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Carbon dioxide, a greenhouse gas and renewable resource, is unattractive as a feedstock for biocatalysis due to inefficiencies in metabolic pathways for CO2 fixation. The creation of a novel enzyme with the ability to convert formaldehyde to dihydroxyacetone (DHA), a reaction not known to occur naturally, allows the development of a unique CO2 fixation pathway that will feed directly into E. coli central metabolism. Strains expressing this pathway can be modified for the production of fuels such as ethanol, enabling the conversion of CO2 to fuels and decreasing the amount of net CO2 produced by fuel consumption. In collaboration with the Baker group in Biochemistry and the Klavins group in Electrical Engineering, we are engineering such a strain that can transform CO2 and glycerol to ethanol.
The novel enzyme, formolase, was created by the Baker group using their computational protein design program, Rosetta. Work is continuing to increase catalytic efficiency of this enzyme.
The proposed pathway utilizes formolase for the conversion of CO2 to ethanol or other fuels. First, CO2 is converted to formaldehyde and then to DHA in four steps: (1) carbonic anhydrase, (2) formate dehydrogenase, (3) acetyl CoA synthase and acylating acetaldehyde dehydrogenase), (4) formolase. Next, DHA and an energy source (in this case, glycerol) are converted to ethanol and CO2. The overall pathway is redox balanced by the conversion of glycerol to DHA, which supplies the necessary 6 reducing equivalents. This CO2 fixation pathway is more economical energetically than other CO2 fixation pathways such as the Calvin-Benson cycle and the reverse Krebs cycle, which require 6 reducing equivalents and 8 ATPs and 6 reducing equivalents and 1.5 ATP, respectively. Future work would replace glycerol with H2, further reducing CO2 production. For the conversion of CO2 and glycerol to ethanol, the proposed pathway can be combined with an engineered E. coli strain that requires 1 NADH consumed per ethanol created [1]. Compared to ethanol production from glucose, this pathway represents 14% increase in theoretical ethanol yield to 58% (g/g) with a 34% decrease in CO2 release to 32% (g/g). The construction and optimization of this pathway will be guided by metabolite flux and 13C labeling experiments. In addition, strain evolution will be utilized. The Klavins group has developed a turbidostat that will enable the placement of selective pressure on the strain, causing it to evolve such that a robust strain is generated capable of chemical production.
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