Yuan, Chenyang; Ma, Zhiguo; Li, Yixin; Zhang, Jingxian; Liu, Xiangcen; Han, Suwan; Du, Guilin; Shi, Jiping; Sun, Junsong; Zhang, Baoguo

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

2023, 107, 1432-0614

Yuan, Chenyang; Ma, Zhiguo; Li, Yixin; Zhang, Jingxian; Liu, Xiangcen; Han, Suwan; Du, Guilin; Shi, Jiping; Sun, Junsong; Zhang, Baoguo

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Delta(1)-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17 beta-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Delta(1)-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry.