Inactivation of the oxidase gene mppG results in the selective loss of orange azaphilone pigments in Monascus purpureus

Monascus species are filamentous ascomycetes fungi and produce azaphilone (Az) pigment that is a well-known food colorant. Az is a class of fungal polyketides that bears a highly oxygenated pyranoquinone bicyclic core and is produced by a nonreducing fungal polyketide synthase with a reductive release domain (NR-fPKS-R). MpPKS5 encodes an NR-fPKS-R for Monascus Az (MAz) and is clustered with four oxidoreductase genes including mppG; mpp designates Monascus pigment production. MAz pigments are classified as yellow and orange MAz, and their structures differ in two hydride reductions with yellow MAz as the reduced type. The biosynthesis of yellow MAz (monascin, Y-1 and ankaflavin, Y-2) is completed by a reductive pathway involving a reductase gene mppE. This reductive pathway is diverged from a common MAz pathway involving two other reductase genes of mppA and mppC. This suggests that the biosynthesis of orange MAz (rubropunctatin, O-1 and monascorubrin, O-2) is completed by an oxidative branch pathway and the cognate oxidative role of mppG is genetically characterized in the present study. A targeted gene inactivation mutant of Delta mppG displayed a severe impairment in the production of orange MAz with no significant alteration in the level of yellow MAz. The feeding experiment with Y-1 in Delta MpPKS5 indicated that Y-1 could not be converted into O-1, which excludes the possibility that mppG mediates the conversion of yellow into orange MAz. This study supports the existence of divergent pathways in MAz biosynthesis and creates a recombinant strain for the selective production of yellow MAz.