Fig. 3

FAD supply in engineered 9-OHAD-producing strains. a Schematic illustration of the biosynthesis pathway of FAD production in prokaryotes. Enzymes encoded by the genes were as follows: ribA, GTP cyclohydrolase II; ribB, 3,4-dihydroxy-2-butanone 4-phosphate synthase; ribD, fused pyrimidine deaminase/uracil reductase; ribH, lumazine synthase; ribF, riboflavin synthase; ribC, riboflavin kinase/FMN adenylyltransferase. The metabolic intermediates were as follows: R5P, ribulose-5-phosphate; DHBP, ribulose-5-phosphate; ARPP, 5-amino-6-(5-phospho-D-ribitylamino)-uracil; Arp, 5-amino-6-(D-ribitylamino) uracil; DARPP, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5’-phosphate; GTP, guanosine-triphosphate; DRL, 6,7-dimethyl-8-(1-D-ribityl) lumazine. b Intracellular FAD concentration and 9-OHAD production in engineered strains in which ribA, ribD, ribB, ribH, ribC, and ribF was overexpressed individually. c Intracellular FAD concentration and cell growth and d 9-OHAD production in engineered strains with combined overexpression of ribB and ribC. All engineered strains were cultivated in fermentation medium containing 4 g/L phytosterols at 30 °C for 144 h. The data represent the mean ± standard deviation of three measurements