Scheme 14.

Artificially developed CETCH cycle applied to the synthesis of malate. The multienzyme cascade employs 17 enzymes catalyzing 13 linear enzymatic steps including two reductive carboxylation steps catalyzed by CCR, and additional auxiliary steps for cofactor recycling. A 520 μL reaction was capable of fixing 1080 μM CO₂ and afforded 540 μM malate in 90 min. ^[23] PCO=propionyl‐CoA oxidase; CCR=crotonyl‐CoA carboxylase/reductase; MCL=β‐methylmalyl‐CoA lyase; MAS=malate synthase. Propionyl‐CoA: 60; acrylyl‐CoA: 70; methylmalonyl‐CoA: 70 a; crotonyl‐CoA: 46; ethylmalonyl‐CoA: 46 a; methylmalyl‐CoA: 71; glyoxylate:72; malate: 73.