Fig. 3

Modular design strategies of synthetic bidirectional promoters (sBDPs) in P. pastoris via bidirectionalization and fusions of MDPs yield sBDPs extending the repertoire of ratios and regulatory profiles. a Bidirectionalization of MDPs by addition of core promoters (Fig. 2b) yielded functional BDPs in most cases, but few designs gave high expression. The core promoters (CPs) indicated were fused to the indicated MDPs. The length of the MDPs is given in bp, selection criteria are outlined in Supplementary Note 4. Asterisk (*): In case of the PMP20 promoter, slightly varying sequences from the CBS7435 and the GS115 strain were tested (Supplementary Note 4). Strains were grown on glucose media for 60 h and subsequently induced with methanol for 48 h. b Fusions of differently regulated MDPs yield BDPs with different regulatory profiles on each side. Fusions of methanol-inducible MDPs provide a set of strong, tightly regulated, sequence-diversified BDPs allowing co-expression of up to 10 genes without reusing any sequence (Supplementary Note 4 and Supplementary Table 1 for details on the MDPs). For P_HTA1 and P_HTB1, the truncated versions shown in Fig. 2b and Supplementary Fig. 4 were used. Asterisk (*): Only the fusion of P_DAS2-699+P_DAS1-552 is shown, for additional comparisons see Supplementary Fig. 2. c Fusing deletion variants of P_DAS1 and P_DAS2 offers strong inducible BDPs with different expression ratios demonstrating that variants of MDPs can be combined into BDPs maintaining their properties on each side. The rationale for the selection of the deletions in P_DAS1 and P_DAS2 and the measurements of the separate promoters are shown and explained in Supplementary Fig. 2/Supplementary Note 3. Fluorescence was measured after 48 h methanol induction and shown as the percentage of the unmodified fusion promoter (P_DAS2-1000+P_DAS1-1000). The bidirectionalized and fusion BDPs maintained the regulatory modes of the respective MDPs^30,31: methanol inducible and tightly glucose/glycerol repressed (P_AOX1, P_PMP20, P_DAS1/2 [and deletion variants thereof], P_FBA2, P_TAL2, P_AOX2), derepressed and methanol inducible (P_CAT1, P_FLD1, P_FDH1), and constitutive (P_GAP, P_TEF1, P_ADH2, P_{HTX1 [HTA1-HTB1]}). In all panels of this figure, mean values and standard deviations of normalized (using the normalization factor calculated in Supplementary Fig. 1) reporter protein fluorescence measurements of biological quadruplicates grown on the respective carbon sources are shown (see Supplementary Data 2 for the exact values)