Fig. 4: Identification and functional expression of the flavoprotein berberine bridge enzyme (BBE).

a Enzymatic reaction catalyzed by reticuline oxidases. b The clustering of reticuline oxidases in SSN of the BBE family, leading to the identification of two BBEs derived from C. japonica and M. cordata. c Characterization of BBE variants from different species and those modified with signal peptide truncation and/or an N-terminal MBP fusion tag in the RET202 strain. The significance of scoulerine titer differences was calculated using two-way ANOVA followed by Tukey’s multiple comparisons test. d Subcellular localization and expression of CjBBE, McBBE, and their variants by fusing EGFP at the C-terminus, visualized through fluorescence confocal microscopy. Micrographs were representative of at least two independent experiments. e Intermediate and product accumulation upon integration of MBP-tMcBBE in the SAN220-tsINT strain. Abbreviations not previously defined in the text are as follows: NOR, (S)-norcoclaurine; RET, (S)-reticuline; SCO, (S)-scoulerine; CHE, (S)-cheilanthifoline; PRO, protopine; SAN, sanguinarine. The significance of sanguinarine titer differences was calculated using two-way ANOVA followed by Sidak’s multiple comparisons test. Data are presented as mean ± s.d. (n  =  3 biologically independent samples). Source data are provided as a Source Data file.