Fig. 2

Combinatorial optimization of the (2S)-pinocembrin pathway through the Design/Build/Test/Learn cycle. a A biosynthetic pathway composed of four enzymes (PAL, 4CL, CHS, and CHI; see Supplementary Table 2) was initially selected²¹. In the first 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 (pSC101 and p15a). Through the application of statistical DoE, the designed library was reduced to 16 representative constructs. This pathway library was assembled and expressed in E. coli DH5α to test pinocembrin titers. Statistical analysis was then used to assess the relative effects of the different design factors tested. b In the second DBTL cycle, a new focused combinatorial library was designed, based on experimental factors from the first cycle which correlated with pinocembrin titer. For this second full-factorial library, PAL was fixed at the end of the pathway and CHI at the beginning, while CHS and 4CL were allowed to exchange positions with or without promoter parts