| 1. |
- Jansson, Ronnie, et al.
(författare)
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Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris
- 2016
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Ingår i: Biotechnology Journal. - : Wiley-VCH Verlagsgesellschaft. - 1860-6768 .- 1860-7314. ; 11:5, s. 687-699
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Tidskriftsartikel (refereegranskat)abstract
- Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.
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| 2. |
- Bergman, Alexandra Linda, 1985, et al.
(författare)
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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
- 2019
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized. Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses. Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.
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| 3. |
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| 4. |
- Kanagarajan, Selvaraju, et al.
(författare)
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Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers
- 2021
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Ingår i: International Journal of Biological Macromolecules. - : Elsevier BV. - 0141-8130 .- 1879-0003. ; 184, s. 955-966
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Tidskriftsartikel (refereegranskat)abstract
- Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.
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| 5. |
- Gustafsson, Kerstin, et al.
(författare)
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Direct and indirect effects of the fungicide azoxystrobin in outdoor brackish water microcosms
- 2010
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Ingår i: Ecotoxicology. - : Springer Science and Business Media LLC. - 0963-9292 .- 1573-3017. ; 19:2, s. 431-444
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Tidskriftsartikel (refereegranskat)abstract
- The effects of the strobilurin fungicide azoxystrobin were studied in brackish water microcosms, with natural plankton communities and sediment. Two experiments were conducted: Experiment 1 (nominal conc. 0, 15 and 60 mu g/L, 24-L outdoor microcosms for 21 days) and a second, follow-up, Experiment 2 (nominal conc. 0, 3, 7.5, 15 mu g/L, 4-L indoor microcosms for 12 days). The microcosms represent a simplified brackish water community found in shallow semi-enclosed coastal areas in agricultural districts in the Baltic Sea region. Measured water concentrations of the fungicide (Experiment 1) were, on average, 83 and 62% of nominal concentrations directly after application, and 25 and 30% after 21 days, for the low and high dose treatments, respectively, corresponding to mean DT50-values of 15.1 and 25.8 days, for low and high dose treatments, respectively. In Experiment 1, direct toxic effects on calanoid copepods at both test concentrations were observed. Similarly, in Experiment 2, the copepod abundance was significantly reduced at all tested concentrations. There were also significant secondary effects on zooplankton and phytoplankton community structure, standing stocks and primary production. Very few ecotoxicological studies have investigated effects of plant protection products on Baltic organisms in general and effects on community structure and function specifically. Our results show that azoxystrobin is toxic to brackish water copepods at considerably lower concentrations than previously reported from single species tests on freshwater crustaceans, and that direct toxic effects on this ecologically important group may lead to cascade effects altering lower food webs and ecosystem functioning.
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| 6. |
- Brunius, Carl, 1974, et al.
(författare)
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Prediction and modeling of pre-analytical sampling errors as a strategy to improve plasma NMR metabolomics data
- 2017
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1460-2059 .- 1367-4811. ; 33:22, s. 3567-3574
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Tidskriftsartikel (refereegranskat)abstract
- Biobanks are important infrastructures for life science research. Optimal sample handling regarding e.g. collection and processing of biological samples is highly complex, with many variables that could alter sample integrity and even more complex when considering multiple study centers or using legacy samples with limited documentation on sample management. Novel means to understand and take into account such variability would enable high-quality research on archived samples. This study investigated whether pre-analytical sample variability could be predicted and reduced by modeling alterations in the plasma metabolome, measured by NMR, as a function of pre-centrifugation conditions (1-36 h pre-centrifugation delay time at 4 A degrees C and 22 A degrees C) in 16 individuals. Pre-centrifugation temperature and delay times were predicted using random forest modeling and performance was validated on independent samples. Alterations in the metabolome were modeled at each temperature using a cluster-based approach, revealing reproducible effects of delay time on energy metabolism intermediates at both temperatures, but more pronounced at 22 A degrees C. Moreover, pre-centrifugation delay at 4 A degrees C resulted in large, specific variability at 3 h, predominantly of lipids. Pre-analytical sample handling error correction resulted in significant improvement of data quality, particularly at 22 A degrees C. This approach offers the possibility to predict pre-centrifugation delay temperature and time in biobanked samples before use in costly downstream applications. Moreover, the results suggest potential to decrease the impact of undesired, delay-induced variability. However, these findings need to be validated in multiple, large sample sets and with analytical techniques covering a wider range of the metabolome, such as LC-MS.
