Publikationsserver
Implementation of CO2 fixation pathways into Methylorubrum extorquens AM1
Growth is a prerequisite for life and metabolism plays a central role to achieve and sustain growth. Its function is to obtain energy needed for metabolic (inter)conversions as well as converting nutrients into cellular building blocks used to synthesize macromolecules and thereby the cell itself. T...
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Beteiligte: | |
| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2020
|
| Schlagworte: | |
| Online Zugang: | PDF-Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Zusammenfassung: | Growth is a prerequisite for life and metabolism plays a central role to achieve and sustain growth. Its function is to obtain energy needed for metabolic (inter)conversions as well as converting nutrients into cellular building blocks used to synthesize macromolecules and thereby the cell itself. The metabolic pathways present in an organism define and limit its growth capabilities. Nowadays, an organism’s core metabolic network might be altered with relative ease through genetic engineering. The modification of biological systems to expand their growth potential was at the core of this thesis with an emphasis on the Alphaproteobacterium Methylorubrum extorquens AM1.
The following results were achieved:
1. Novel inducible promoters were designed for M. extorquens AM1 based on the LacI lacO system that are tight before induction and exhibit good dynamic range. The expression level achieved with these promoters exceeds that of the standard PmxaF promoter making them the strongest promoters described to date for this organism. New extrachromosomal elements termed ‘mini-chromosomes’ based on repABC cassettes were also identified, which maintain a stable unit copy number and faithful inheritance even in the absence of selection. These new DNA vehicles are compatible with each other and with the broad host plasmid used for M. extorquens AM1 so far. A conditionally unstable replicon, Mex CM4, showed the behavior needed to establish CRISPR Cas and similar techniques for which transient expression and fast extinction of the replicon are prerequisites.
2. The Calvin-Benson-Bassham (CBB) cycle was introduced into M. extorquens AM1 for heterologous CO2 fixation and assimilation of methanol was disrupted to provide the cell with energy consistent with (chemo)organoautotrophic growth. The operation of a functional CBB cycle was confirmed with 13C tracer analysis in the engineered strain. Furthermore, a positive growth phenotype and increased cell viability was found dependent on a functional RubisCO.
3. M. extorquens AM1 was rationally engineered and further evolved by serial transfer to improve growth on the novel substrate ribose. Mutations throughout the evolutionary landscape were identified by whole genome (re)sequencing and further characterized genetically and biochemically. The CBB cycle was introduced into ribose evolved clones for simultaneous CO2 fixation and acetate was provided for additional energy. These strains were evolved in semi chemostat conditions to promote carbon fixation via the CBB cycle.
4. In vivo implementation of a synthetic CO2 fixation cycle, the crotonyl CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, was explored in M. extorquens AM1. Suitable candidate enzymes were identified and characterized biochemically for every step of the crotonyl CoA regeneration module. These new candidates were combined to close the CETCH cycle and various M. extorquens AM1 strains were created to test growth based selection schemes. |
|---|---|
| Umfang: | 196 Seiten |
| DOI: | 10.17192/z2020.0473 |