1. Prediction of key genes in the AICAR biosynthetic pathway of endophytic Fusarium solani
Previous study showed that overexpression of the global regulator VeA mediated a significant increase in the antitumor activity of F. solani, with a marked enrichment of differential metabolites including AICAR. To elucidate the molecular regulatory mechanism by which veAOE14 contributed to the increased synthesis of AICAR, we obtained the predicted synthetic pathway of AICAR in F. solani by analyzing the veAOE14 transcriptome and metabolome data, with a total of 10 steps in the major synthetic reactions. Based on the previous findings and predicted metabolic pathways, VeA overexpression was demonstrated to not only enhance AICAR production but also upregulate PRPS2 transcriptional levels. These results suggest that the global regulator VeA may play a pivotal regulatory role in modulating PRPS2 expression, a key gene involved in AICAR biosynthesis. Numerous studies have demonstrated that PRPS2 functions as a critical rate-limiting enzyme in the second enzymatic reaction of nucleic acid precursor biosynthesis. This process is constrained by the energy availability of ATP and catalyzed ribulose 5-phosphate (R5P) into the critical metabolic intermediate, ribulose 5-phosphate-1-pyrophosphate (PRPP). Among 26 candidate key genes identified as consistent with the overexpression pattern of VeA, this analysis revealed that PRPS2 may play a critical role in the AICAR synthesis pathway in F. solani. Therefore, PRPS2 was selected as the key gene responsible for catalyzing AICAR synthesis and proceeded with further validation.
2. Functional characterization of PRPS2 and transcription factor MtfA and analysis of their regulatory mechanisms in catalyzing AICAR biosynthesis
Through integrated approaches including bioinformatics analysis, gene overexpression, gene knockout, HPLC quantification, and yeast one-hybrid assays, this study identified PRPS2 as the key enzyme catalyzing AICAR biosynthesis. The transcription factor MtfA was shown to negatively regulate PRPS2-mediated AICAR biosynthesis. Therefore, the research revealed a novel molecular regulatory mechanism in F. solani whereby VeA negatively modulates MtfA, which in turn suppresses PRPS2 transcriptional levels to regulate AICAR biosynthesis.
This study elucidates the mechanism underlying VeA-mediated enhancement of AICAR production through VeA overexpression in endophytic F. solani. The results reveal a molecular regulatory mechanism involving the global regulator VeA, C2H2-type transcription factor MtfA, and PRPS2 in mediating AICAR biosynthesis in F. solani at the molecular level. This breakthrough addresses the challenge of constructing engineered filamentous fungal strains for AICAR production and provides new experimental evidence for microbial fermentation-based AICAR production.
See the article: MtfA, a C2H2 transcriptional regulator, negatively regulates PRPS2-mediated biosynthesis of the adenosine analogue acadesine in Fusarium solani
