## 3 Results
### Functional Analysis of Differential Expression Genes  
GO annotation analysis of differentially expressed genes revealed that genes with significant expression changes in the treatment group were primarily enriched in metabolic processes, cellular processes, cellular anatomical entities, catalytic activity, and binding compared to the control group (Figure 3). A smaller subset of genes was associated with transporter activity, transcription regulator activity, biological regulation, and ATP-dependent activity. The GO annotation results demonstrated that the addition of glutamine and hypoxanthine, which increased adenosine production, significantly altered the expression of functional genes in _Bacillus subtilis_ involved in metabolic synthesis, enzyme expression, and intracellular metabolite transport. These changes may influence the types and levels of secondary metabolites biosynthesized by the bacterium and directly regulate adenosine biosynthesis. Figure 1: The fermentation yield of _Bacillus subtilis_ Q40-2-37 under different treatments. This study included two groups: cultured in different media and fermentation medium supplemented with glutamine and hypoxanthine. Building upon previous analyses, further GO enrichment analysis revealed that DEGs were predominantly enriched in small-molecule metabolic processes and multiple purine-related pathways, including purine-containing compound metabolic/biosynthetic processes, purine nucleotide metabolic/biosynthetic processes, and eight specific pathways associated with purine nucleoside metabolism (Figure 4). Additional enriched pathways encompassed inosine monophosphate (IMP) biosynthesis, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism, IMP metabolism metabolism, IMP  
de novo synthesis, nicotinamide adenine dinucleotide (NAD) binding, and glutamate synthesis/metabolism. Notably, the initial carbon metabolic flux from glucose remained largely unaffected by the addition of glutamine and hypoxanthine, indicating that these substrates primarily influenced downstream purine synthesis pathways dependent on 5-phosphoribosyl-1-pyrophosphate (PRPP) rather than altering upstream glucose utilization. This suggests that the enhanced adenosine production induced by glutamine and hypoxanthine supplementation is mechanistically linked to the targeted modulation of PRPP-driven purine biosynthesis. KEGG functional annotation analysis of differentially expressed genes was conducted to identify their involvement in major biochemical, metabolic, and signal transduction pathways. Statistical analysis of gene enrichment across metabolic pathways (Figure 5) demonstrated that the treatment group utilizing hypoxanthine and glutamine as carbon sources showed significant enrichment of differentially expressed genes in 17 metabolic categories. The most prominently enriched pathways included carbohydrate metabolism, amino acid metabolism, and energy metabolism, with additional enrichment observed in nucleotide metabolism, cofactor and vitamin metabolism, glycan biosynthesis and metabolism, signal transduction, and terpenoid and polyketide metabolism. These results systematically map the regulatory effects of hypoxanthine and glutamine supplementation on adenosine-producing _Bacillus subtilis_, highlighting their targeted influence on carbon flux partitioning and nucleotide biosynthesis networks. Figure 4: GO enrichment analysis. The size of the dots indicates the number of genes in the GO term, and the different colors indicate different levels of significance.