| 1. |
- Guevara-Martínez, Mónica, 1989-, et al.
(författare)
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Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation
- 2015
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Ingår i: Frontiers in Microbiology. - : Frontiers Research Foundation. - 1664-302X. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coil strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetylCoA to 3HB also in E coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L-1 h(-1), was seen for phosphate-limitation.
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| 2. |
- Azouri, Kristian, et al.
(författare)
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Hotellrapport Stockholm
- 2011
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Rapport (populärvet., debatt m.m.)abstract
- Stockholm är Sveriges största stad och marknadsför sig som ”The Capital of Scandinavia”. De flesta resenärer som besöker Sverige åker till just Stockholm. År 2010 nådde Stockholms län 10 miljoner kommersiella övernattningar. Det är lätt att ta sig till Stockholm med både flyg, tåg, båt, bil och buss. Stockholm har ett brett utbud av kongress- och mässanläggningar. År 2009 var Stockholm den sjunde mest populära staden i världen att förlägga kongresser på. I dagsläget är Sverige på väg ur den lågkonjunktur som startade år 2008. Sveriges BNP och valuta är på väg att stabiliseras och stärkas. Regeringen har även lagt in ett förslag om att sänka tjänstemomsen inom hotell- och restaurangbranschen. I denna rapport har vi valt att avgränsa oss till ett eget utvalt område inom Stockholm. Detta område innefattar 143 hotell med 20 054 hotellrum.De prognoser vi gjort gällande staden Stockholm och segmenten affärsresenärer och fritidsresenärer pekar på en ökning av besökare till staden. Detta kommer resultera i att hotellens beläggning, snittpris och RevPAR kommer att öka framtill år 2013. Efter år 2013 tror vi att segmentet affärsresenärer kommer att minska något procentuellt och kurvan för belagda nätter kommer att stabiliseras. Dock tror vi att segmentet fritidsresenärer kommer att öka procentuellt efter år 2013 och antalet belagda hotellrum kommer att successivt öka mellan åren 2011 – 2015. Vi tror att snittpris och RevPAR kommer att sjunka på grund av att en fritidsresenär generellt sätt inte betalar lika mycket för ett hotellrum som en affärsresenär gör.
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| 3. |
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| 4. |
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| 5. |
- Berggren, Gustav, et al.
(författare)
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Compounds with capacity to quench the tyrosyl radical in Pseudomonas aeruginosa ribonucleotide reductase
- 2019
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 841-848
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) has been extensively probed as a target enzyme in the search for selective antibiotics. Here we report on the mechanism of inhibition of nine compounds, serving as representative examples of three different inhibitor classes previously identified by us to efficiently inhibit RNR. The interaction between the inhibitors and Pseudomonas aeruginosa RNR was elucidated using a combination of electron paramagnetic resonance spectroscopy and thermal shift analysis. All nine inhibitors were found to efficiently quench the tyrosyl radical present in RNR, required for catalysis. Three different mechanisms of radical quenching were identified, and shown to depend on reduction potential of the assay solution and quaternary structure of the protein complex. These results form a good foundation for further development of P. aeruginosa selective antibiotics. Moreover, this study underscores the complex nature of RNR inhibition and the need for detailed spectroscopic studies to unravel the mechanism of RNR inhibitors.
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| 6. |
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| 7. |
- Berggren, Gustav, et al.
(författare)
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Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate
- 2014
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:46, s. 31940-31949
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Tidskriftsartikel (refereegranskat)abstract
- Background: Activation of ribonucleotide reductase Ib depends on the flavodoxin-like maturase NrdI.Results: The redox properties of Bacillus anthracis NrdI allow isolation of the semiquinone state, NrdI(sq), which can catalyze formation of the manganese-tyrosyl radical cofactor.Conclusion: The maturation capacity of NrdI(sq) provides evidence that Mn-NrdF is activated via a superoxide radical.Significance: Novel antibiotics may be designed to selectively target the maturation mechanism.
