Acetylation of lysine residues in proteins is a dynamically regulated, reversible and evolutionarily conserved posttranslational modification mediated by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). To comprehensively characterize acetylated proteins, lysine acetylomes have been determined for a number of eukaryotic and prokaryotic organisms, including human [[1], [2], [3]], Arabidopsis [4,5], Saccharomyces cerevisiae [6], Aspergillus flavus [7,8], and Escherichia coli [9]. These data suggest that protein acetylation plays important roles in many cellular physiological processes, including cell cycle regulation and apoptosis [10]. cell morphology [2], protein synthesis [2,11], and metabolic pathways [[12], [13], [14]]. Thus, protein acetylation has become a key regulator of various physiological functions in organisms and deserves to be studied in depth.

Lysine acetylation can be classified as histone acetylation or nonhistone acetylation [15]. A large body of evidence suggests that histone acetylation plays an important role in transcription [16,17]. For example, acetylation of H3K9 at the FLOWERING LOCUS C (FLC) site enhances its expression, ultimately leading to delayed flowering [18]. Compared with histone acetylation, nonhistone acetylation has gradually become a research topic of considerable interest due to its important regulatory mechanism and extensive applications [[19], [20], [21]]. Acetylation of different proteins can cause different physiological functions, for example, acetylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) increases its enzymatic activity and eventually promotes cell proliferation and tumor formation [22]. Acetylation of heat shock factor 1 (HSF1) inhibits its binding to the HSP70 promoter and accelerates cell damage [23]. Therefore, further identification of novel acetylated target proteins is crucial for resolving the function of acetylation.

Reactive oxygen species (ROS) are important intracellular signaling molecules that are involved in the regulation of a variety of intracellular biological processes, including cell proliferation, metabolism, and stress responses [24,25]. The homeostasis of intracellular ROS plays an important role in the maintenance of normal cellular physiology and metabolism [26]. To ensure that ROS levels are tightly regulated, cells have various antioxidant defense mechanisms. Major antioxidant enzymes include superoxide dismutase, catalase, and glutathione peroxidase. To date, several substrates for the regulation of redox homeostasis by acetylation have been identified [27]. For instance, acetylation positively regulates NADPH oxidase activity and enhances ROS production [28]. The mitochondrial deacetylase SIRT3 increases SOD2 activity through deacetylation, thereby scavenging intracellular ROS [29]. Overall, these data suggest that an important role of changes in the acetylation status of different proteins in regulating intracellular ROS homeostasis. Such studies are vital for further elucidating how organisms regulate ROS homeostasis. Therefore, further studies are needed to discover novel redox-associated proteins regulated by acetylation to determine the effects of acetylation on the ROS signaling pathway.

Ganoderma lucidum is an important medicinal fungus that is widely utilized in Asian countries to improve health and promote longevity [30]. As a secondary metabolite, ganoderic acid (GA) is considered to be the main bioactive component of G. lucidum. With the establishment of a genetic manipulation system in G. lucidum and the elucidation of the mechanism underlying the environmental regulation of GA biosynthesis [31], G. lucidum has become a well-studied model for research on the environmental regulation of secondary metabolite biosynthesis. Our previous study revealed that the deacetylase Glsirt1 has a positive regulatory role in GA biosynthesis, and further studies showed that this regulatory role is related to protein acetylation [32]. Therefore, identifying acetylated target proteins has become the key to resolving the mechanism of GA biosynthesis.

In this work, based on the acetylomics and immunoprecipitation-mass spectrometry, we identified 2802 acetylation-modified proteins, of which GlCAT was further identified as the target protein directly mediated by Glsirt1. Our results demonstrate that Glsirt1 directly deacetylates GlCAT and show that hypoacetylation of GlCAT reduces its activity, causes ROS accumulation and positively regulates the biosynthesis of GA. These findings contribute to a better understanding of the mechanism by which acetylation modulates the ROS signaling pathway and shed light on the environmental regulatory mechanisms of secondary metabolite biosynthesis in G. lucidum.