Metabolomic insights into homogentisic acid-mediated regulation of tocopherol enhancement in Monascus purpureus fermentation.

Targeted enrichment of tocopherols in fungal fermentation systems remains a significant metabolic engineering challenge. This study presented a novel strategy employing homogentisic acid (HGA) modulation to efficiently enhance tocopherol production in the M. purpureus-fermented coix seed (MFCS) system. Three strategies of exogenous HGA supplementation (Exo-HGA), endogenous HGA accumulation (End-HGA), and the combination of exogenous HGA supplementation and endogenous HGA accumulation (Exo + End-HGA) were systematically investigated. While End-HGA optimally promoted lovastatin, pigment, and biomass, the Exo + End-HGA strategy (supplemented with 250 μg/mL Exo-HGA) achieved superior tocopherol enrichment. The contents of total, α-, and γ-tocopherol reached 225.407, 64.022, and 161.385 μg/mL, respectively, corresponding to a 1.86-fold increase in total tocopherol compared with the control group. A total of 2425 differentially expressed metabolites were identified. The significant accumulation of HGA, concomitant increases in γ-tocotrienol and menatetrenone (VK2), along with a decline in ubiquinone precursors such as 4-hydroxybenzoic acid derivatives, suggested that HGA specifically activated the tocopherol biosynthesis pathway. KEGG pathway analysis further revealed that HGA upregulated the shikimate and methylerythritol phosphate/mevalonate (MEP/MVA) pathways, thereby coordinating the redistribution of carbon flux and repressing competing routes such as phenylpropanoid and alkaloid biosynthesis, ultimately facilitating tocopherol enrichment. This study elucidated the metabolic network underlying HGA-mediated tocopherol enrichment in MFCS, providing a the oretical foundation and potential targets for enhancing tocopherol production through microbial metabolic engineering strategies.