Engineering 2‐oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by optimizing consecutive components

Abstract Multienzyme complexes have the potential for green catalysis of sequential reactions. The Escherichia coli 2‐oxoglutarate dehydrogenase complex (OGDHc) was converted from a 2‐ oxoglutarate dehydrogenase to a 2‐oxo aliphatic dehydrogenase complex by engineering consecutive components. OGDHc catalyzes succinyl‐CoA synthesis in the Krebs cycle. OGDHc is composed of three components: E1o, 2‐oxoglutarate dehydrogenase; E2o, dihydrolipoylsuccinyl transferase; E3, dihydrolipoyl dehydrogenase. There are three substrate checkpoints. One is in E1o and two in E2o. OGDHc was reprogrammed to accept alternative substrates by evolving the E1o and E2o components. Wt‐ODGHc does not accept aliphatic substrates. E1o was previously engineered to accept a non‐natural aliphatic substrate, 2‐oxovalerate (2‐OV). E2o also required engineering to accept 2‐OV in the overall reaction. Hence, saturation mutagenesis libraries of E2o were screened for 2‐OV activity. E2o‐S333M, E2o‐H348F, E2o‐H348Q, and E2o‐H348Y were identified to show activity for 2‐OV in the reconstituted complex. Variants also displayed activity for larger aliphatic substrates.