Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production.

Metabolic engineering aims to balance intracellular pathways and increase the precursor supply. However, some heterologous enzymes are not evolved to support high flux. To remove the limitation, the catalytic properties of rate-limiting enzymes must be enhanced. Here, we engineered carotenoid cleavage dioxygenase 1 (CCD1), whose intrinsic promiscuity and low activity limited the production of α-ionone in . Site-directed mutagenesis was carried out to mutate three structural elements of CCD1: an active site loop, η-helices, and α-helices. Furthermore, mutated CCD1 was fused with lycopene ε-cyclase to facilitate substrate channelling. Collectively, these methods improved the α-ionone concentration by >2.5-fold compared to our previously optimized strain. Lastly, the engineered enzyme was used in conjunction with the metabolic engineering strategy to further boost the α-ionone concentration by another 20%. This work deepens our understanding of CCD1 catalytic properties and proves that integrating enzyme and metabolic engineering can be synergistic for a higher microbial production yield.