Evaluation of inducible promoter–riboswitch constructs for heterologous protein expression in the cyanobacterial species Anabaena sp. PCC 7120

Abstract

Abstract Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.