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| 8. |
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| 9. |
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| 10. |
- Hultman, Elin
(författare)
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Bristande vattenföring förbi Gävleborgs regleringsdammar
- 2010
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- Gävleborgs regionala miljömål8.6 Godtagbar minimivattenföring,lyder”2010 ska 50 % av alla kraftverk och regleringsdammar ha en, ur biologiskmångfald, godtagbar minimivattenföring genom eller förbi respektive anläggning”.Måletsyftar till att främja den biologiska mångfalden genom att förbättra vandringsmöjligheter ochlivsmiljöer för fisk och övriga vattenlevande växter och djur.Vattenkraft är en ren och förnybar energikälla med låga utsläpp och mycket litenklimatpåverkan men konsekvenserna för landskapet och organismerna i och kring ett utbyggtvattendrag är stora. Vissa områden torrläggs, medan andra överdäms, forsar försvinner ochdet blir svårare för fiskar och andra djur och växter att överleva. De flesta stora svenskakraftverken byggdes under en tid då samhället inte prioriterade naturvårdsfrågor.Länsstyrelsens uppföljning av miljömålet visar att situationen för länets vattendrag är mycketallvarlig. Resultatet visar att det vid endast 16 av 142 kraftverksdammar finns en godtagbarminimivattenföring. Det motsvarar 11 % vilket är långt ifrån det uppsatta målet om 50 %.Vi har i den här studien definierat att en damm har en biologiskt godtagbarminimivattenföring när dammens minimitappning är större än eller lika stor som vattendragetslågvattenföring. För att urskilja de dammar som har en godtagbar minimivattenföring, harstudien gjorts i två steg utifrån länets 142 regleringsdammar. I det första steget bedömdes dedammar som har nolltappninginte ha en biologiskt godtagbar vattenföring. Nolltappningbetyder att inget vatten alls behöver tappas förbi anläggningen. De 65 urskiljdaregleringsdammarna bedömdes sedan utifrån dess minimitappning i jämförelse medvattendragets normala lågvattenföring. Minimitappning är den minsta vattenföring somregleringsdammen måste släppa förbi.För att på sikt nå målet krävs ett aktivt arbete från Länsstyrelsen, kommuner ochKammarkollegiet för att ta fram underlag och att driva omprövningar av gamla vattendomar isyfte att få till stånd ökade minimitappningar förbi dammar och kraftverk. Arbetet bör ske idialog med kraftbolagen. Berörda myndigheter bör också tillsammans med kraftbolagenidentifiera de dammar som idag inte ger någon större nytta för kraftproduktion och åtgärdadessa, t.ex. genom utrivning, så att vattenföringen kan återgå till det ursprungliga flödet.
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| 11. |
- Jarmander, Johan, et al.
(författare)
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Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli
- 2015
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 14:1, s. 51-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundLignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40 % of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose.ResultsThe Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (< 2 mg g-1 h-1) compared to carbon excess (25 mg g-1 h-1), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g-1)during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g-1 h-1) and to increase the yield of 3HB/cell dry weight to 1.38 g g-1. Nitrogen-limited fed-batch process design lead to further increased specific productivity (38 mg g-1 h-1) but also to additional cell growth (Y3HB/CDW = 0.16 g g-1). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid.ConclusionsIt was demonstrated that by using the strain E. coli PPA652ara it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB by E. coli reported here from glucose, xylose and arabinose is further comparable to the current state of the art for production of 3HB from glucose sources.
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| 12. |
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| 13. |
- Karlsson, Gunnar, 1955-, et al.
