| 1. |
- Gustavsson, Martin, 1984-, et al.
(författare)
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Biocatalysis on the surface of Escherichia coli : melanin pigmentation of the cell exterior
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.
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| 2. |
- Hörnström, David, 1988-
(författare)
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Engineering and applications of surface displayed tyrosinase on Escherichia coli
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The rise of biotechnology has provided a toolbox to deal with major challenges related to pollution and health. Microbial enzymes constitute powerful macromolecules with applications in environmental technology, and industrial and medical production. The display of enzymes on cellular surfaces promotes external access to reactants, thereby simplifying production and cost-effectiveness of bioprocesses. To this end, a system for the surface display of the oxidative enzyme tyrosinase was developed, optimized and implemented. The first part of the thesis focused on developing tyrosinase surface-display via autotransport-based secretion in Escherichia coli. Initially, the presence of active surface-displayed tyrosinase, catalyzing the oxidation, of L-tyrosine was verified. Next, the components of the surface expression system were systematically engineered to yield an optimized tyrosinase-displaying strain with five times higher biomass-specific tyrosinase activity. The second half of the thesis applied the surface-displayed tyrosinase for wastewater treatment and biosensor development. It was found that the catalyzed oxidation of L- tyrosine resulted in the deposition of melanin at the E. coli cell surface. The resulting melanized cells were used in a membrane bioreactor for adsorption of the pharmaceutical chloroquine from an aqueous solution, with a specific binding capacity of 140 mg/g cells and allowed simple cell regeneration by lowering the pH. In a second application, the tyrosinase- display system was integrated into a genetic circuit with regulated oxidation and production of L-tyrosine in response to specific toxins. By employing the resulting cells in an electrochemical cell, the circuit generated a means to directly and selectively link biological information to an electrical output. Overall, the results in this thesis highlight the functionality of the surface expression methodology and demonstrates its versatile applicability.
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| 3. |
- Hörnström, David, et al.
(författare)
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Molecular optimization of autotransporter-based tyrosinase surface display
- 2019
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : ELSEVIER SCIENCE BV. - 0005-2736 .- 1879-2642. ; 1862:2, s. 486-494
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Tidskriftsartikel (refereegranskat)abstract
- Display of recombinant enzymes on the cell surface of Gram-negative bacteria is a desirable feature with applications in whole-cell biocatalysis, affinity screening and degradation of environmental pollutants. One common technique for recombinant protein display on the Escherichia colt surface is autotransport. Successful autotransport of an enzyme largely depends on the following: (1) the size, sequence and structure of the displayed protein, (2) the cultivation conditions, and (3) the choice of the autotransporter expression system. Common problems with autotransporter-mediated surface display include low expression levels and truncated fusion proteins, which both limit the cell-specific activity. The present study investigated an autotransporter expression system for improved display of tyrosinase on the surface of E. coli by evaluating different variants of the autotransporter vector including: promoter region, signal peptide, the recombinant passenger, linker regions, and the autotransporter translocation unit itself. The impact of these changes on translocation to the cell surface was monitored by the cell-specific activity as well as antibody-based flow cytometric analysis of full-length and degraded passenger. Applying these strategies, the amount of displayed full-length tyrosinase on the cell surface was increased, resulting in an overall 5-fold increase of activity as compared to the initial autotransport expression system. Surprisingly, heterologous expression using 7 different translocation units all resulted in functional expression and only differed 1.6-fold in activity. This study provides a basis for broadening of the range of proteins that can be surface displayed and the development of new autotransporter-based processes in industrial-scale whole-cell biocatalysis.
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| 4. |
- Lindroos, Magnus, et al.
(författare)
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Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
- 2019
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Ingår i: Journal of Hazardous Materials. - : ELSEVIER SCIENCE BV. - 0304-3894 .- 1873-3336. ; 365, s. 74-80
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Tidskriftsartikel (refereegranskat)abstract
- Environmental release and accumulation of pharmaceuticals and personal care products is a global concern in view of increased awareness of ecotoxicological effects. Adsorbent properties make the biopolymer melanin an interesting alternative to remove micropollutants from water. Recently, tyrosinase-surface-displaying Escherichia coli was shown to be an interesting self-replicating production system for melanin-covered cells for batch-wise absorption of the model pharmaceutical chloroquine. This work explores the suitability of these melanin-covered E. coli for the continuous removal of pharmaceuticals from wastewater. A continuous-flow membrane bioreactor containing melanized E. coli cells was used for adsorption of chloroquine from the influent until saturation and subsequent regeneration. At a low loading of cells (10 g/L) and high influent concentration of chloroquine (0.1 mM), chloroquine adsorbed until saturation after 26 +/- 2 treated reactor volumes (39 +/- 3 L). The average effluent concentration during the first 20 h was 0.0018 mM, corresponding to 98.2% removal. Up to 140 +/- 6 mg chloroquine bound per gram of cells following mixed homo- and heterogeneous adsorption kinetics. In situ low pH regeneration released all chloroquine without apparent capacity loss over three consecutive cycles. This shows the potential of melanized cells for treatment of conventional wastewater or highly concentrated upstream sources such as hospitals or manufacturing sites.
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| 5. |
- Pöschel, Laura, et al.
(författare)
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Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
- 2023
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Nature. - 0175-7598 .- 1432-0614. ; 107:20, s. 6219-6236
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches. Key points:• Substitutions at position F35 of YciAHI changed the productivity of YciA-based release of carboxylic acid products in M. extorquens AM1 and E. coli.• YciAHI F35N and F35L are improved variants for dicarboxylic production of 2-methylsuccinic acid and mesaconic acid with M. extorquens AM1.• In vitro enzyme assays did not reveal superior properties of the optimized protein variants.
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| 6. |
- 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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| 7. |
- 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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