Fig. 4: Elucidation of the upstream pathway of ECH biosynthesis.
a Expression heatmap of candidate upstream genes and the identified downstream genes in C. tubulosa cell suspension cultures treated with 6% PEG6000. b Decarboxylation reaction catalysed by CtTyDC towards L-tyrosine (1) or L-dopa (3) to form tyramine (2) or dopamine (4), respectively. c Combined decarboxylation-deamination reaction catalysed by the mutant CtTyDCY347F in the presence of PLP towards L-tyrosine (1) to form 4-HPAA (7), which was further reduced to tyrosol (9) by NaBH4, or L-dopa (3) to 3,4-diHPAA (8), which was further reduced to hydroxytyrosol (10) by NaBH4. d The amino transfer reaction of L-tyrosine (1) to form 4-HPPA (5) or L-dopa (3) to form 3,4-diHPPA (6), respectively, catalysed by CtTAT in the presence of PLP and α-ketoglutarate. e With the addition of TPP, CtPPDC could decarboxylate 4-HPPA (5) to form 4-HPAA (7), which was further reduced to tyrosol (9) by NaBH4, or decarboxylate 3,4-diHPPA (6) to form 3,4-diHPAA (8), which was further reduced to hydroxytyrosol (10) by NaBH4. f Ct4HPAR could catalyse the reduction of 4-HPAA (7) or 3,4-diHPAA (8) to generate tyrosol (9) or hydroxytyrosol (10), respectively, using NADH as the cofactor. (b-f) HRESI-MS and MS2 spectra of the products are listed in Supplementary Fig. 36. g Heterologous transient expression of different combinations of upstream-related genes (Comb. 1–Comb. 7 annotated in Supplementary Table 8) in N. benthamiana. The detection wavelength was set at 280 nm. Source data are provided as a Source Data file.
