Discovery and Characterization of Native Deinococcus radiodurans Promoters for Tunable Gene Expression

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in Of these top eight, two promoter regions, and , were stronger and more consistent than the most widely used promoter sequence in , Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in or were specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in The tolerance of the extremophile, , to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in , very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in The promoters identified in this study will facilitate metabolic engineering of and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.