Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli

• Verfasst von: Mattozzi, Matthew [VerfasserIn]
• Verfasst von: Ziesack, Marika [VerfasserIn]
• Verfasst von: Voges, Mathias J. [VerfasserIn]
• Verfasst von: Silver, Pamela A. [VerfasserIn]
• Verfasst von: Way, Jeffrey C. [VerfasserIn]
• Titel: Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli
• Titelzusatz: toward horizontal transfer of autotrophic growth
• Verf.angabe: Matthew d. Mattozzi, Marika Ziesack, Mathias J. Voges, Pamela A. Silver, Jeffrey C. Way
• E-Jahr: 2013
• Jahr: 29 January 2013
• Umfang: 10 S.
• Teil: volume:16
• Teil: year:2013
• Teil: month:03
• Teil: pages:130-139
• Teil: extent:10
• Fussnoten: Gesehen am 29.06.2021
• Titel Quelle: Enthalten in: Metabolic engineering
• Ort Quelle: Orlando, Fla. : Academic Press, 1999
• Jahr Quelle: 2013
• Band/Heft Quelle: 16(2013) vom: März, Seite 130-139
• ISSN Quelle: 1096-7184
• DOI: doi:10.1016/j.ymben.2013.01.005
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: 3-hydroxypropionate
• Sach-SW: Carbon fixation
• Sach-SW: Diaminopimelic acid
• Sach-SW: Metabolic engineering
• Sach-SW: Propionate biosensor
• Sach-SW: Propionate toxicity
• K10plus-PPN: 176146390X

Abstract

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.