Lysine butyrylation modulates central carbon metabolism and photosynthesis in cyanobacteria.

Cyanobacteria, evolutionarily ancient photoautotrophs, serve as key model organisms for studying diverse biological processes. Lysine butyrylation (Kbu) is an emerging post-translational modification implicated in diverse biological processes from bacteria to mammals. However, the distribution and functional significance of Kbu in cyanobacteria have yet to be elucidated. In this study, we present the first proteome-wide analysis of Kbu in the model cyanobacterium Synechococcus sp. strain PCC 7002 using peptide pre-fractionation, butyrylation antibody enrichment and high-accuracy mass spectrometry analysis. In total, we identified 132 high-confidence Kbu sites across 93 proteins in Syn7002. These butyrylated proteins were predominantly enriched in carbon metabolism and photosynthesis pathways, which are tightly interconnected in cyanobacteria. Immunoblotting revealed that global Kbu levels in Syn7002 varied dynamically under different growth conditions, and the change in Kbu levels of the allophycocyanin α subunit were further confirmed using parallel reaction monitoring. Among the butyrylated metabolic proteins, phosphoenolpyruvate synthase (PPSA) was found to be butyrylated at the K143 residue. By employing genetic code expansion to site-specifically incorporate Nε-butyryl-lysine at position 143 (K143bu), we demonstrated that butyrylation at this site significantly affects the enzymatic activity of PPSA in vitro. Consistently, the K143R mutant of PPSA in Syn7002 displayed impaired growth and reduced photosynthetic activity under high light conditions compared with the wild type, suggesting a functional role for Kbu in vivo. Together, our results expand the understanding of Kbu and suggest its potential involvement in regulating cyanobacterial carbon metabolism and photosynthesis.