Evidence for the Formation of Metabolons Surrounding the Pyruvate Node in the Supercritical CO<sub>2</sub>‑Tolerant <i>Priestia megaterium</i> SR7

Carbon fixation and metabolic efficiency in extremophiles offer promising avenues for sustainable bioproduction. <i>Priestia megaterium</i> SR7 is a supercritical CO₂-tolerant extremophile, whose central carbon metabolism may be particularly well adapted toward efficient heterotrophic CO₂ fixation. However, the regulatory organization of carbon flux in this organism remains unclear, especially around the pyruvate node, a central metabolic hub that directs carbon toward energy generation, metabolic replenishment, and biosynthesis. In this work, we proposed and substantiated the formation of multiple potential metabolons, associated enzyme complexes that channel metabolites between sequential reactions, surrounding the pyruvate node in <i>P. megaterium</i> SR7, which serves as an important regulatory mechanism in central carbon metabolism by enabling the channeling of metabolites toward different pathways. Cross-linking experiments, together with subsequent proteomics, provided <i>in vitro</i> evidence of metabolon formation among phosphoenolpyruvate carboxylase (PPC), NAD+-dependent malic enzyme (NDME), lactate dehydrogenase (LDH), and pyruvate kinase (PYK) protein-protein interactions. <i>In silico</i> simulations of protein-protein interactions revealed possible interacting residues between different proteins. Furthermore, observed dilution of metabolite pools from ¹³C isotopic labeling experiments provides <i>in vivo</i> support for metabolon formation. These insights suggested a possible novel regulatory mechanism and provided additional avenues for future metabolic engineering efforts that leverage enzyme organization to enhance CO₂-linked bioproduction.