Dissecting the metabolic plasticity of Actinobacteria

• Applicants: Professor Dr. Michael Bott; Professor Dr. Bernhard Eikmanns
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 406563304

Actinobacteria form one of the most ancient and diverse bacterial phyla and include important human pathogens like Mycobacterium tuberculosis, invaluable sources of antibiotics like Streptomyces, and major industrial cell factories like Corynebacterium glutamicum. There is a strong interest and an increasing need to improve our understanding of the actinobacterial molecular physiology, both for therapeutic and biotechnological purposes, as present knowledge mostly comes from non-actinobacterial species like Escherichia coli or Bacillus subtilis. Recent studies have uncovered unique features in the way how key enzymes in metabolism are structurally organized and regulated in Actinobacteria. In the METACTINO proposal, we use an integrative approach combining molecular microbiology, protein biochemistry, and structural biology to obtain a detailed understanding of the molecular mechanisms and signalling pathways that control carbon flux at the pyruvate and 2-oxoglutarate nodes in Actinobacteria, using C. glutamicum as model organism. Previous studies of the applicants revealed that the pyruvate dehydrogenase complex (PDH) and the 2-oxoglutarate dehydrogenase complex (ODH) form a unique supercomplex with major structural differences to known PDHs and ODHs. Furthermore, ODH activity was found to be inhibited by a FHA domain-containing protein, called OdhI in Corynebacterium and GarA in Mycobacterium, with the effect of directing 2-oxoglutarate flux towards L-glutamate and nitrogen assimilation. Phosphorylation of OdhI/GarA by Ser/Thr protein kinases, in particular PknG, prevents inhibition of ODH, but the signals triggering phosphorylation are poorly understood. In the first topic of METACTINO, the objective is therefore to decipher the signal transduction cascade that controls PknG activity, and thus the OdhI phosphorylation status. In the second topic targeting the pyruvate node, we aim to identify allosteric effectors controlling PDH activity and study the cross-influence of these effectors on ODH activity, and likewise analyse the effect of allosteric ODH effectors on PDH. Furthermore, the physiological function and regulation of pyruvate: menaquinone oxidoreductase (PQO) will be studied, an enigmatic enzyme enabling an alternative route for pyruvate oxidation. The third topic aims at an integrative structural analysis of the PDH-ODH supercomplex in order to unravel the mechanistic basis of allosteric and phosphorylation-mediated regulation of pyruvate and 2-oxoglutarate oxidation by a single complex. In addition, structural analysis of actinobacterial PQO is addressed in this topic. The results obtained in METACTINO will offer new targets for the rational improvement of metabolite production with the cell factories Corynebacterium and Streptomyces and they will pave the way to novel therapeutic approaches against M. tuberculosis, which metabolic plasticity is key to maintain infection in the human host.

• DFG Programme: Research Grants
• International Connection: France
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Privatdozent Marco Bellinzoni, Ph.D.