A comprehensive genetic toolkit for Zymomonas mobilis; allowing rapid, high-resolution, combinatorial engineering of metabolic pathways.

The limited availability of reliable genetic tools has constrained the precise engineering of Zymomonas mobilis, a promising bacterium for the sustainable bioproduction of fuels and commodity chemicals. Here, we present a comprehensive, modular genetic toolkit that enables high-resolution, combinatorial control of gene expression in Z. mobilis. We constructed and characterised a synthetic promoter library spanning the broadest dynamic range reported for this organism, alongside a suite of strong transcriptional terminators that effectively insulate genetic cassettes and prevent transcriptional readthrough. To achieve predictable translation across diverse genetic contexts, we further implemented bicistronic design (BCD) elements, allowing context-independent control of expression. Promoter activities were shown to be stable under both aerobic and anaerobic conditions, highlighting the robustness of the regulatory parts across physiologically relevant environments. The toolkit was integrated into the Start-Stop Assembly system, facilitating high-throughput construction of multi-gene pathways. We demonstrate its utility through the rapid assembly and testing of the possible expression space of genes encoding a 2,3-butanediol biosynthetic pathway. Collectively, this genetic toolkit now allows the high-resolution engineering of multi-enzyme pathways in Z. mobilis for industrial biotechnology applications.