Abstract: This study investigated high-throughput screening of inhibitors for the key enzyme CitE in the synthesis of red yeast citrinin through computer simulation. First, Alphafold 2 was used to build the homology model of CitE. and it was aligned with similar sequences to predict the ligand binding region. Subsequently, a ligand library of 20 000 compounds was constructed by searching for natural ligands and flavonoid skeleton analogues in the Arthur database. Afterwards, molecular docking of the CitE protein to the library of small molecule compounds was performed using Maestro to select potential CitE ligands. Gromacs kinetic simulation was used to evaluate the binding stability, and the binding free energy was calculated using MM/PBSA. The protein-ligand binding mode was analyzed, confirming that six small molecule ligands including quercetin, luteolin, apigenin, genistein, 5,4’-dihydroxyflavone, and fisetin had strong CitE binding activities. The results from solid and liquid state fermentation showed that the above compounds exhibited significant inhibitory activity against Monascus citrinin production. Specifically, the addition of these compounds in solid state fermentation (20 mg/28 g of dry material) resulted in a reduction of Monascus citrinin production by 42.52%, 48.81%, 32.54%, 32.57%, 21.02%, and 13.67%, respectively, while their addition in liquid state fermentation (0.1 g/L) led to a reduction by 33.77%, 15.58%, 33.33%, 62.34%, 58.87%, and 50.22%, respectively. In summary, this study is of reference significance for establishing an efficient and safe Monasucs fermentation system.

Key words: Monascus; citrinin; inhibitors; molecular docking; molecular dynamics simulation; CitE