Overexpression of purine nucleoside phosphorylase increases the adenosine content in Ganoderma lucidum

Abstract

Background: 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 adenosine biosynthetic gene is an optimized way to increase the production of adenosine in submerged culture of Ganoderma lucidum. Results: In this study, we correlated the expression of adenosine synthase genes (including GlATIC, GlPNP, GlADK) with the adenosine content in mycelium at different fermentation time points. 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 and PI of 5.89. The GlPNP displayed a trimeric quaternary structure by theoretically modelling with SWISS-MODEL. The transcript levels of GlPNP overexpression transformants (namely OE::GlPNP-5 and OE::GlPNP-7) 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. In addition, the GlPNP overexpression strains showed decreased colony growth and reduced biomass in submerged cultivation. Conclusions: GlPNP gene overexpression is an effective strategy to improve the production of adenosine in G. lucidum. This study is the first report about the manipulation of adenosine biosynthesis in medicinal fungi.