Enhancing production and purity of 9‐OH‐AD from phytosterols by balancing metabolic flux of the side‐chain degradation and 9‐position hydroxylation in Mycobacterium neoaurum

Abstract 9α‐Hydroxyandroster‐4‐ene‐3,17‐dione (9‐OH‐AD) is a representative steroid drug intermediate that can be prepared by phytosterols (PS) biotransformation with mycobacteria in a resting cell‐cyclodextrin system. In this study, over‐expression of 17β‐hydroxysteroid dehydrogenase (Hsd4A) was testified to enhance the side‐chain degradation of PS and to reduce the incomplete degradation by‐products. Meanwhile, the complete degradation product 4‐androstene‐3,17‐dione (AD) was increased due to the lack of 3‐Ketosteroid 9α‐Hydroxylase (KshA1) activities. To increase the production and purity of 9‐OH‐AD, the metabolic pathway of the side‐chain degradation of PS and 9‐position hydroxylation was modulated by balancing the over‐expression of Hsd4A and KshA1 in mycobacteria and reducing the bioconversion rate via lowering the ratio of PS and cyclodextrin. The production and purity of 9‐OH‐AD in broth were improved from 22.18 g L −1 and 77.13% to 28.27 g L −1 and 87.84%, with a molar yield of 78.32%.