(författare)
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The experience of guilt and shame : A phenomenological-psychological study
- 2009
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Ingår i: Human Studies. - Dordrecht The Netherlands : Springer. - 0163-8548 .- 1572-851X. ; 32:3, s. 335-355
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Tidskriftsartikel (refereegranskat)abstract
- This study aims at discovering the essential constituents involved in the experiences of guilt and shame. Guilt concerns a subject's action or omission of action and has a clear temporal unfolding entailing a moment in which the subject lives in a care-free way. Afterwards, this moment undergoes a reconstruction, in the moment of guilt, which constitutes the moment of negligence. The reconstruction is a comprehensive transformation of one's attitude with respect to one's ego; one's action; the object of guilt and the temporal-existential experience. The main constituents concerning shame are its anchorage in the situation to which it refers; its public side involving the experience of being perceptually objectified; the exclusion of social community; the bodily experience; the revelation of an undesired self; and the genesis of shame in terms of a history of frozen now-ness. The article ends with a comparison between guilt and shame. Adapted from the source document.
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| 14. |
- Liljegren, Gustav, 1973-
(författare)
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Development and Investigations of Novel Sample Preparation Techniques : Electrochemical Extraction and Evaluation of Miniaturized Analytical Devices Coupled to Mass Spectrometry
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Different sample preparation steps prior to a detection method are often essential in analytical chemistry. In this thesis, both static extractions and on-line coupled solid-phase extractions have been studied in combination with different detection techniques. Aspects of performing sample preparations in miniaturized analytical devices and the development of poly(dimethylsiloxane) (PDMS) microchips are discussed. Polypyrrole was also evaluated as an electrochemically controllable stationary phase for solid-phase microextraction (SPME) and solid-phase extraction (SPE).The first part of this thesis describes the extraction of an organic compound from a very complex solid matrix utilizing the pressurized-fluid extraction (PFE) technique. The presented results show that PFE is easily optimized and enables rapid extractions and extracts relatively free from interferences.An integrated three-electrode device, which enabled electrochemical (EC) SPME under potential control, was developed. With this device, both anions and cations could be extracted employing two types of polypyrrole films. Planar micro band electrodes positioned at the end of a capillary were also used to electrochemically extract and detect anions in a miniaturized flow system. Different analyte concentrations and preconcentration times were examined, and good linear correlations were found between the extraction time and the detection response. The on-line coupling of a thin layer EC cell, with a polypyrrole coated working electrode, to different mass spectrometric (MS) techniques is also described and evaluated. The results show that EC-SPE, employing polypyrrole as stationary phase, can be used as a preconcentration step prior to detection.In addition, this thesis describes the development and on-line coupling of a microelectrode array equipped PDMS microchip with an integrated graphite electrospray emitter to electrospray ionization (ESI) MS. The system enabled short transfer times and an EC conversion efficiency of 30% at a flow rate of 0.5 μL/min. The on-line EC/ESI-MS experiments were significantly simplified using a wireless Bluetooth battery-powered EC instrument.
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| 15. |
- Lindell-Frantz, Eva, et al.
(författare)
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Tolkning av försäkringsavtal
- 2019
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Ingår i: Festskrift till Göran Millqvist. - 1651-8918. - 9789172237476 ; :32
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 16. |
- Lundin, Daniel, 1965-, et al.
(författare)
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The Origin and Evolution of Ribonucleotide Reduction
- 2015
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Ingår i: Life. - : MDPI. - 2075-1729. ; 5:1, s. 604-636
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reduction is the only pathway for de novo synthesis of deoxyribonucleotides in extant organisms. This chemically demanding reaction, which proceeds via a carbon-centered free radical, is catalyzed by ribonucleotide reductase (RNR). The mechanism has been deemed unlikely to be catalyzed by a ribozyme, creating an enigma regarding how the building blocks for DNA were synthesized at the transition from RNA- to DNA-encoded genomes. While it is entirely possible that a different pathway was later replaced with the modern mechanism, here we explore the evolutionary and biochemical limits for an origin of the mechanism in the RNA + protein world and suggest a model for a prototypical ribonucleotide reductase (protoRNR). From the protoRNR evolved the ancestor to modern RNRs, the urRNR, which diversified into the modern three classes. Since the initial radical generation differs between the three modern classes, it is difficult to establish how it was generated in the urRNR. Here we suggest a model that is similar to the B12-dependent mechanism in modern class II RNRs.
