Enhanced biocatalytic production of cortisol by protein engineering and process engineering

Cortisol, the primary glucocorticoid in humans, plays crucial physiological functions and serves as an intermediate for synthesizing other glucocorticoids. Currently, cortisol production mainly relies on a semi-synthetic route, where the key step of introducing 11β-OH into 11-deoxycortisol is catalyzed by the filamentous fungi Curvularia lunata and Absidia orchidis. This method, however, generates by-products and involves lengthy cultivation. To achieve specific and efficient production of cortisol, we constructed a recombinant biocatalyst by expressing and engineering the human mitochondrial 11β-hydroxylase CYP11B1 in Escherichia coli. Firstly, the balance between CYP11B1 and its redox partners AdR and Adx was regulated through ribosome binding site (RBS) engineering, resulting in a slight increase in cortisol productivity (from 344±19 mg·L⁻¹·d⁻¹ to 407±7 mg·L⁻¹·d⁻¹). Subsequently, the heterologous expression of CYP11B1 was improved through application of the computational design tool PROSS, generating a triple mutant S169V/H354D/L463F with 87.5 % higher cortisol yield than the wild type. Finally, the catalytic performance was improved by optimizing the recombinant protein expression conditions and enhancing the substrate solubility in the reaction system, further elevating the productivity of cortisol to 2.8±0.1 g·L⁻¹·d⁻¹. To our knowledge, this is the highest ever reported cortisol productivity using a human 11β-hydroxylase-based biocatalyst.