Fig. 2: The biosynthesis pathway from biflavonoids 1 to its methyl modified derives in G. biloba.

For GbOMT characterization, crude enzymes generated from BL21(DE3) carrying the empty vector pET28a were used as a negative control. a Expression patterns of screened O-methyltransferases in different organs. The figure displayed the values as log₂FPKM. b LC-MS/MS analysis of G. biloba OMTs chr7.385 (GbOMT1) and chr8.315 (GbOMT2) activities on substrates 1 in vitro. GbOMT1 and GbOMT2 catalyzed substrate 1 to produce bilobetin (2). MS² fragmentation spectra for authentic 2 and products of GbOMT1 and GbOMT2 were shown on the right. c LC-MS/MS analysis of G. biloba OMTs chr1.879 (GbOMT3), chr10.1253 (GbOMT4) and chr12.613 (GbOMT5) activities on substrates 2 in vitro. GbOMT3 catalyzed 2 to produce ginkgetin (3), GbOMT4 and GbOMT5 catalyzed 2 to produce isoginkgetin (4). d LC-MS/MS analysis of G. biloba OMTs chr1.879 (GbOMT3), chr10.1253 (GbOMT4), and chr12.613 (GbOMT5) activities on substrate 1 in vitro. GbOMT4 and GbOMT5 catalyzed substrates 1 to podocarpusflavone A (8). GbOMT3 catalyzed 1 to sequoiaflavone (9). MS² fragmentation spectra of products 8 and 9 were shown on the right side. e LC-MS/MS analysis of G. biloba OMTs chr7.385 (GbOMT1) activities on substrates 2 in vitro. GbOMT1 catalyzed 2 to 10, which was speculated as 4,7”-O-methylamentoflavone. f The biosynthetic pathway of biflavonoid 1 and its methylated derivatives 2–4 and 8–10 in G. biloba. GbCYP90J6 dimerized 5 to generate 1, which served as the common precursor for 2–4 and 8–10. GbOMT1 and GbOMT2 catalyze the conversion of 1 to 2. Subsequently, GbOMT3 can modify 2 to produce 3, while GbOMT4 and GbOMT5 convert 2 into 4. 8–10 are shunt products.