Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Engineering and applications of surface displayed tyrosinase on Escherichia coli
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.ORCID iD: 0000-0003-1442-6689
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Abstract [sv]

Forskning och utveckling inom bioteknologi har gett det moderna samhället en rad unika biologiska verktyg för att hantera problem inom miljö och folkhälsa. Enzymer är kraftfulla makromolekyler som kan nyttjas inom både miljöteknik och läkemedelsproduktion. Strategier för att presentera proteiner på cellytor kan användas för att utveckla smartare och mer kostnadseffektiva bioprocesser. I denna avhandling utvecklas, optimeras och tillämpas ett system för bakteriellt ytuttryck av enzymet tyrosinas. I den första delen av avhandlingen hanteras metoder för tyrosinas-ytuttryck i Escherichia coli. Studierna visade att man kunde uttrycka ett aktivt yttermembran-förankrat tyrosinas, som kan oxidera aminosyran L-tyrosin till polymeren melanin. För att förbättra det utvecklade uttryckssystemet, med avseende på aktivitet, optimerades de olika komponenterna i expressionsvektorn systematiskt. I slutändan resulterade detta i en E. coli stam med fem gånger högre specifik aktivitet. Den andra delen av avhandlingen är mer fokuserad på att implementera det bakteriella enzymsystemet i miljötekniska situationer. I den etablerade bioprocessen skapades melanintäckta bakterier genom oxidering av L-tyrosin vid cellytan. De melanintäckta bakterierna nyttjades i ett membran-bioreaktorsystem för att, via adsorption, rena vatten från ett läkemedel. En annan applikation som testades var att nyttja tyrosinasytuttryck som en sensor mot utvalda toxiner och bekämpningsmedel. Generna för tyrosinas-ytuttryck integrerades i en krets för att både producera och oxidera tyrosin. Denna ”genetiska krets” skapade ett system för att sammanlänka, vanligtvis otillgänglig, biologisk information till en elektrisk kvantifierbar signal. Sammanfattat så har denna avhandling etablerat nytänkande idéer och koncept som demonstrerar värdet och flexibiliteten i att kunna presentera enzymer på cellytor, samt hur detta kan nyttjas mot både miljöinriktade och industriella användningsområden.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. , p. 77
Series
TRITA-CBH-FOU ; 2019:55
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-263038ISBN: 978-91-7873-340-8 (print)OAI: oai:DiVA.org:kth-263038DiVA, id: diva2:1366082
Public defence
2019-11-29, F3, Lindstedtsvägen 26, Stockholm, 14:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, VR-621-2014-5293
Note

QC 2019-10-28

Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-10-28Bibliographically approved
List of papers
1. Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior
Open this publication in new window or tab >>Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior
Show others...
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 36117Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
Keywords
Expression, Autotransporter, Purification, Tyrosinase, Proteins, Enzymes, Design, Gene
National Category
Bioprocess Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-194418 (URN)10.1038/srep36117 (DOI)000386212200001 ()2-s2.0-84992597799 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20161031

Available from: 2016-10-27 Created: 2016-10-27 Last updated: 2019-10-28Bibliographically approved
2. Molecular optimization of autotransporter-based tyrosinase surface display
Open this publication in new window or tab >>Molecular optimization of autotransporter-based tyrosinase surface display
2019 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1862, no 2, p. 486-494Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Autotransport, Whole-cell biocatalysis, Tyrosinase, Protein engineering, Escherichia coli
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-244107 (URN)10.1016/j.bbamem.2018.11.012 (DOI)000456764100014 ()30521785 (PubMedID)2-s2.0-85058059281 (Scopus ID)
Funder
Swedish Research Council, VR-621-2014-5293
Note

QC 20190219

Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-10-28Bibliographically approved
3. Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
Open this publication in new window or tab >>Continuous removal of the model pharmaceutical chloroquine from water using melanin-covered Escherichia coli in a membrane bioreactor
Show others...
2019 (English)In: Journal of Hazardous Materials, ISSN 0304-3894, E-ISSN 1873-3336, Vol. 365, p. 74-80Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Wastewater treatment, Pharmaceuticals, Membrane bioreactor, Adsorption, Surface expression
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-244080 (URN)10.1016/j.jhazmat.2018.10.081 (DOI)000456761000009 ()30412809 (PubMedID)2-s2.0-85055974597 (Scopus ID)
Note

QC 20190219

Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-10-28Bibliographically approved
4. Development of a tyrosine-tyrosinase it for connecting cellular communication with electronic networks
Open this publication in new window or tab >>Development of a tyrosine-tyrosinase it for connecting cellular communication with electronic networks
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-263036 (URN)
Note

QC 20191115

Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-11-15Bibliographically approved

Open Access in DiVA

fulltext(6606 kB)33 downloads
File information
File name FULLTEXT01.pdfFile size 6606 kBChecksum SHA-512
d19119f9b3b9bc9b1d94b142be65ab2be96540a0e68135235772bdf02eb5f26ee6ebcf0aa1aa928a0b54235fc339d5d9f7c361242eafa135d67694141849d977
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Hörnström, David
By organisation
Industrial Biotechnology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 33 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 231 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf