Full title: Gene Network Centrality Analysis Identifies Key Regulators Coordinating Day-Night Metabolic Transitions in Synechococcus elongatus PCC 7942 Despite Limited Accuracy in Predicting Direct Regulator-Gene Interactions

Zachary Johnson ^1,2 David Anderson ¹ Margaret Cheung ^3,4 Pavlo Bohutskyi ^1,2*

• ¹ Division of Biological Science, Pacific Northwest National Laboratory (DOE), Richland, United States
• ² Washington State University, Pullman, Washington, United States
• ³ Environmental Molecular Sciences Laboratory, Environmental Molecular Sciences Division, Pacific Northwest National Laboratory (DOE), Richland, Washington, United States
• ⁴ Department of Physics, College of Arts and Sciences, University of Washington, Seattle, Washington, United States

Synechococcus elongatus PCC 7942 is a model organism for studying circadian regulation and bioproduction, where precise temporal control of metabolism significantly impacts photosynthetic efficiency and CO2-to-bioproduct conversion. Despite extensive research on core clock components, our understanding of the broader regulatory network orchestrating genome-wide metabolic transitions remains incomplete. We address this gap by applying machine learning tools and network analysis to investigate the transcriptional architecture governing circadian-controlled gene expression. While our approach showed moderate accuracy in predicting individual transcription factor-gene interactions -a common challenge with real expression data -network-level topological analysis successfully revealed the organizational principles of circadian regulation. Our analysis identified distinct regulatory modules coordinating daynight metabolic transitions, with photosynthesis and carbon/nitrogen metabolism controlled by day-phase regulators, while nighttime modules orchestrate glycogen mobilization and redox metabolism. Through network centrality analysis, we identified potentially significant but previously understudied transcriptional regulators: HimA as a putative DNA architecture regulator, and TetR and SrrB as potential coordinators of nighttime metabolism, working alongside established global regulators RpaA and RpaB. This work demonstrates how network-level analysis can extract biologically meaningful insights despite limitations in predicting direct regulatory interactions. The regulatory principles uncovered here advance our understanding of how cyanobacteria coordinate complex metabolic transitions and may inform metabolic engineering strategies for enhanced photosynthetic bioproduction from CO2.