Dynamic regulation of Monascus azaphilones biosynthesis by the binary MrPigE-MrPigF oxidoreductase system.

Monascus azaphilones (MAs) have been extensively applied as natural food coloring agents. MAs are classified into three categories: yellow MAs (YMAs), orange MAs (OMAs), and red MAs with various biological activities. However, the exact biosynthetic mechanism of OMAs and YMAs are not thoroughly elucidated. Firstly, we identified four DNA-binding residues of transcription factor MrPigB and constructed a multi-site saturation mutagenesis library of MrPigB. Then, comparative metabolite and gene expression of the mutants revealed that two oxidoreductases MrPigE and MrPigF were responsible for the formation of YMAs and OMAs. Finally, the in vitro and in vivo assays demonstrated the opposite roles of MrPigE and MrPigF in conversion of OMAs to YMAs. To our knowledge, this is the first report of a binary oxidoreductase system for dynamic regulation of fungal secondary metabolite biosynthesis. Broadly, our work also demonstrates the transcription factor engineering strategy for elucidating the biosynthetic pathway of secondary metabolite. KEY POINTS: • MrPigE converts orange Monascus azaphilones to yellow Monascus azaphilones • MrPigF oxidizes intermediates to afford orange Monascus azaphilones • MrPigE and MrPigF constitute a binary system in Monascus azaphilones biosynthesis.