Riboflavin is converted to the flavocoenzymes FMN and FAD, which serve as ubiquitous redox cofactors and also participate in other non-redox processes, such as the protection of DNA against UV ligh, light sensing, phototropism, circadian time keeping and bioluminiscence. GTP is the immediate precursor for riboflavin biosynthesis and its formation through the purine pathway is subject to several regulatory mechanisms at transcriptional and metabolic levels. Extracellular purines repress the transcription of most genes required for de novo AMP and GMP synthesis, thus avoiding energy waste. Additionally, three enzymes of the purine pathway, PRPP amidotransferase, adenylosuccinate synthase and IMP dehydrogenase, are subject to feedback inhibition by their end products.
In this work we use the filamentous hemiascomycete Ashbya gossypii, which naturally overproduces riboflavin as a detoxifying and protective mechanism and is currently in use for industrial vitamin B2 production. Indeed, A. gossypii is considered as a paradigm of the environmentally friendly “white” biotechnology with regard to industrial riboflavin overproduction. Here we manipulate the purine pathway of A. gossypii and eliminate the mechanisms of regulation affecting the committed step of the pathway, the synthesis of PRA, which is catalyzed by the enzyme PRPP amidotranferase. We report successful engineering of A. gossypii strains whose regulatory properties had been modified as described above to significantly enhance metabolic flow through the purine pathway and to increase the production of riboflavin ten-fold over the wild-type strain.