title: Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth.
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.
authors: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. matt.mattozzi@wyss.harvard.edu
authors: Matthew d
authors: Md
authors: Mattozzi
authors: Marika
authors: M
authors: Ziesack
authors: Mathias J
authors: MJ
authors: Voges
authors: Pamela A
authors: PA
authors: Silver
authors: Jeffrey C
authors: JC
authors: Way
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D041861:Chloroflexus / Q000201:enzymology / Q000235:genetics*; D004798:Enzymes / Q000096:biosynthesis* / Q000235:genetics; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004798:Enzymes; D011994:Recombinant Proteins; D019344:Lactic Acid; C031601:hydracrylic acid