidative pentose phosphate pathway (PPP) genes enables efficient growth on xylose and xylose fermentation, provided that the initial xylose pathway is expressed at a high level. When comparing the two xylose utilization pathways, higher ethanol productivity was achieved using the XR-XDH pathway, whereas higher ethanol yield was achieved with the XI pathway. The industrial xylose-fermenting S. cerevisiae strain TMB 3400, which has been previously generated by mutagenesis and selection, was tested for fermentation of lignocellulose hydrolysate. TMB 3400 displayed significantly better fermentation performance compared to the laboratory strains tested, highlighting the need for robust industrial strains in lignocellulose fermentation. TMB 3400 was also characterized by proteome analysis using difference in-gel 2-D electrophoresis. Consistently with the results obtained in other studies, increased activities of XR, XDH and a PPP enzyme TKL were found. The bacterial arabinose utilization pathway was introduced into TMB 3400, which resulted in the novel glucose, xylose and arabinose co-fermenting strain TMB 3063, with ethanol, xylitol and arabitol as the main fermentation products

”Käymme yhdessä ain, käymme aina rinnakkain.” : Päiväkodin henkilöstön ja lasten vanhempien yhdessä luoma arvopohja yksikkökohtaisen varhaiskasvatussuunnitelman lähtökohtana

Varhaiskasvatus on pienten lasten eri elämänpiireissä tapahtuvaa suunnitelmallista ja tavoitteellista kasvatuksellista vuorovaikutusta, joka edistää lasten tasapainoista kasvua, kehitystä ja oppimista yhdessä vanhempien ja kasvatuksen ammattilaisten kanssa. Päiväkodin varhaiskasvatussuunnitelma ohjaa toiminnan sisällöllistä kehittämistä ja luo osaltaan mahdollisuudet varhaiskasvatuksen laadun kehittämiselle. Kehittämistehtäväni tavoitteena on nimetä ja määrittää Päiväkoti Mainingin keskeiset arvot (3-5 arvoa) yhdessä päiväkodin henkilökunnan ja lasten vanhempien kanssa. Opinnäytetyöni myötä luomme päiväkodillemme toimintaamme ohjaavan arvopohjan yhdessä lasten vanhempien kanssa. Arvopohja on myös ehdoton edellytys yksikkömme varhaiskasvatussuunnitelman laatimisen lähtökohdaksi, sillä työyhteisön on kyettävä kertomaan selkeästi, mikä sille on tärkeää ja mihin se uskoo. Opinnäytetyöni on laadullinen tutkimus, jossa käytin aineistonkeruumenetelmänä henkilökunnalle aivoriihi-ideointimenetelmää arvojen nimeämiseksi ja määrittämiseksi. Lasten vanhemmat osallistuivat hankkeeseen avoimen kyselylomaketutkimuksen muodossa. Näin sain aineiston alustavan päärungon (arvot) henkilöstöltä, mutta sitä tukevan varsinaisen tutkimusaineiston lasten vanhemmilta. Tällöin pääasiallisina tiedonlähteinä toimivat vanhempien tuottamat aineistot, joita täydensivät henkilöstön ideoimat arvomääritelmät. Päiväkodin kehittämistehtävän avulla valikoidut arvot, turvallisuus, yksilöllisyys, luottamus ja leikki, ovat reaalimaailmaan liittyviä, toimintaa suuntaavia merkityksiä, jotka näkyvät ihmisten asenteissa, mielipiteissä ja teoissa. Arvoissa piilee yhteinen ymmärrys siitä, mikä on tärkeää ja mitä kohti kannattaa mennä. Arvoihin turvaudutaan vaikeissa tilanteissa, joita ei voida ratkaista toimimalla kuten ennen sekä tilanteissa, joita ei ole ohjeistettu. Henkilöstön ja vanhempien muodostamista ajatuksista kokosin tiivistetysti näiden arvojen merkityksen päiväkotiyhteisöllemme. Arvopäiväkirja voisi olla tulevaisuudessa yksi hyvä väline arvokeskustelujen pohjaksi työntekijän tai työtiimin kehityskeskusteluissa tai koko työyhteisön kehittämispäivillä tietyin sovituin väliajoin. Myös henkilöstölle ja vanhemmille tehtävän arvobarometrin avulla voitaisiin arvioida kunkin arvon toteutumista asteikolla 1-5. Arvobarometrin pohjalta voitaisiin järjestää keskustelu, jossa kiinnitettäisiin huomiota parhaiten ja heikoiten toimiviin arvoihin.“We always go together, we always go side by side” : The basic values shared by the staff of a kindergarten and the parents as a starting point for the unit-specific early childhood education plan Early childhood education refers to systematic and target-oriented educational interaction which takes place in various educational milieus and which promotes the children's balanced growth, development and learning together with the parents and the professionals. The early childhood education plan of a kindergarten directs the developing of contents of the operation and creates possibilities for the developing of the quality of early childhood education. The main purpose of this thesis was to name and to determine the central values of the kin-dergarten Maininki (3-5 values) together with the staff of the kindergarten and the parents. The aim was to provide our kindergarten with a value basis to direct our operation. The values are also an absolute precondition for the drawing up of the early childhood education plan of the kindergarten of our unit because the work community must be able to state clear-ly what is important to it and what it believes. The study was qualitative. I used brainstorming as the data-collecting medhod. The staff named the values and gave them a definition. The parents participated in the project by answering the open-ended questions of a questionnaire. This way I got the material from the staff, which was supported by the material based on the responses of the parents. With the help of the development project of the kindergarten the selected values - safety, individuality, confidence and play- refer to meanings which are related to the real world and direct operation are seen in the people's attitudes, opinions and acts. The values refer to the common understanding, what is important and in which direction the organization should go. It is re-sorted to the values in the difficult situations which cannot be solved by operating, such as in before and the situations which have not been instructed. From the thoughts formed by staff and parents I collected the significance of these values for our kindergarten in brief. The value diary could be one good tool in the future as a basis of value discussions in the development discussions of a worker or work team or in the developing days of the whole work community with certain agreed intervals. Also the value barometer which is done to staff and parents could be used to estimate how the values are seen in daily activities on scale 1-5. A discussion in which attention would be best paid and worst to functional values could be arranged based on the value barometer