Fig. 4.

Combinatorial optimization of the (S)-reticuline/(S)-scoulerine pathway through the Design/Build/Test/Learn cycle. a A biosynthetic pathway composed of four enzymes from Coptis japonica (6OMT, CNMT, 4′OMT, and BBE; see Supplementary Table 10) was selected. For this DBTL cycle, a combinatorial library totaling 2592 pathway configurations was designed by varying the order of pathway genes, promoter parts (P_trc and P_lacUV5) and plasmid copy numbers (pBBR1 and ColE1 origins). Through the application of statistical DoE, the designed library was reduced to 16 representative constructs, of which 14 were successfully assembled and tested. This pathway library was expressed in E. coli DH5α and reticuline titers were quantified. Statistical analysis was then used to assess the relative effects of the different design factors tested. b Quantification of scoulerine titers observed for the same 14 constructs and statistical analysis of the relative effects of the different design factors