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| 7. |
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| 8. |
- Önneby, Karin
(författare)
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Bioaugmentation for reduction of diffuse pesticide contamination : a bioprophylactic concept
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pesticides and their residues frequently contaminate surface waters and groundwater so consequently there is a great need to identify methods and practices that reduce such contamination. This thesis examined the potential of a 'bioprophylactic' concept based on the hypothesis that diffuse contamination after application in the field can be significantly reduced if pesticides are degraded as rapidly as possible after the intended effect has been achieved. This involves adding pesticide degraders together with the pesticide at the time of application. Such enhanced degradation is particularly important for pesticides used on non-agricultural soils with low degradation and high leaching potential (e.g. roadsides, railway embankments, paths, farmyards, urban land). The herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) was used as a model herbicide in this thesis, together with its degrader Sphingobium sp. T51. Soil inoculation with Sphingobium sp. T51 in microcosms significantly enhanced the degradation of MCPA compared with an uninoculated soil, without losing the herbicidal effect. In sand, 105 cells g-1 dry weight of soil reduced the MCPA concentration by 79-86% within one day, while only 0-31% reduction was observed in the uninoculated control after three days. However, Sphingobium sp. T51 needs to be formulated to retain its viability and degradation activity during prolonged storage and also for protection from the harsh environmental stresses associated with its application and functioning in the field. Both freeze drying and fluidised bed drying of Sphingobium sp. T51 resulted in high initial cell survival rates, of 67-85%. The storage stability of formulated Sphingobium sp. T51 was dependent on the formulation excipient/carrier used, storage temperature and atmospheric conditions. Sphingobium sp. T51 demonstrated approximately 50% survival in a freeze-dried sucrose formulation after six months of storage under partial vacuum at 25 °C. A fluidised bed-dried formulation with cottonseed flour as carrier resulted in the highest MCPA degradation in microcosms, but the storage stability was insufficient. Formulated and long-term stored Sphingobium sp. T51 reduced pesticide leaching by 85-94% of the total amount added in sand columns. Future research needs regarding practical implementation of the concept are discussed.
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| 9. |
- Ståhlberg, Jerry
(författare)
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Product Binding Varies Dramatically between Processive and Nonprocessive Cellulase Enzymes
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287, s. 24807-24813
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Tidskriftsartikel (refereegranskat)abstract
- Cellulases hydrolyze beta-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate-and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.
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| 10. |
- Kreuger, Emma, et al.
(författare)
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Biogas från Skånsk betblast - potential, teknik och ekonomi
- 2014
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Rapport (populärvet., debatt m.m.)abstract
- Sammanfattning I Skåne odlas 34 500 ha sockerbetor för sockerutvinning och i Sverige 36 000 ha (Jordbruksstatistik årsbok 2014). Vid insamling och rötning av blasten från betorna i Skåne skulle drygt 200 GWh biogas kunna produceras per år och driva minst 19 000 bilar eller 1000 bussar. Men idag lämnas den i fält. Tidigare studier har visat att betblast från sockerproduktion är på gränsen till lönsamt att skörda och använda för biogasproduktion (Lantz, 2013b). I det här projektet har flera forskare, en biogasproducent och en representant för betodlarna gemensamt tagit fram och undersökt ett par förslag för hur skörd, lagring och rötning av betblast kan genomföras och hur olika tillvägagångssätt påverkar kostnader och klimatpåverkan. Dessa innefattar en jämförelse av två olika skördekedjor och undersökning av effekterna av att fraktionera betblast före lagring och rötning på; biogasproduktion, ekonomi och klimatpåverkan. För dessa