Summary

Carboxylic acid reductases (CARs) are attractive catalysts for the synthesis of valuable aldehyde building blocks. However, the application of CARs still remains a challenge because CARs depend on essential NAD(P)H and ATP cofactors that need to be regenerated. To overcome this limitation, locally immobilized multi-enzyme constructs will be designed with superior catalytic activity and stability in E. coli compared to unfused enzymes. To this end, catalytic partners will be fused with optimized peptidic linkers using iterative cycles of structural bioinformatics, simulations of diffusion and reaction systems, and experimental in vitro and whole-cell validation. From an applied viewpoint, the project shall provide transferable rational knowledge to guide the integration of CARs into self-sufficient multi-enzyme fusion assemblies. From a basic research viewpoint, the project contributes to elucidating the impact of fusion on the performance of multienzyme fusion assemblies.