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CETCH Cycle

The crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a synthetic carbon fixation pathway created through biological retrosynthesis. The most prevalent carbon fixation pathway in nature is the Calvin-Benson-Bassham (CBB) cycle, which uses RuBisCo as the predominant enzyme to fix carbon. RuBisCo is the most abundant enzyme on earth, however, it arose during a time when there was no oxygen. In the presence of oxygen, RuBisCo participates in side reactions which reduce its efficiency at fixing carbon. The CETCH cycle pathway was designed to be a more efficient and controlled way to fix carbon. It was designed in a bottom-up process, where scientists created a variety of theoretical cycles, piecing together enzymatic reactions that they thought could lead to an efficient carbon fixation pathway. The CETCH cycle was analyzed and deemed the most feasible pathway to synthesize. The first model of the pathway had 17 enzymes derived from 9 different organisms spanning all three domains. The initial test was a successful as a proof of principle. There were bottlenecks and low affinity for some substrates. The pathway was optimized by enzyme engineering. One example mutated an enzyme to increase oxygen tolerance making it more suitable for the environment. A second example replaced an enzyme with another that had a higher affinity for the substrate. The purpose of engineering a new cycle was to increase current CO₂ fixation rates. The CETCH cycle has many potential biotechnological applications. In vivo transplant of CETCH cycle into lithotrophic or photosynthetic organisms could improve CO₂ fixation. It could be used to develop artificial photosynthetic processes and or combined with photovoltaics or artificial leaves. It could also aid in the design of self sustained, completely synthetic carbon metabolism for artificial or minimal cells. The CETCH cycle continues to be improved and studied.

Metabolic Retrosynthesis Process

Synthetic biology techniques allow for the recombination of enzymes and their respective functions from different organisms. The synthetic pathways are thermodynamically and or kinetically favorable. Schwander et al. identified that carboxylating enzyme efficiency was a limiting step of the pathway. They decided to focus on the coenzyme A (CoA)–dependent carboxylases, and enoyl-CoA carboxylases/reductases (ECRs). Enzymes were identified through use of the Enzyme Commission Numbers assigned by the International Union of Biochemistry and Molecular Biology.

These enzymes were purified, tested, and characterized before used in testing. To test the experimental design cofactors and intermediates were synthesized.  Many of the coA-thioesters were synthesized from their anhydrides and the other enzymes were extracted by expressing them in different E-coli strains and then purifying them. The enzymes were added stepwise and activity assays were used to determine the enzyme functionality.

The crotonyl–coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle

The CETCH cycle is a synthetic carbon fixation pathway created through biological retrosynthesis. The cycle was published as a success in vitro, but has yet to have published in vivo success. It was created in 2018 by Schwander et al. looking to improve the efficiency of carbon fixation. The cycle includes 17 different enzymes from 9 different organisms spanning all three domains.