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

Joydeep Chakraborty, Natalia S. Nemeria, Xu Zhang, Pradeep R. Nareddy, Michal Szostak, Edgardo Farinas, Frank Jordan

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere16769
JournalAIChE Journal
Volume66
Issue number3
DOIs
StatePublished - Mar 1 2020

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All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Environmental Engineering
  • Chemical Engineering(all)

Keywords

  • directed evolution
  • green chemistry
  • multienzyme complex
  • protein engineering
  • thioester synthesis

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