Fig. 4
From: Engineered bidirectional promoters enable rapid multi-gene co-expression optimization
Modularly designed and exceptionally short bidirectional hybrid promoters (179–457 bp) achieve the highest expression strength per bp of promoter length matching the strong monodirectional AOX1 promoter (940 bp length). The bidirectional hybrid promoters were assembled from histone core promoters (Fig. 2b) and CRMs of methanol-regulated promoters (Supplementary Fig. 2, Supplementary Fig. 4). The detailed color code for the regulatory elements/abbreviations used is provided in Supplementary Fig. 4; a list of the exact designs of shBDP1–31 is provided in Supplementary Data 2. Yellow boxes indicate experimentally confirmed Mxr1p (methanol master regulator) binding sites in PAOX1 and PDAS2 (Supplementary Fig. 2, Supplementary Fig. 4); red boxes: TATA boxes. Additional bidirectional variants, controls, and extended discussion are provided in Supplementary Fig. 4 and Supplementary Note 5. PAOX1 is a reference of a monodirectional, strong, methanol-inducible promoter. PAOX1 was cloned in forward and reverse orientation in the bidirectional reporter vector, therefore the values shown are derived from separate constructs and not from bidirectional activity. CP core promoter, CRM cis-regulatory module. “HHT2-T3” is the truncated side of a bidirectional histone promoter (see Fig. 2c, d) used to generate hybrid promoters with growth-associated expression from one side. Strains were grown on glucose media for 60 h subsequently induced with methanol for 48 h. 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). All elements used (except for the non-regulated core promoters and constitutive HHT2-T3) are methanol inducible