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| 17. |
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| 18. |
- Mårback, Sebastian, et al.
(författare)
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Uhm... What's going on? An EEG study on perception of filled pauses in spontaneous Swedish speech
- 2009
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Ingår i: Proceedings of the 22nd Swedish Phonetics Conference. ; , s. 92-95, s. 92-95
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Konferensbidrag (refereegranskat)abstract
- Filled pauses have been shown to play a significant role in comprehension and long-term storage of speech. Behavioral and neurophysiological studies suggest that filled pauses can help mitigate semantic and/or syntactic incongruity in spoken language. The purpose of the present study was to explore how filled pauses affect the processing of spontaneous speech in the listener. Brain activation of eight subjects was measured by electroencephalography (EEG), while they listened to recordings of Wizard-of-Oz travel booking dialogues.The results show a P300 component in the Primary Motor Cortex, but not in the Broca or Wernicke areas. A possible interpretation could be that the listener is preparing to engage in speech. However, a larger sample is currently being collected.
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| 19. |
- Nyberg, Lena, 1979, et al.
(författare)
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Optical mapping of single DNA molecules in nanochannels: A novel method for identification and characterization of antibiotic resistance
- 2015
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1045-1047
-
Konferensbidrag (refereegranskat)abstract
- The use, and overuse, of antibiotics has during the last decade led to a dramatic increase in antibiotic resistance and there is a crying need for novel methods for fast identification of antibiotic resistance genes. We here demonstrate how our previously developed assay for optical mapping of DNA in nanochannels can be used for characterization of resistance genes located on plasmid DNA from bacteria. The assay requires extremely small sample volumes and does neither rely on PCR, nor culturing of bacteria, which greatly reduces the time for analysis.
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| 20. |
- Perez-Zabaleta, Mariel, 1987-, et al.
(författare)
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Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
- 2016
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Ingår i: Microbial Cell Factories. - : Springer. - 1475-2859. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. Results: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V-max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q(3HB) = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y-3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L-1, which was 50 % higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L-1 and a productivity of 0.42 g L-1 h(-1), which is comparable to maximum values found in recombinant E. coli. Conclusions: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.
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| 21. |
- Rozman Grinberg, Inna, et al.
(författare)
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A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant
- 2018
-
Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 293:41, s. 15889-15900
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Tidskriftsartikel (refereegranskat)abstract
- Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor MnIII/MnIV. We show that NrdA from F. ignava can form a catalytically competent RNR with the MnIII/MnIV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis. In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.
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| 22. |
- Rozman Grinberg, Inna, et al.
(författare)
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Class Id ribonucleotide reductase utilizes a Mn-2(IV,III) cofactor and undergoes large conformational changes on metal loading
- 2019
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 24:6, s. 863-877
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Tidskriftsartikel (refereegranskat)abstract
- Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn-2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn-2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn-2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2 center dot-). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide.
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| 23. |
- Rozman Grinberg, Inna, et al.
(författare)
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Novel ATP-cone-driven allosteric regulation of ribonucleotide reductase via the radical-generating subunit
- 2018
-
Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 A crystal structure. We also present evidence for an (MnMnIV)-Mn-III metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.
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| 24. |
- Sjöberg, Gustav, 1991-, et al.
(författare)
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Characterization of volatile fatty acid utilization in Escherichia coli aiming for robust valorisation of food residues
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight chain VFAs (C2-C6) in the mixture were readily consumed simultaneously. At the dilution rate 0.1 h-1, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h-1. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon supplied from VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.
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| 25. |
- Sjöberg, Gustav, et al.
