A Synthetic Promoter Library Spanning a Wide Dynamic Range for Metabolic Pathway Engineering in Zymomonas mobilis

Abstract The limited availability of reliable genetic tools has hindered the engineering of Zymomonas mobilis, a promising bacterium for the sustainable bioproduction of commodity chemicals. Here, we present a synthetic promoter library that enables high-resolution control of gene expression in Z. mobilis. We constructed and characterized 29 promoters spanning the broadest dynamic range reported for this organism to date, and 25 were identified with expression levels higher than the previously reported strongest native constitutive promoter, Pgap, including some up to 15-fold higher. Additionally, a set of strong heterologous and synthetic transcriptional terminators that effectively insulate genetic cassettes was validated in Z. mobilis. Promoters were stable under both aerobic and anaerobic conditions, highlighting their robustness in industrially relevant conditions. Surprisingly, all of the promoters identified contained non-canonical promoter element sequences that adhered poorly to the proposed consensus sequences. The promoter library and terminators were integrated into the Type IIS-based, hierarchical start−stop assembly system, facilitating high-throughput construction of multi-cistronic designs. We demonstrate their utility through the rapid construction, testing, and optimization of a 2,3-butanediol biosynthetic pathway. This resource substantially expands the accessible expression space for metabolic engineering in Z. mobilis, supporting precise tuning of multi-step pathways and genetic circuits.