Unlocking Industrial Testosterone Production: Engineering <em>Aspergillus nidulans</em> Strain Deficient in Steroid 11-α-Hydroxylase Activity and Expressing 17-β-Hydroxysteroid Dehydrogenase Enzyme

Testosterone holds significant medical and economic importance, with the global market for testosterone replacement therapies valued at approximately $1.9 billion in 2023. This hormone is essential for the development and maintenance of male sexual characteristics as well as bone and muscle health. It plays a key role in conditions such as hypogonadism, muscle disorders, and andropause. However, the industrial production of testosterone often involves complex chemical processes that result in low yields, high costs, and environmental damage. Microbial biotransformation of steroids presents an eco-friendly alternative to traditional chemical synthesis. A knockout strain of Aspergillus nidulans deficient in steroid 11-α-hydroxylase activity was developed, rendering it incapable of hydroxylating androstenedione, progesterone, and testosterone. In this strain, two newly identified CYP450 enzymes—CYP68L1 from A. nidulans and CYP68L8 from Aspergillus ochraceus—were expressed to confirm their roles as steroid 11-α-hydroxylases. These enzymes successfully hydroxylated steroids such as androstenedione, progesterone, and testosterone. The availability of these 11-α-hydroxylases represents significant progress toward achieving efficient single-step steroid fermentation. Furthermore, the A. nidulans knockout strain is proposed as an effective model for studying the conversion of androstenedione to testosterone owing to its inability to hydroxylate the testosterone produced during bioconversion.