## 4 Discussion  
Adenoise plays essential roles in physiological processes and is found in most organisms. Adenosine can be used to produce food additives, such as adenosine monophosphate (AMP) and adenosine diphosphate (ADP), demonstrating significant economic potential. Currently, the primary method for adenosine production is microbial fermentation [24]. However, adenosine biosynthesis involves complex metabolic pathways, and a substantial portion of adenosine is further converted into energy carriers (e.g., ATP) or genetic material during synthesis, making its effective accumulation in fermentation broth challenging [25]. To address this, genetic engineering strategies are required to optimize microbial strains by reducing growth or metabolic burdens, necessitating the identification of key regulatory genes and their expression patterns in adenosine biosynthesis. Our findings confirm that supplementing fermentation media with glutamine and hypoxanthine significantly enhances adenosine yield. Hypoxanthine is a hydrolysis product of nucleotides, and glutamine, derived from glucose, can be catabolized into glutamate. Both compounds exhibit relatively straightforward downstream metabolic pathways in bacteria and minimally impact cellular growth yet significantly enhance adenosine synthesis [26; 27]. Compared to the direct supplementation of carbon or nitrogen sources, hypoxanthine and glutamine enable more precise regulation for conducting transcriptome analysis of adenosine synthesis. However, due to their high production costs, these compounds are generally not recommended for industrial-scale adenosine production. Instead, selecting low-cost raw materials (for example, using ammonium sulfate as a substitute for glutamine) and engineering bacterial strains to utilize these substrates more efficiently represents a more viable approach. Building on transcriptome analysis, this study identified and screened differentially expressed genes, providing robust reference data for developing metabolically engineered strains optimized for adenosine biosynthesis. We identified 105 DEGs potentially associated with adenosine biosynthesis, including 69 downregulated and 36 upregulated genes. These DEGs were predominantly enriched in pathways, such as purine metabolism and glutamate metabolism, with key adenosine-related genes clustered in the purine metabolic pathway downstream of 5-phosphoribosyl-1-pyrophosph (PRPP). The \(pur\) operon plays a critical regulatory role in nucleotide synthesis; transcriptomic analysis revealed a significant downregulation of the core biosynthetic genes (\(purD\), \(guaC\), \(purH\), and \(purN\)) of the \(pur\) operon in high-yield strains, suggesting that adenosine accumulation may suppress their expression via negative feedback regulation. However, we observed no significant differential expression in \(purR\) (the repressor gene of the _pur_ operon). This indicates that the expression of _purD_, _purH_, and _purN_ is independently regulated rather than co-expressed as part of the operon. The _guaC_ gene encodes guanosine monophosphate (GMP) reductase, which catalyzes the reduction of IMP to GMP. Intriguingly, under conditions of adenosine overproduction, _guaC_ expression was significantly suppressed. This repression redirects carbon flux toward adenosine synthesis, providing a novel strategy for industrial strain engineering. Cell biomass and residual glucose were monitored during fermentation, showing that biomass peaked at 48 h (OD\({}_{600}\) = 0.82), with residual glucose at 18.7 g/L (consumption rate: 73.28%). Continued fermentation further depleted glucose but reduced biomass, indicating that glucose levels were insufficient to sustain continued microbial growth, in turn suggesting that carbon source supplementation requires attention in industrial-scale operations. Gene expression analysis demonstrated that exogenous supplementation of hypoxanthine (6 g/L) and glutamine (10 g/L) markedly inhibited _purD_, _guaC_, _purH_, and _purN_ expression.