National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, People's Republic of China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, People's Republic of China.
Biotechnol J. 2024 Nov;19(11):e202400489. doi: 10.1002/biot.202400489.
Boldenone (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-Δ-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.
宝丹酮（BD），一种蛋白质同化激素，常用于治疗运动员的肌肉损伤、骨质疏松症和淡季肌肉建设。传统的 BD 合成方法依赖于化学过程，成本高且对环境有影响。因此，开发更可持续和经济的生物合成途径对于 BD 的生产至关重要。本研究旨在实现 BD 的高效生产。首先，通过酶工程提高了 17β-羟甾脱氢酶和 3-酮甾体-Δ-脱氢酶的催化性能，并通过代谢工程增强了它们在新分枝杆菌中的表达。这些改进使 BD 的产量显著提高到 4.05 g/L，同时副产物的生成显著减少。为了进一步提高产量，构建了多酶融合表达系统，并敲除了关键的细胞壁基因 kasB，使 BD 的时空产率达到 1.02 g/（L·d）。随后对转化系统进行优化，使 BD 的产量进一步提高到 5.56 g/L，时空产率达到 1.39 g/（L·d）。本研究开发的植物甾醇一步转化为 BD 的绿色生物合成途径为工业生产奠定了基础。
