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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 17\u03b2\u2010hydroxysteroid dehydrogenase and 3\u2010ketosteroid\u2010\u0394<jats:sup>1<\/jats:sup>\u2010dehydrogenase was improved through enzyme engineering, and their expression in the new strain of <jats:italic>Mycobacterium neoaurum<\/jats:italic> was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05\u00a0g\/L, with a significant decrease in by\u2010product generation. To further increase the yield, a multi\u2010enzyme fusion expression system was constructed, and a key cell wall gene <jats:italic>kasB<\/jats:italic> was knocked out, resulting in a spatial\u2010time yield of BD reaching 1.02\u00a0g\/(L\u00b7d). Subsequent optimization of the transformation system further increased the BD production to 5.56\u00a0g\/L, with a spatiotemporal yield of 1.39\u00a0g\/(L\u00b7d). The green biosynthetic route of phytosterol one\u2010step conversion to BD developed in this study lays the foundation for industrial production.<\/jats:p>","DOI":"10.1002\/biot.202400489","type":"journal-article","created":{"date-parts":[[2024,11,18]],"date-time":"2024-11-18T00:56:57Z","timestamp":1731891417000},"source":"Crossref","is-referenced-by-count":5,"title":["Construction of a Cell Factory for the Targeted and Efficient Production of Phytosterol to Boldenone in <i>Mycobacterium neoaurum<\/i>"],"prefix":"10.1002","volume":"19","author":[{"given":"Bo","family":"Zhang","sequence":"first","affiliation":[{"name":"National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals Zhejiang University of Technology  Hangzhou People's Republic of China"},{"name":"Key Laboratory of Bioorganic Synthesis of Zhejiang Province College of Biotechnology and Bioengineering Zhejiang University of Technology  Hangzhou People's Republic of 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