Construction of a Promoter Library for Use in Zymomonas Mobilis

The bacterium Zymomonas mobilis is of interest for the commercial production of lignocellulosic biofuels due to its ability to produce the biofuel ethanol at near theoretical yields. Additionally, Z. mobilis displays the “uncoupled growth” phenomenon, meaning it metabolizes sugars rapidly regardless of its requirements for growth. It then converts less than 3% of these sugars into biomass. These characteristics make Z. mobilis an ideal candidate not only for ethanol production, but for heterologous product production as well. However, genetic tools for this organism are limited, and as a result, few recombinant strains have emerged. Specifically, there exists no standard set of promoters for the precise tuning of heterologous gene expression in Z. mobilis. Therefore, we have used error-prone PCR as well as rational design techniques to construct a library of promoters of varying strengths based on the strong and constitutive Z. mobilis pyruvate decarboxylase (pdc) promoter. We characterized our promoter library using transcriptional fusions to the fluorescent protein Venus and have shown that it leads to varying levels of gene expression in Z. mobilis. These promoters can be used for efficient pathway engineering in the strain for the production of value-added products.