Unexpected extensive lysine acetylation in the trump-card antibiotic producer Streptomyces roseosporus revealed by proteome-wide profiling

Lysine acetylation is emerging as a ubiquitous and conserved posttranslational modification in living cells. While the role of lysine acetylation in regulating primary metabolism is well-established, its function in secondary metabolism remains largely elusive. To gain insight into the nature, extent and biological function of lysine acetylation in Streptomyces reseosporus, a producer of daptomycin, dubiously dubbed as the trump card antibiotic, we used immunoaffinity-based acetyllysine peptide enrichment integrated with high resolution mass spectrometry to comprehensively characterize lysine acetylated proteins in this microbe. We identified a total of 667 proteins with 1143 unique sites, representing the largest acetylproteome reported to date in bacteria. Acetylated proteins belong to various functional classes such as metabolism and gene expression according to the gene ontology. We demonstrated for the first time that proteins involved in the biosynthesis of diverse secondary metabolites are acetylated, such as a nonribosomal peptide synthetase, enzymes essential for hydroxamate siderophore and phosphinic acid natural products biosynthesis, implying an important role of acetylation in these processes. Taken together, this proteomic analysis revealed a surprising breadth of cellular processes affected by lysine acetylation and also furnishes some fresh intervention nodes for the rational improvement of the antibiotic producer.

Biological significance: Despite considerable efforts have been devoted to elucidating the mechanism underlying secondary metabolism in Streptomyces, which are prolific producers of secondary metabolites with diverse biological activities, such as bacteriocides and antitumors, the full map of regulation and corresponding network is still far from perfect. Protein lysine acetylation is an evolutionarily conserved protein post-translational modification, abundantly existing in proteins with diverse biological context. We took advantage of integrated high throughput PTM proteomics followed by intensive bioinformatic analysis to profile lysine acetylome of Streptomyces roseosporus. In total, 1134 unique Kac sites in 667 lysine acetylated substrates were identified, representing the largest aceylomics in prokaryotes to date. Significantly, a nonribosomal peptide synthetase, an enzyme essential for hydroxamate siderophore and phosphinic acid natural products biosynthesis, was found to be acetylated. Given the conservation of these enzymes in biosynthesis of diverse secondary metabolites, lysine acetylation likely plays an important role in regulating secondary metabolism in Streptomyces.