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
Development of a tyrosine-tyrosinase it for connecting cellular communication with electronic networks
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
Show others and affiliations
(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: urn:nbn:se:kth:diva-263036OAI: oai:DiVA.org:kth-263036DiVA, id: diva2:1366069
Note

QC 20191115

Available from: 2019-10-28 Created: 2019-10-28 Last updated: 2019-11-15Bibliographically approved
In thesis
1. Engineering and applications of surface displayed tyrosinase on Escherichia coli
Open this publication in new window or tab >>Engineering and applications of surface displayed tyrosinase on Escherichia coli
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:nbn:se:kth:diva-263038 (URN)978-91-7873-340-8 (ISBN)
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

Open Access in DiVA

No full text in DiVA

Search in DiVA

By author/editor
Hörnström, DavidSjöberg, GustavVan Maris, Antonius J.
By organisation
Industrial Biotechnology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 109 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