Molecular Mechanism of Mok I Gene Overexpression in Enhancing Monacolin K Production in Monascus pilosus

<i>Monascus</i> species are capable of producing various active metabolites, including monacolin K (MK) and pigments. Studies have shown that the overexpression of the <i>mok I</i> gene from the MK synthesis gene cluster in <i>Monascus</i> species can significantly increase MK production; however, the molecular mechanism has not yet been fully elucidated. Therefore, this study focused on the <i>mok I</i> gene of <i>Monascus pilosus</i> to construct overexpression strains of the <i>mok I</i> gene, resulting in high-yield MK production. Sixteen positive transformants were obtained, seven of which produced 9.63% to 41.39% more MK than the original strain, with no citrinin detected in any of the transformants. The qRT-PCR results revealed that the expression levels of <i>mok I</i> in the transformed strains TI-13, TI-24, and TI-25 increased by more than 50% compared to the original strain at various fermentation times, with the highest increase being 10.9-fold. Furthermore, multi-omics techniques were used to analyze the molecular mechanisms underlying enhanced MK production in transformed strains. The results indicated that <i>mok I</i> overexpression may enhance MK synthesis in <i>M. pilosus</i> by regulating the expression of key genes (such as <i>MAO</i>, <i>HPD</i>, <i>ACX</i>, and <i>PLC</i>) and the synthesis levels of key metabolites (such as delta-tocopherol and alpha-linolenic acid) in pathways linked to the biosynthesis of cofactors, the biosynthesis of unsaturated fatty acids, tyrosine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, alpha-linolenic acid metabolism, and glycerophospholipid metabolism. These findings provide a theoretical basis for further study of the metabolic regulation of MK in <i>Monascus</i> species and for effectively enhancing their MK production.