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New central carbon metabolic pathways in Alphaproteobacteria: from natural to synthetic metabolism
CO2 is one of the main greenhouse gases driving global warming, and plays a fundamental role in the global carbon cycle. The threat posed by climate change needs to be addressed during this century, if its worst effects are to be prevented or at least mitigated. While new (bio)technological solutio...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2022
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| Online Zugang: | PDF-Volltext |
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| Zusammenfassung: | CO2 is one of the main greenhouse gases driving global warming, and plays a fundamental role in the global carbon cycle. The threat posed by climate change needs to be addressed during this century, if its worst effects are to be prevented or at least mitigated.
While new (bio)technological solutions are essential to successfully face this herculean challenge, continuously improving the understanding of the sources, sinks and fluxes of the global carbon cycle is required as well, in order to accurately build models and predict the effects of human intervention on the climate. This thesis aims at contributing to both approaches, i. e. furthering knowledge of the global carbon cycle, and providing biotechnological tools to convert CO2 into value-added products.
The first part of this thesis describes the β-hydroxyaspartate cycle, a glyoxylate assimilation pathway that was originally proposed nearly 60 years ago, but until now never fully elucidated. The gene cluster encoding the regulator and the four enzymes of the cycle was identified in Paracoccus denitrificans, and their biochemical properties were characterised. In the process, a rare primary imine reductase was discovered, structurally elucidated and assigned to a novel family within the ornithine cyclodeaminase/µ-crystalline superfamily. Additional phylogenetics analyses revealed that the β-hydroxyaspartate cycle is widespread among marine Alphaproteobacteria, especially in the Rhizobiales and Rhodobacterales orders, and is also ubiquitously present in oceanic metagenomes, suggesting that it fulfills a relevant ecological role in this habitat. Field studies supported this hypothesis and established that the β-hydroxyaspartate cycle is involved in assimilation of glycolate, an abundant phytoplankton exudate, by heterotrophic marine bacteria, therefore revealing a new dimension of the marine carbon cycle.
The second part of this thesis focuses on re-engineering the central carbon metabolism of Methylorubrum extorquens AM1 for production of valuable compounds from methanol, which can be sustainably obtained from CO2 and hydrogen. In order to access acyl-CoAester intermediates from the ethylmalonyl-CoA pathway, which is essential for growth on C1 and C2 compounds, rewiring of the metabolic network of M. extorquens AM1 was undertaken. Heterologous implementation of the glyoxylate cycle, followed by adaptive laboratory evolution, yielded a strain which was able to growth on methanol without using the native ethylmalonyl-CoA pathway. Characterisation of the engineered and evolved strain by whole-genome resequencing, proteomics and targeted metabolomics revealed that two mutations in the isocitrate dehydrogenase putative promoter region and coding sequence were responsible for the overall rebalancing of the cellular carbon flux from the TCA cycle towards the glyoxylate cycle. Furthermore, proof-of-principle production of crotonic acid from methanol and acetate was demonstrated, and the key bottlenecks of the process, including undesired flux into polyhydroxybutyrate synthesis, were identified. |
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| Umfang: | 141 Seiten |
| DOI: | 10.17192/z2022.0099 |