(författare)
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Characterization of volatile fatty-acid utilization in Escherichia coli aiming for robust valorisation of food residues
- 2020
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Ingår i: AMB Express. - : Springer Nature. - 2191-0855. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Valorisation of food residues would greatly benefit from development of robust processes that create added value compared to current feed- and biogas applications. Recent advances in membrane-bioreactor-based open mixed microbial cultures, enable robust conversion of fluctuating streams of food residues to a mixture of volatile fatty acids (VFAs). In this study, such a mixed stream of VFAs was investigated as a substrate for Escherichia coli, a well-studied organism suitable for application in further conversion of the acids into compounds of higher value, and/or that are easier to separate from the aqueous medium. E. coli was cultured in batch on a VFA-rich anaerobic digest of food residues, tolerating up to 40 mM of total VFAs without any reduction in growth rate. In carbon-limited chemostats of E. coli W3110 ΔFadR on a simulated VFA mixture, the straight-chain VFAs (C2-C6) in the mixture were readily consumed simultaneously. At a dilution rate of 0.1 h−1, mainly acetic-, propionic- and caproic acid were consumed, while consumption of all the provided acids were observed at 0.05 h−1. Interestingly, also the branched isovaleric acid was consumed through a hitherto unknown mechanism. In total, up to 80% of the carbon from the supplied VFAs was consumed by the cells, and approximately 2.7% was excreted as nucleotide precursors in the medium. These results suggest that VFAs derived from food residues are a promising substrate for E. coli.
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| 26. |
- Sjöberg, Gustav, 1991-
(författare)
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Engineering short-chain carboxylic-acid metabolism in the model microorganism Escherichia coli
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ever-increasing concern about carbon dioxide emissions has created an urgent need to develop alternative methods to cheaply and renewably produce materials, chemicals and fuels. The biorefinery is uniquely suited to deliver these products from sustainable biomass. However, cheaply and efficiently converting the dispersed, heterogenous and recalcitrant biomass to useful products requires further technical development. To address some of these challenges, the aim of this thesis was to investigate methods to improve the economic viability of the microbial biorefinery by evaluating short chain carboxylic acids as substrates (volatile fatty acids) and products ((R)-3-hydroxybutyrate, 3HB).Initially, two renewable and cheap sources of carbon were investigated as substrates for E. coli. It was determined that E. coli is a suitable microorganism for valorization of volatile fatty acids derived from food waste. Also, it was shown that lignocellulosic sugars with a composition based on a hydrolysate of wheat straw can be converted to 3HB in E. coli with similar yields and productivities as from pure glucose. To improve the yield of the model product 3HB, and thereby the potential gross profit, substrate depletion was used as a strategy throughout the thesis to control bioprocesses. Specifically, nutrient depletion was shown to decouple growth from 3HB production in nitrogen and phosphorous depleted batches, increasing the yield of 3HB. To further improve 3HB production, metabolic engineering was used to improve the availability of NADPH. Additionally, the bacterial artificial chromosome (BAC) was investigated as a robust single-copy vector for metabolic engineering in E. coli. The expression of a large operon from the BAC was shown to be comparable to chromosomal expression. Then, the specific growth rate, productivity and yield of 3HB producing strains was increased by expression of the 3HB production pathway from the BAC instead of a multi-copy plasmid. Finally, the BAC was shown to be a useful tool for the optimization of enzyme expression levels in metabolic pathways. While directly beneficial for 3HB production, the methods and strategies employed in this thesis are broadly applicable to increase the economic viability of microbial biorefineries.
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| 27. |
- Sjöberg, Gustav, et al.
