Integrating Enzyme and Metabolic Engineering for Apocarotenoids 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. Previously, we have demonstrated the use of fusion-partners to improve the expression of carotenoid cleavage dioxygenase 1 (CCD1) in Escherichia coli. Here, we further engineered CCD1 whose intrinsic promiscuity and low activity limited the production of α-ionone in Escherichia coli. 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 epsilon-cyclase to facilitate substrate channelling. Collectively, these methods improved α-ionone concentration by > 2.5-fold as compared to our previously optimized strain. Lastly, the engineered enzyme was used in conjunction with metabolic engineering strategy to further boost α-ionone concentration by another 20%. This work deepens our understanding of CCD1 catalytic properties and proves integrating enzyme and metabolic engineering can be synergistic for higher microbial production yield.

(1) Zhang, C.; Chen, X.; Lindley, N. D.; Too, H.-P. A “Plug-n-Play” Modular Metabolic System for the Production of Apocarotenoids. Biotechnology and Bioengineering 2018, 115 (1), 174–183. https://doi.org/10.1002/bit.26462.

(2) Chen, X.; Shukal, S.; Zhang, C. Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production. J. Agric. Food Chem. 2019. https://doi.org/10.1021/acs.jafc.9b00860.