beräkningar antogs att endast betblast rötades i en biogasanläggning med en årlig produktion om 172 TJ (48 GWh) metan. Effekterna av att introducera icke fraktionerad och fraktionerad betblast i en samrötningsanläggning analyserades också. Dessutom arrangerades en skördedemonstration i oktober 2013 i samarbete med Skånska Biobränslebolaget (länk till video). Analysen av skördeteknik har begränsats till skörd av blast från betor odlade för sockerproduktion, vilket är det som görs i Sverige idag. Om sockerbetor odlas endast för biogasproduktion kan andra skördetekniker för betor och blast vara aktuella. Studien har visat att när biogas från betblast ersatte fossil energi som drivmedel så sänktes utsläppen av klimatgaser kraftigt, med 80 %. Därmed uppfylldes EUs hållbarhetskriterier för biodrivmedel, både enligt dagens direktiv (35 % reduktion) och föreslagna framtida (60 % reduktion). Viktigt i detta sammanhang är att blasten är en restprodukt och den konkurrerar inte om åkermark för livsmedelsproduktion. I Skåne skulle ca 200 GWh biogas kunna produceras från betblast vid dagens sockerbetsproduktion. Men, även för den andel av blasten som skördas under september (motsvarande ca 40 GWh), då det är mer gynnsamt än vid senare skörd, är det svårt att hitta ekonomisk hållbarhet. Studien tyder på att kostnader och klimatpåverkan är de samma om betblast fraktioneras eller ej. I fallstudien framkom att fraktionering av betblasten gav praktisk möjlighet att ta emot mer material i den studerade samrötningsanläggningen. Vätskefraktionen kunde då ersätta vatten i förbehandlingen och mera torrsubstans (TS) kunde tas emot med den fasta fraktionen innan uppehållstiden begränsade mängden i rötningsprocessen. Att ersätta vatten i förbehandlingsanläggningen ger mindre kapitalkostnader per producerad MWh jämfört med om man skulle röta denna fraktion i en dedikerad anläggning. Men, inte heller i fallstudien medförde fraktionering lägre kostnader per producerad mängd metan. Blastskörden visade sig vara högre i september, 3,6 ton torrsubstans per hektar (t TS/ha), än i oktober, 3,2 t TS/ha, vilket gör det fördelaktigare att samla in blast i september än oktober. Av de skörde- och transportkedjor som teoretiskt utvärderades i projektet var det ekonomiskt mest fördelaktigt med en skördekedja där en mindre mängd blast samlades in (55 % av tillgänglig mängd) för att minimera maskinernas väntetider. Alternativet har dock nackdelen att en större andel kvarlämnad blast gör att en större andel av fältets ytafår ojämn förfruktseffekt i efterföljande gröda jämfört med ett scenario då större andel av blasten samlas in. Priset för skörd (i september) och lagring beräknades till 1,7–2,1 kr/kg TS både med och utan fraktionering. Detta är högre än det pris som tidigare beräknats (Gissén et al. 2014), vilket bedöms som underskattat. Tester av fraktionering av betblast gjordes i liten skala med en äppeljuicepress. Metanpotentialtester gjordes på de olika fraktionerna. Pressning av strimlad blast (13 % TS) gav en vätskefraktion (7 % TS) motsvarande en fjärdedel av våtvikten och 3 fjärdedelar återstod som fast fraktion (15 % TS). Den fasta fraktionen gav dubbelt så högt metanutbyte per kg våtvikt som vätskefraktionen, men ingen signifikant skillnad i metanutbyte per kg organiskt material. Ingen inverkan av sortval på betblastskörden eller metanutbyte per kg organiskt material kunde hittas vid test av fem sockerbetssorter som förädlats fram för sockerproduktion. När fraktionerad blast används kan möjlighet finnas att dubbelanvända lager för den våta fraktionen och rötrest. Det gäller även för andra flytande substrat som behöver lagras. Studien visar att dubbelanvändning kan påverka investeringskostnaderna för rötrestlagret signifikant och en närmare undersökning av om det är praktiskt möjligt vore intressant. När flera positiva faktorer samspelar kan det finnas möjlighet att med dagens förhållanden producera biogas som biodrivmedel från betblast på ett ekonomiskt hållbart sätt. Exempel på identifierade positiva faktorer är: högt blastutbyte, användning av underutnyttjade jordbruksredskap, rötning i befintliga anläggningar för att fylla ut substratluckor, korta transportsträckor och direktanvändning av färsk betblast utan lagring. Det är troligtvis endast för en liten del av den totala mängden blast som tillräckligt många positiva faktorer samspelar för att den idag ska kunna vara ekonomiskt intressant att använda för biogasproduktion.
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