Integrating Synergistic Mutagenesis and Targeted Metabolic Engineering with Process Control for the Hyper-Production of Pyrroloquinoline Quinone in Hyphomicrobium denitrificans

Abstract Pyrroloquinoline quinone (PQQ), a redox-active quinone cofactor with diverse physiological functions, has attracted increasing attention owing to its expanding applications in pharmaceuticals, agriculture, and electrochemical sensing. Microbial fermentation provides a sustainable and scalable approach for PQQ production. In this study, an elite mutant strain, WX17A, was obtained through combined mutagenesis and adaptive laboratory evolution. To further improve PQQ biosynthesis, key fermentation conditions were systematically optimized, and response surface methodology was subsequently employed to optimize the supplementation of exogenous amino acids, metal ions, and hydrogen peroxide. Targeted metabolic engineering further identified PqqC and PqqE as key engineering targets in the PQQ biosynthetic pathway. Rational overexpression of these genes increased the PQQ titer to 134.77 mg/L. Finally, a fed-batch fermentation strategy coupled with two-stage regulation of pH and dissolved oxygen was established in a 5 L bioreactor, resulting in a final PQQ titer of 385.83 mg/L. Overall, the integration of strain improvement, fermentation optimization, metabolic engineering, and process control substantially enhanced PQQ production, providing a promising basis for the development of efficient and scalable PQQ biomanufacturing.