Fig. 5

Cyclic regeneration of FAD and NAD⁺. (a) Schematic illustration of the cyclic regeneration of FAD and NAD⁺. The conversion of phytosterols into 9-OHAD was accompanied by the transformation of NAD⁺ into NADH and FAD into FADH₂ in Mycolicibacterium. The combined expression of key genes ribB and ribC in FAD biosynthesis pathway resulted in the supply of intracellular FAD. The NADH-FAD(H2)-dependent styrene monooxygenase SMO (in yellow color) was expressed to promote the cyclic regeneration of FAD and NAD⁺ including the catalytic mechanism of the self-sufficient SMO. FAD regeneration was performed by the NAD⁺ (in red dashed) and oxygen (in solid purple line) consumption and production of NADH (in blue) and H₂O₂ (in purple), respectively. NAD⁺ regeneration was performed by the FAD consumption (in red dashed) and FADH₂ production (in solid blue line). (b) Effect of overexpressing the flavincontaining monooxygenase genes RoStyA2B (GenBank: ANS29975.1), PaStyA2B (GenBank: ABM10099.1), and NfStyA2B (GenBank: BAD56094.1) on 9-OHAD production and cell growth; All engineered strains were cultivated in fermentation medium containing 4 g/L phytosterols at 30℃ for 144 h. The data represent the mean ± standard deviation of three measurements. The data represent the mean ± standard deviation of three measurements.