Dokument: Development of Escherichia coli-based biocatalysts using CRISPR/Cas9-assisted genome engineering
| Titel: | Development of Escherichia coli-based biocatalysts using CRISPR/Cas9-assisted genome engineering | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=71810 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20260209-142644-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Lülf, Uwe Joost [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Urlacher, Vlada B. [Gutachter] Prof. Dr. Schmitt, Lutz [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Since the first steps in biocatalysis, the introduction of molecular biological tools has revolutionized the field many times. The introduction of recombinant DNA has allowed the transfer of foreign genes and thus new functions into fast-growing host organisms like the workhorse Escherichia coli. Furthermore, the properties of enzymes including selectivity, activity, and stability can be changed using protein engineering. In 2012, the discovery and subsequent application of CRISPR/Cas as a programmable genome editing tool enabled scar- less genome engineering including chromosomal integration, deletion and substitution of any DNA fragment ranging from single nucleotides to whole metabolic pathways.
In the course of this dissertation, various applications of CRISPR/Cas-assisted genome engineering of E. coli in the context of the entire spectrum of biocatalysis were examined. This includes applications in fermentation with growing cells, biotransformation with resting cells and catalysis with isolated enzymes. Part of this work focuses on the oxyfunctionalization of non-activated C-H bonds catalyzed by different oxygenases (cytochrome P450 monooxygenases and peroxygenases as well as unspecific peroxygenases), which is of particular interest for organic synthesis. Here, the knockout of two E. coli genes encoding catalases enabled the use of resting cells and cell-free extracts for H2O2-driven biocatalysis with peroxygenases, omitting the need for enzyme purification. Further, the effects of chromosomal integration on the catalytic performance of a multi-component P450 system to produce a human drug metabolite with resting E. coli cells were evaluated. In contrast to plasmid-based expression systems, the use of selectable markers as antibiotic resistance could be avoided after chromosomal integration and a more stable expression could be achieved. In order to facilitate the transfer from plasmid-based to plasmid-free chromosomally integrated biocatalysts, a versatile toolbox was designed and established which can be applied in various E. coli strains.
Apart from oxyfunctionalization reactions catalyzed by a single enzyme, the multi-step synthesis of valuable plant secondary metabolites is a particularly interesting field of application for biocatalysis. Many active pharmaceutical ingredients are derived from scarce natural resources which can be substituted by biotechnological solutions. For this reason, an artificial biosynthetic pathway for the plasmid-free production of the high value key lignan pinoresinol from the relatively inexpensive phenylpropanoid ferulic acid was reconstituted in growing E. coli cells by combining exogenous and endogenous enzymes.
Finally, a proof-of-concept study for combinatorial promoter substitution after chromosomal integration of heterologous genes in E. coli is presented. This aims to address the challenges of potential bottlenecks in the construction of multi-step reaction cascades in vivo.
In summary, this thesis explores various possible biocatalytic applications of CRISPR/Cas- assisted genome engineering in E. coli. These include the prevention of side reactions by gene knockouts, the transfer of plasmid-based to plasmid-free expression systems as well as the reconstitution and optimization of an artificial biosynthetic pathway for biocatalysis both in vivo and in vitro. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Biochemie | |||||||
| Dokument erstellt am: | 09.02.2026 | |||||||
| Dateien geändert am: | 09.02.2026 | |||||||
| Promotionsantrag am: | 22.05.2025 | |||||||
| Datum der Promotion: | 20.11.2025 |
