Physiological characterization of a pyrimidine auxotroph exposes link between uracil phosphoribosyltransferase regulation and riboflavin production in Ashbya gossypii

The blockage of the de novo pyrimidine biosynthetic pathway at the orotidine-5-phosphate decarboxylase level was previously demonstrated to affect riboflavin production in the industrial producer fungus Ashbya gossypii. However, the molecular basis for the unusual sensitivity to uracil displayed by the pyrimidine auxotroph A. gossypii Agura3 was unknown. Here, uridine was shown to be the only intermediate of the pyrimidine salvage pathway able to fully restore this mutants growth. Conversely, uracil, which is routinely used to rescue pyrimidine auxotrophs, had a dose-dependent growth-inhibitory effect. Uracil phosphoribosyltransferase (UPRT) is the pyrimidine salvage pathway enzyme responsible for converting uracil to uridine monophosphate in the presence of phosphoribosyl pyrophosphate (PRPP). Characterization of the A. gossypii UPRT, as produced and purified from Escherichia coli, revealed that uracil concentrations above 1mM negatively affected its activity, thus explaining the hypersensitivity of the Agura3 mutant to uracil. Accordingly, overexpression of the AgUPRT encoding-gene in A. gossypii Agura3 led to similar growth on rich medium containing 5mM uracil or uridine. Decreased UPRT activity ultimately favors the preservation of PRPP, which otherwise may be directed to other pathways. In A. gossypii, increased PRPP availability promotes overproduction of riboflavin. Thus, this UPRT modulation mechanism reveals a putative means of saving precursors essential for riboflavin overproduction by this fungus. A similar uracil-mediated regulation mechanism of the UPRT activity is reported only in two protozoan parasites, whose survival depends on the availability of PRPP. Physiological evidence here discussed indicate that it may be extended to other distantly related flavinogenic fungi.This study was supported by the Portuguese Foundation for Science and technology-FCT(strategic funding UID/BIO/04469/2013, Post-Doc grant SFRH/BPD/110640/2015 and PhD grant PD/BD/113812/2015), and European Regional Development Fund under the scope of Compete2020 (POCI-01-0145-FEDER-006684) and Norte2020 Programa Operacional Regional do Norte (BioTecNorte operation NORTE-01-0145-FEDER-000004).