Construction of a Cell Factory for the Targeted and Efficient Production of Phytosterol to Boldenone in Mycobacterium neoaurum

Abstract

ABSTRACTBoldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off‐season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β‐hydroxysteroid dehydrogenase and 3‐ketosteroid‐Δ1‐dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by‐product generation. To further increase the yield, a multi‐enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial‐time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one‐step conversion to BD developed in this study lays the foundation for industrial production.