β-Cryptoxanthin Production in Escherichia coli by Optimization of the Cytochrome P450 CYP97H1 Activity

Cytochromes P450, forming a superfamily of monooxygenases containing heme as a cofactor, show great versatility in substrate specificity. Metabolic engineering can take advantage of this feature to unlock novel metabolic pathways. However, the cytochromes P450 often show difficulty being expressed in a heterologous chassis. As a case study in the prokaryotic host Escherichia coli, the heterologous synthesis of fi-cryptoxanthin was addressed. This carotenoid intermediate is difficult to produce, as its synthesis requires a monoterminal hydroxylation of fi-carotene whereas most of the classic carotene hydroxylases are dihydroxylases. This study was focused on the optimization of the in vivo activity of CYP97H1, an original P450 fi-carotene monohydroxylase. Engineering the N-terminal part of CYP97H1, identifying the matching redox partners, defining the optimal cellular background and adjusting the culture and induction conditions improved the production by 400 times compared to that of the initial strain, representing 2.7 mg/L fi-cryptoxanthin and 20% of the total carotenoids produced.

Automated summary

Cytochromes P450, a superfamily of monooxygenases, have versatile substrate specificity. Metabolic engineering can exploit this feature to unlock new metabolic pathways. However, expressing cytochromes P450 in a different host can be challenging. This study focused on optimizing the in vivo activity of CYP97H1 in Escherichia coli to produce fi-cryptoxanthin, a difficult carotenoid intermediate. Through engineering the N-terminal region of CYP97H1, identifying compatible redox partners, defining the optimal cellular background, and adjusting culture and induction conditions, the production of fi-cryptoxanthin increased by 400 times compared to the initial strain, reaching 2.7 mg/L and accounting for 20% of total carotenoid production.