Identification and functional characterization of ammonium transporters in Penicillium purpurogenum

Abstract

The filamentous fungus Penicillium purpurogenum IAM15392 produces a nitrogen-containing azaphilone pigment, (10Z)-12-carboxyl-monascorubramine (PP-V), which is a potentially valuable natural food colorant. Because ammonium is used as a nitrogen source, and because ammonium uptake is the first step in the synthesis of PP-V, ammonium transporters of P. purpurogenum were identified and characterized. The P. purpurogenum genome was found to contain four putative ammonium transporter genes, designated amtA, amtB, amtC, and amtD, which encode 11 transmembrane proteins of 479, 567, 452, and 475 amino acid residues, respectively. These genes were tested for their ability to complement mutations in the ammonium transporter genes of the fission yeast Schizosaccharomyces pombe. The phenotypes of mutants included defects in growth on low ammonium medium, methylammonium sensitivity, ammonium uptake from the culture medium, and ammonium limitation-induced invasive growth. Furthermore, the transcription of the amt genes was examined in P. purpurogenum grown under different ammonium concentrations. The results suggest that AmtB plays a major role in growth using ammonium as a nitrogen source, whereas AmtA and possibly AmtD function at low ammonium concentrations. Because a medium used for the production of PP-V contains a high concentration of ammonium, our functional characterization of the P. purpurogenum ammonium transporters suggests that AmtB is a potential target of bioengineering for increased PP-V production.