Engineering a two-enzyme system in Mycolicibacterium neoaurum for efficient biotransformation of phytosterols to dihydrotestosterone

Dihydrotestosterone (DHT) is a valuable steroid drug with widespread clinical applications, but traditional chemical synthesis is environmentally harmful and requires complex reaction conditions. This study introduces a one-step microbial transformation method for the production of DHT from inexpensive phytosterols (PS) using engineered Mycolicibacterium neoaurum (MNR). The heterologous expression of 5 alpha-reductase (5 alpha-R) and 17(3hydroxysteroid dehydrogenase (17(3-HSD) in MNR enabled the efficient conversion of PS to DHT. To further enhance 5 alpha-R activity, semi-rational mutagenesis was employed, which significantly increasing DHT production. Molecular dynamics simulations provided insights into the underlying mechanisms driving this enhancement. Additionally, the incorporation of a PntAB-based NADPH regeneration system further improved DHT yield. Process optimization resulted in a maximum DHT concentration of 1.123 g & sdot;L- 1, representing the first gram-scale microbial production of DHT. Compared to traditional chemical synthesis, this biotransformation method offers milder reaction conditions, reduced environmental impact, and eliminates the need for toxic catalysts. This work demonstrates a sustainable and efficient microbial method for DHT production, with significant industrial potential for the greener manufacturing of steroid hormone drugs.