High-efficiency secretion expression of FAD-dependent glucose dehydrogenase in Pichia pastoris via signal peptide optimization and redox cofactor enhancement.

FAD-dependent glucose dehydrogenase (FAD-GDH) is a promising glucose sensing enzymes owing to its excellent thermostability and substrate specificity. To produce recombinant FAD-GDH with higher enzymatic activity by Pichia pastoris to meet commercial demands, three complementary strategies were proposed. First, the OST signal peptide, which outperformed ADV and MSB, replaced α-MF to yield much more efficient secretion, enhancing enzyme activity and protein yield up to 201.4% and 214.0% compared to the levels obtained with the original secretion signal, respectively, in shake-flask cultures. Second, to address the low specific activity of FAD-GDH, mixed feeding with riboflavin, adenosine, and yeast extract during induction period of fermentation had improved enzyme activity and specific activity to196.6% and 161.4%, respectively, while maintaining cell viability, compared with the control without additional feeding. Third, to further enhance specific activity of FAD-GDH, endogenous FAD synthase was ectopically overexpressed under the control of either the GAP or AOX1 promoter via CRISPR-Cas9-mediated genomic integration. The strain with AOX1-drivenFAD synthase (WCG-AOX1-FAD1) exhibited the most significant improvements, with enzyme activity of 1023.46 ± 95.85 U/mL (239.4% of WCG-OST) and specific activity of 886.31 ± 25.42 U/mg (233.4% of WCG-OST), both representing relatively high levels among similar studies. The synergistic application of these three strategies provides a reference for the industrial production of various cofactor-dependent enzymes.