9α-Hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a representative precursor in steroid drug synthesis.However, during the 9-OHAD production from phytosterol in Mycolicibacterium neoaurum (M. neoaurum), the current manufacturing processes suffer from low productivity and purity due to the generation of a variety of byproducts.In this study, we genetically modified the phytosterol catabolism in M. neoaurum and achieved 13.4 g/L and 91.1 % molar yield of 9-OHAD with no detectable byproducts at a high phytosterol concentration of 20 g/L in shake flask.It was achieved by inactivating Opccr, SalA, TeB, and overexpressing KshA1 and ChsH1-2 to eliminate the production of 20-hydroxymethyl-9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HBC), 4-androstene-3,17-dione (4-AD) and 3-oxo-4-pregnene-9-OH-20-carboxylic Me ester (9-OH-3-OPCM) as well as overcoming the production limitations of 9-OHAD.Notably, when scaled up in a 5 L bioreactor with 45 g/L phytosterol concentration, the modified M. neoaurum achieved 24.5 g/L and 74.5 % molar conversion of 9-OHAD by using soybean oil and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) as a co-solvent and antifoam agent.Therefore, our findings demonstrate a method to improve the efficiency and purity of 9-OHAD biosynthesis in M. neoaurum.