Biosynthesis of non-natural progestogen in yeast

Steroid hormones comprise hundreds of clinically used drugs, and their de novo biosynthesis from simple bio-based carbon sources holds great potential for advancing novel green industries. As a vital class of steroid hormones, progestogens are widely used to treat menstrual disorders, breast cancer, and pregnancy-related conditions. Here, we identified a novel steroid dehydrogenase from Marsdenia tenacissima , Mt CYP82BQ1, which catalyzes Δ 6 -dehydrogenation of progesterone, leading to the synthesis of 6-dehydroprogesterone, a non-natural progestogen with therapeutic potential. Through homology modeling and site-directed mutagenesis experiments, the key residues governing the catalytic activity of Mt CYP82BQ1 were identified. In order to construct an efficient cell factory for the production of 6-dehydroprogesterone. Initially, the best performing P450 sterol side chain cleavage enzyme (P450scc) Cc CYP87N1 and 3 β -hydroxysteroid dehydrogenase/Δ 5 -Δ 4 ketosteroid isomerase (3 β HSD) Cc HSD6 from Cynanchum chinense were screened for progesterone synthesis, a key precursor in the synthesis of 6-dehydroprogesterone. Subsequently, semi-rational design of Cc CYP87N1 obtained the Cc CYP87N1 F282W mutant with 74.4 % increased activity, which further improved progesterone titer by 14.0 %. Through further metabolic regulation and fermentation optimization, the progesterone titer reached 1.34 g/L in a 5 L bioreactor. Furthermore, advantageous Mt CYP82BQ1 variants were employed to construct the 6-dehydroprogesterone-producing strains. The optimization of heme metabolism boosted the 6-dehydroprogesterone titer by 107.3 %, achieving 26.1 mg/L. Finally, homologous enzymes of Ps CYP82BQ2 from Periploca sepium , exhibiting a high substrate conversion rate of 90.4 %, were screened and utilized to improve the 6-dehydroprogesterone production. The 6-dehydroprogesterone titer was further increased by 28.9 %, and a final titer of 0.72 g/L was achieved through fed-batch fermentation. This work provides a sustainable platform for the industrial-scale production of progesterone derivatives.