Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: toward horizontal transfer of autotrophic growth
The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relat...
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| Hauptverfasser: | , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
29 January 2013
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| In: |
Metabolic engineering
Year: 2013, Jahrgang: 16, Pages: 130-139 |
| ISSN: | 1096-7184 |
| DOI: | 10.1016/j.ymben.2013.01.005 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1016/j.ymben.2013.01.005 Verlag, lizenzpflichtig, Volltext: https://www.sciencedirect.com/science/article/pii/S1096717613000086 
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| Verfasserangaben: | Matthew d. Mattozzi, Marika Ziesack, Mathias J. Voges, Pamela A. Silver, Jeffrey C. Way |
| Zusammenfassung: | The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD− double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC−(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer. |
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| Beschreibung: | Gesehen am 29.06.2021 |
| Beschreibung: | Online Resource |
| ISSN: | 1096-7184 |
| DOI: | 10.1016/j.ymben.2013.01.005 |