Adenosine recognized for its immunomodulatory, anti-inflammatory, and anti-cancer properties, has limited clinical and commercial applications due to low production levels in Ganoderma lucidum. Enhancing the expression of key biosynthetic genes presents a viable strategy to boost adenosine production through microbial fermentation. This study focused on the cloning, characterization, and overexpression of the key biosynthetic gene 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase ( GlATIC) in G. lucidum. The 1803-bp GlATIC cDNA was inserted into the plasmid pCAMBIA1302 and introduced into G. lucidum protoplasts via PEG-mediated transformation. Protoplasts were prepared using lywallzyme and driselase. Transformants were analyzed for GlATIC expression levels and adenosine content. The transformant OE- GlATIC-14 exhibited a 7.4-fold increase in GlATIC expression compared to the wild-type (WT) strain on the fourth day of culture. Adenosine content in OE- GlATIC-14 increased by 130.6%, reaching 3970.89 µg/g, compared to the WT strain. Additionally, AMP and guanosine levels were elevated in OE- GlATIC-14. The adenosine enhancement rate achieved in this study surpasses that of genetically engineered Bacillus subtilis strains, with the adenosine content being the highest reported among G. lucidum and other fungi. Overexpression of GlATIC in G. lucidum significantly enhances adenosine production and represents a promising strategy for optimizing microbial fermentation of adenosine in G. lucidum.

Adenosine has been getting increasing attention due to its positive role in immunomodulation, anti-inflammation, and anti-cancer, etc.. The low production of Ganoderma adenosine is a bottleneck for clinical trials and commercial applications. Regulating the expression of key biosynthetic gene is an optimized way to increase the adenosine production in submerged culture of Ganoderma lucidum. In this study, we correlated the expression of adenosine synthase genes (including GlATIC, GlPNP, GlADK) with the adenosine content. The results showed that GlPNP was positively correlated with Ganoderma adenosine contents. Then the key biosynthetic gene GlPNP was cloned, characterized and overexpressed in G. lucidum. The cDNA of GlPNP gene was 969-bp in length, with a predicted molecular weight of 34.6 kDa. The GlPNP displayed a trimeric quaternary structure. The transcript levels of GlPNP overexpression transformants were approximately 2.9-3.9-fold higher than those of the WT strains on day 4, while the adenosine contents were increased by 78% and 63%, respectively, by compared with vector-containing strain. The GlPNP overexpression strains showed decreased colony growth and reduced biomass. In conclusion, GlPNP gene overexpression is an effective strategy to improve the production of adenosine. This study is the first report about the manipulation of adenosine biosynthesis in medicinal fungi.