Abstract
Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.
Highlights
Efficient and well-tuned co-expression of multiple genes is a common challenge in metabolic engineering and synthetic biology, wherein protein components must be optimized in terms of cumulative expression, expression ratios, and regulation[1,2,3,4]
Our study began by searching for Natural BDPs (nBDPs) that might satisfy various engineering needs (Fig. 1a), targeting our search to the yeast P. pastoris
Bioinformatics approaches (Supplementary Data 1, Supplementary Note 1) identified 1462 putative bidirectional promoters (BDPs) in P. pastoris’ genome (Fig. 1b), with a subset of 40 BDPs selected for detailed characterization due to their expected high expression as housekeeping genes or previous application as monodirectional promoters (MDPs) (Fig. 1c, see Supplementary Data 2 for a list of the promoters tested)
Summary
Efficient and well-tuned co-expression of multiple genes is a common challenge in metabolic engineering and synthetic biology, wherein protein components must be optimized in terms of cumulative expression, expression ratios, and regulation[1,2,3,4]. NBDPs with non-cryptic expression in both orientations frequently co-regulate functionally related genes[20,21] Inspired by these circuits, biological engineers have recently utilized BDPs to improve designs for gene coexpression in Escherichia coli[22], Saccharomyces cerevisiae[23], plants[24], and mammals[25,26]. Our library covers a 79-fold range of cumulative expression, has variable expression ratios ranging from parity to a 61-fold difference between sides, and combines different regulatory profiles per side including the possibility for consecutive induction The utility of these BDPs is demonstrated through the optimization of multi-gene co-expression, and the conserved nature of the framework histone promoters suggests the generalizability of this approach for other eukaryotes
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