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The enzymes of Krebs tricarboxylic acid (TCA) cycle form multi‐enzyme complexes mediating metabolite (substrate) channeling. As metabolite channeling can enhance the pathway reactions, the multi‐enzyme complexes are postulated to regulate metabolic flux by dynamic association and dissociation. Especially, the interaction between malate dehydrogenase (MDH) and citrate synthase (CS) is expected to have significant impact on the TCA cycle flux since the forward reaction of MDH to produce oxaloacetate from malate is thermodynamically infeasible with physiological metabolite concentrations. However, the dynamics of this multi‐enzyme complex and its relationship with metabolic pathway fluxes have not been demonstrated in vivo. To this end, plate reader‐based split‐luciferase assay was developed to monitor the dynamics of the protein‐protein interaction in living yeast cells and was applied to observe the dynamics of MDH/CS multi‐enzyme complex during the transition of metabolic status. The sequences coding N‐ and C‐ terminus of highly sensitive NanoLUC luciferase were introduced into the yeast genome by homologous recombination to fuse these peptides to the C‐terminus of CS (CIT1) and MDH (MDH1) enzymes, respectively. The resulted yeast strain showed decent fluorescent signals while wild‐type cells showed no background signal. The substrate concentration and cell concentration in the culture were optimized to obtain constant signals for 120 min of measurement. Using this system, the interaction of MDH1/CIT1 complex was monitored following the changes of growth condition which are expected to induce the alteration in the TCA cycle metabolic flux. In the preliminary results, we observed the alteration of luminescence signal in some conditions which are known to modulate the TCA cycle flux. We are currently testing various conditions to analyze the relationship between the dynamics of the multi‐enzyme complex and metabolic fluxes in the TCA cycle and adjacent pathways. We are also trying to identify the factors affecting the interaction of the enzymes. Support or Funding Information This study is supported by National Science Foundation, Faculty Early Career Development Program 1845451 to T.O.

WhiB‐like (Wbl) proteins are a unique group of iron‐sulfur cluster ([4Fe4S])‐containing transcription factors exclusive to actinobacteria. They play crucial roles in the virulence, survival, and propagation of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Seven Wbl proteins (WhiB1‐WhiB7) are found in Mtbwith diverse regulatory roles. All Mtb Wbl proteins except for WhiB5 have been shown to interact with the conserved region 4 of the primary sigma factor (SigAr4) in RNA polymerase holoenzyme. Our recent structural and biochemical analyses indicate that two Wbl proteins, WhiB1 and WhiB7, bind to the same site on SigAr4 unexpectedly through tight hydrophobic interactions involving several conserved aromatic residues of the Wbl proteins and His516 of SigAr4. Substitution of these aromatic residues in either WhiB1 or WhiB7 at the molecular interface abolishes their binding to SigAr4. Here, we hypothesize that all Mtb Wbl proteins share a similar molecular interface when in complex with SigAr4, based on the primary sequence analysis and structural modeling. Using the site‐directed mutagenesis and the in vitro protein‐protein interaction assays, we confirm that all Mtb Wbl proteins, including WhiB5, bind to the same site on SigA centered on His516, and the conserved aromatic residues within the Fe‐S cluster binding pocket are required for SigA binding. The results from this study will offer insights into how the Wbl proteins are utilized in Mtb to orchestra gene expression in response to environmental cues in the host.

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