Driving the conversion of phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide.

Background: The conversion of phytosterols to steroid synthons by engineered Mycolicibacteria comprises one of the core steps in the commercial production of steroid hormones. This is a complex oxidative catabolic process, and taking the production of androstenones as example, it requires about 10 equivalent flavin adenine dinucleotide (FAD). As the high demand for FAD, the insufficient supply of FAD may be a common issue limiting the conversion process.
Results: We substantiated, using the production of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) as a model, that increasing intracellular FAD supply could effectively increase the conversion of phytosterols into 9-OHAD. Overexpressing ribB and ribC, two key genes involving in FAD synthesis, could significantly enhance the amount of intracellular FAD by 167.4% and the production of 9-OHAD by 25.6%. Subsequently, styrene monooxygenase NfStyA2B from Nocardia farcinica was employed to promote the cyclic regeneration of FAD by coupling the oxidation of nicotinamide adenine dinucleotide (NADH) to NAD ⁺ , and the production of 9-OHAD was further enhanced by 9.4%. However, the viable cell numbers decreased by 20.1%, which was attributed to sharply increased levels of H ₂ O ₂ because of the regeneration of FAD from FADH ₂ . Thus, we tried to resolve the conflict between FAD regeneration and cell growth by the overexpression of catalase and promotor replacement. Finally, a robust strain NF-P2 was obtained, which could produce 9.02 g/L 9-OHAD after adding 15 g/L phytosterols with productivity of 0.075 g/(L h), which was 66.7% higher than that produced by the original strain.
Conclusions: This study highlighted that the cofactor engineering, including the supply and recycling of FAD and NAD ⁺ in Mycolicibacterium, should be adopted as a parallel strategy with pathway engineering to improve the productivity of the industrial strains in the conversion of phytosterols into steroid synthons.
(© 2023. The Author(s).)