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Here, we report a streamlined and sustainable \u201ctwo\u2010stage\u201d bioconversion strategy for direct testosterone production from phytosterols. In the first stage,\n                    <jats:italic>Mycobacterium neoaurum<\/jats:italic>\n                    NRRL B\u20103805 was metabolically reprogrammed to maximize 4\u2010androstenedione accumulation by deleting\n                    <jats:italic>kstd1<\/jats:italic>\n                    and\n                    <jats:italic>kshB1<\/jats:italic>\n                    and overexpressing\n                    <jats:italic>hsd4A<\/jats:italic>\n                    , generating strain MnB\n                    <jats:italic>\n                      <jats:sup>\u0394kstd1\/\u0394kshB1\/p261\u2010hsd4A<\/jats:sup>\n                    <\/jats:italic>\n                    . This engineered strain produced 12.3\u00a0g L\n                    <jats:sup>\u2212<\/jats:sup>\n                    <jats:sup>1<\/jats:sup>\n                    of 4\u2010androstenedione within 6 days, corresponding to a molar yield exceeding 88%. In the second stage, structure\u2010guided redesign of ketoreductase markedly enhanced its catalytic efficiency toward 4\u2010androstenedione. Molecular docking and dynamics analyses identified residues G141 and M215 as critical contributors to substrate recognition. The G141A substitution optimized the local binding environment by modulating hydrophobic and polar interactions, leading to a 5.1\u2010fold increase in catalytic efficiency. Under optimized reaction conditions, the engineered enzyme achieved complete conversion of in situ\u2010generated 4\u2010androstenedione to testosterone in a single bioreactor, yielding 12.05 g\/L of testosterone with an overall molar yield of 86% from phytosterol and delivering a record spatiotemporal productivity of 0.6\u00a0g L\n                    <jats:sup>\u2212<\/jats:sup>\n                    <jats:sup>1<\/jats:sup>\n                    h\n                    <jats:sup>\u2212<\/jats:sup>\n                    <jats:sup>1<\/jats:sup>\n                    . By eliminating intermediate 4\u2010androstenedione extraction and purification, this integrated strategy establishes a cost\u2010effective and environmentally sustainable route for industrial testosterone manufacture. More broadly, our work illustrates how synergistic metabolic engineering and structure\u2010guided enzyme design can be combined to accelerate steroid biomanufacturing.\n                  <\/jats:p>","DOI":"10.1002\/cbdv.202503343","type":"journal-article","created":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T06:35:21Z","timestamp":1779172521000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Direct Conversion of Phytosterol to Testosterone via an Integrated Microbial\u2013Enzymatic Strategy"],"prefix":"10.1002","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-9795-4859","authenticated-orcid":false,"given":"Shikui","family":"Song","sequence":"first","affiliation":[{"name":"Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology, College of Life Sciences Xinjiang Normal University  Urumqi Xinjiang 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