Orf6 gene encoded glyoxalase involved in mycotoxin citrinin biosynthesis in Monascus purpureus YY-1.

Abstract

As traditional edible fungi, Monascus spp. have been widely used as folk medicine, food colorants, and fermentation starters in East Asian countries for more than a thousand years. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, raises suspicions about the safety of Monascus products. Citrinin biosynthesis in Monascus is known to occur via a polyketide pathway and a citrinin biosynthesis gene cluster, which include the characterized polyketide synthetase pksCT. A gene, orf6, encodes a protein that shows significant similarity to glyoxalase and is located between ctnE and orf1. This study analyzed orf6 function, and successfully obtained an orf6 disruption strain (Δorf6). Citrinin production was significantly greater (3.6-fold) in the Δorf6 strain than in the wild-type Monascus purpureus YY-1, and RT-PCR analysis further revealed increased expression of numerous genes of the citrinin biosynthesis gene cluster in Δorf6. Therefore, orf6 proved to be a major inhibitor, directly involved in citrinin biosynthesis. Moreover, pigment production in Δorf6 was reduced by approximately 30%, while the transcription levels of many genes involved in Monascus pigments (MPs) biosynthesis had increased. This dichotomy indicated that MPs and citrinin yields may be improved simultaneously; however, a portion of the pigments was consumed to protect the cells from oxidative damage in the Δorf6 strain. An Δorf6 revertant restored the citrinin and pigment yields to normal levels. This study makes a contribution to explore the citrinin biosynthesis pathway and provides some theoretical guidance to improving the safety of Monascus-related products.