Fig. 1

A library of bidirectional promoters (BDPs) for gene co-expression fine-tuning and bidirectional histone promoters are among the few strong P. pastoris nBDPs. a A library of diversely regulated natural and synthetic BDPs (nBDPs and sBDPs) covering a wide range of regulatory profiles facilitates optimization of dual gene co-expression and the assembly of multi-gene co-expression cassettes (Supplementary Fig. 5, Supplementary Note 6). b The P. pastoris genome harbors 1462 putative nBDPs (gene pairs in divergent head-to-head orientation, Supplementary Data 1, Supplementary Note 1). The distribution of distances between gene pairs is shown in 25 bp intervals. The last bar indicates gene pairs with an intragenic distance >1000 bp. Also convergent tail-to-tail gene pairs (forming putative bidirectional transcription terminators, BDTs) and head-to-tail/tail-to-head gene pairs flanking a monodirectional promoter (MDP) and a monodirectional terminator (MDT) are shown. Genes are illustrated as bold single-line arrows, promoters as filled arrows, terminators as rectangles. c The natural bidirectional DAS1-DAS2 promoter is the only methanol-inducible P. pastoris promoter³⁰ showing strong reporter gene fluorescence on both sides and histone promoters are the strongest nBDPs of several housekeeping gene pairs tested in P. pastoris. All strains were grown on glucose media for 60 h and MUT promoters subsequently induced with methanol for 48 h (for MUT promoters measurements after growth on methanol, for housekeeping genes on glucose are shown, see Supplementary Data 2 for the exact values). The promoters were screened with a single reporter gene in both orientations and bidirectional expression confirmed using two FPs (normalization factor used as determined in Supplementary Fig. 1). Gene names denoted with an asterisk (*) were shortened and are provided in Supplementary Data 2. Mean values and standard deviations of biological quadruplicates are shown. PBI peroxisome biogenesis and import, ROS reactive oxygen species, TX,TL transcription, translation