Toward Heterologous Carbon Fixation in E. Coli with the Chloroflexus 3-Hydroxypropionate Pathway

We aim to produce hydrocarbons using methods theoretically more efficient than natural photosynthesis. In an analogous process, our E. coli cells will fix CO2 and gain their reducing equivalents from a green electrical source. The most common CO2 fixation pathway, the Calvin cycle, depends on the slow enzyme Rubisco (kcat ~ 3/sec). Land plants and cyanobacteria make up for this activity by overexpressing it; in some environments it can make up to 30% of the protein by mass (Ellis 1979). The process is oxygen-sensitive; Rubisco is usually sequestered into organelles. Chloroflexus urantiacus lives commensally in hot springs, and fixes carbon with a unique bicyclic pathway that is insensitive to oxygen and potentially faster (Zarzycki et al2009). We have divided engineering the bicycle into four key pathways: 1) Carbon fixation via fatty acid synthesis; 2) Carbon fixation by propionyl-CoA; 3) Glyoxylate production; and 4) Glyoxylate/propionate assimilation. Through metabolic engineering techniques we have expressed 13 heterologous enzymes separately in E. coli. We demonstrate function of each of the pathways by the use of a novel propionate biosensor (pathway 1), complementation of relevant knockouts for growth on propionate (2, 4) or diaminopimelic acid (3). We are currently integrating all the pathways into a single engineered E. coli strain for autotrophic growth. Future work includes production of a biofuel from these cells, formation of an integrated bioreactor system in which the cells are provided with reducing equivalents from an electrical source, and integrating with a plant carbon fixation system.