Efficient Production of 9,22-Dihydroxy-23,24-bisnorchol-4-ene-3-one from Phytosterols by Modifying Multiple Genes in Mycobacterium fortuitum.

C19 steroids and C22 steroids are vital intermediates for the synthesis of steroid drugs. Compared with C19 steroids, C22 steroids are more suitable for synthesizing progesterone and adrenocortical hormones, albeit less developed. 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one(9-OHBA), due to its substituents at positions C-9 and C-22, is a beneficial and innovative steroid derivative for synthesizing corticosteroids. We focused on the C22 pathway in Mycobacterium fortuitum ATCC 35855, aiming to develop a productive strain that produces 9-OHBA. We used a mutant strain, MFΔkstD, that knocked out kstds from Mycobacterium fortuitum ATCC 35855 named MFKD in this study as the original strain. Hsd4A and FadA5 are key enzymes in controlling the C19 metabolic pathway of steroids in Mycobacterium fortuitum ATCC 35855. After knocking out hsd4A, MFKDΔhsd4A accumulated 81.47% 9-OHBA compared with 4.13% 9-OHBA in the strain MFKD. The double mutant MFKDΔhsd4AΔfadA5 further improved the selectivity of 9-OHBA to 95.13%, and 9α-hydroxy-4-androstenedione (9-OHAD) decreased to 0.90% from 4.19%. In the end, we obtained 6.81 g/L 9-OHBA from 10 g/L phytosterols with a molar yield of 80.33%, which showed the best performance compared with formerly reported strains.

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

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 Δ1-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 17ß-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-Δ1-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. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.