(författare)
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Evaluation of enzyme-constrained genome-scale model through metabolic engineering of anaerobic co-production of 2,3-butanediol and glycerol by Saccharomyces cerevisiae
- 2024
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 82, s. 49-59
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Tidskriftsartikel (refereegranskat)abstract
- Enzyme-constrained genome-scale models (ecGEMs) have potential to predict phenotypes in a variety of conditions, such as growth rates or carbon sources. This study investigated if ecGEMs can guide metabolic engineering efforts to swap anaerobic redox-neutral ATP-providing pathways in yeast from alcoholic fermentation to equimolar co-production of 2,3-butanediol and glycerol. With proven pathways and low product toxicity, the ecGEM solution space aligned well with observed phenotypes. Since this catabolic pathway provides only one-third of the ATP of alcoholic fermentation (2/3 versus 2 ATP per glucose), the ecGEM predicted a growth decrease from 0.36 h−1 in the reference to 0.175 h−1 in the engineered strain. However, this <3-fold decrease would require the specific glucose consumption rate to increase. Surprisingly, after the pathway swap the engineered strain immediately grew at 0.15 h−1 with a glucose consumption rate of 29 mmol (g CDW)−1 h−1, which was indeed higher than reference (23 mmol (g CDW)−1 h−1) and one of the highest reported for S. cerevisiae. The accompanying 2,3-butanediol- (15.8 mmol (g CDW)−1 h−1) and glycerol (19.6 mmol (g CDW)−1 h−1) production rates were close to predicted values. Proteomics confirmed that this increased consumption rate was facilitated by enzyme reallocation from especially ribosomes (from 25.5 to 18.5 %) towards glycolysis (from 28.7 to 43.5 %). Subsequently, 200 generations of sequential transfer did not improve growth of the engineered strain, showing the use of ecGEMs in predicting opportunity space for laboratory evolution. The observations in this study illustrate both the current potential, as well as future improvements, of ecGEMs as a tool for both metabolic engineering and laboratory evolution.
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| 28. |
- Sjöberg, Gustav, et al.
(författare)
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Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3 hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.
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| 29. |
- Sjöberg, Gustav, et al.
(författare)
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Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome
- 2019
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Ingår i: Journal of Biotechnology. - : ELSEVIER. - 0168-1656 .- 1873-4863. ; 305, s. 43-50
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Tidskriftsartikel (refereegranskat)abstract
- In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3-hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.
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| 30. |
- VanArsdale, Eric, et al.
(författare)
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A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks
- 2020
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:5, s. 1117-1128
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Tidskriftsartikel (refereegranskat)abstract
- There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples and cells, one can both electronically obtain a redox "portrait" of a biological system and, conversely, program gene expression. Here, we have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues, producing an output that can be directly recorded via electronics-but without the need for added redox mediators. The process is robust; two key components must act together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. "Catalytic" transducer cells provide for signal-mediated surface expression of tyrosinase. Additionally, "reagent" transducer cells synthesize and export tyrosine, a substrate for tyrosinase. In cocultures, this system enables real-time electrochemical transduction of cell activating molecular cues. To demonstrate, we eavesdrop on quorum sensing signaling molecules that are secreted by Pseudomonas aeruginosa, N-(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.
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| 31. |
- VanArsdale, Eric, et al.
(författare)
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Development of a tyrosine-tyrosinase it for connecting cellular communication with electronic networks
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- There is a growing interest in mediating information transfer between biology and electronics. By the addition of redox mediators to various samples, one can electronically obtain a redox “portrait” of a biological system and additionally program gene expression in suitably engineered cells. We have created a cell-based synthetic biology-electrochemical axis in which engineered cells process molecular cues producing an output that can be directly recorded via electronics – without added redox mediators. The process is robust; two key components must be together to provide a valid signal. The system builds on the tyrosinase-mediated conversion of tyrosine to L-DOPA and L-DOPAquinone, which are both redox active. “Sensor” cells provide for signal-mediated surface expression of tyrosinase. Similarly, “producer” cells synthesize and export tyrosine. In co-cultures, this system enables real-time electrochemical transduction of the original molecular cues. To demonstrate, we eavesdrop on the quorum sensing molecules from Pseudomonas aeruginosa N-(3-oxododecanoyl)-l-homoserine lactone and pyocyanin.
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| 32. |
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