Identifying promoters for gene expression in Clostridium thermocellum

Abstract

A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase (lacZ) and NADPH-alcohol dehydrogenase (adhB) in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB). Keywords: Promoter, Copy number, Structural instability, Rolling circle replication, lacZ, adhB