| Summary: | Citrinin (CIT), a secondary metabolite produced by the filamentous fungi <i>Monascus</i> species, exhibits nephrotoxic, hepatotoxic, and carcinogenic effects in mammals, remarkably restricting the utilization of <i>Monascus</i>-derived products. CIT synthesis is mediated through the <i>pksCT</i> gene and modified by multiple genetic factors. Here, the regulatory effects of two <i>pksCT</i> transcripts, <i>pksCT</i>α, and <i>pksCT</i>β, generated via pre-mRNA alternative splicing (AS), were investigated using hairpin RNA (ihpRNA) interference, and their impact on CIT biosynthesis and the underlying mechanisms were assessed through chemical biology and transcriptome analyses. The CIT yield in ihpRNA-pksCTα and ihpRNA-pksCT (α + β) transformants decreased from 7.2 μg/mL in the wild-type strain to 3.8 μg/mL and 0.08 μg/mL, respectively. Notably, several genes in the CIT biosynthetic gene cluster, specifically <i>mrl3</i>, <i>mrl5</i>, <i>mrr1,</i> and <i>mrr5</i> in the ihpRNA-pksCT (α + β) transformant, were downregulated. Transcriptome results revealed that silencing <i>pksCT</i> has a great impact on carbohydrate metabolism, amino acid metabolism, lipid metabolism, and AS events. The key enzymes in the citrate cycle (TCA cycle) and glycolysis were significantly inhibited in the transformants, leading to a decrease in the production of biosynthetic precursors, such as acetyl-coenzyme-A (acetyl-coA) and malonyl-coenzyme-A (malonyl-coA). Furthermore, the reduction of CIT has a regulatory effect on lipid metabolism via redirecting acetyl-coA from CIT biosynthesis towards lipid biosynthesis. These findings offer insights into the mechanisms underlying CIT biosynthesis and AS in <i>Monascus</i>, thus providing a foundation for future research.
|
|---|