Sustainable biosynthesis of small-molecule natural products (NPs) is often hampered by enzyme instability and high cofactor costs. Herein, we developed a modular bi-functional immobilization system to synthesize two NPs (carvone and vanillyl alcohol). Building on the glyoxyl-agarose technology pioneered by Prof. José M. Guisán, we covalently immobilized an alcohol dehydrogenase (BsADH) to allow for biocatalyst reusability. This system was then coupled with two different cofactor-recycling enzymes (LpNOX for NAD+ regeneration, and CbFDH for NADH regeneration), which were reversibly immobilized on the same support. The co-immobilization enabled 25–50% higher productivity compared to independently immobilized enzymes. This modular system allows the selective desorption of a cofactor-recycling enzyme (LpNOX, >90% desorption) without affecting the covalently attached ADH, and enabling the co-immobilization of another cofactor-recycling enzyme (CbFDH). This flexible and sustainable platform for redox biocatalysis was applied to the consecutive synthesis of NPs of interest for the pharmaceutical and fragrance industries.
