The rrnA promoter as a tool for the improved expression of heterologous genes in cyanobacteria

Abstract

The regulatory sequence of ribosomal RNA A (rrnA) operon from Synechococcus PCC7942 was characterized using green fluorescent protein gene (gfp) as a reporter. The P<inf>R</inf> promoter (nt. -83 to +2) including upstream promoter element and P1 promoter of rrnA exhibited GFP fluorescence intensity about 30-fold higher than full length sequence (nt. -147 to +79). The effects of P<inf>R</inf> promoter arranged in tandem with consensus-σ⁷⁰ promoter (P<inf>S</inf>) of Escherichia coli on the expression of gfp and opd gene encoding organophosphorus hydrolase (OPH) in Synechococcus were investigated. The P<inf>S</inf>-P<inf>R</inf> tandem promoter was superior to all of the other promoters; its GFP fluorescence intensity was a 1.8-fold increase when compared to P<inf>R</inf>-P<inf>R</inf> tandem promoter, a 2.5-fold, 9.5-fold and a 15-fold increase compared to P<inf>R</inf>, P<inf>S</inf> and promoter of tRNA^pro, respectively. The GFP from P<inf>S</inf>-P<inf>R</inf> tandem promoter accounted for about 12% of its total extracted proteins. OPH activity of Synechococcus harboring opd gene under the control of P<inf>S</inf>-P<inf>R</inf> tandem promoter was 738±128units/OD<inf>730</inf>. We demonstrated that the tandem promoters remarkably enhanced the GFP and OPH production which were detected on SDS-PAGE stained with Coomassie blue. The promoter system in this study could be generally applied to production of valuable organic products from cyanobacteria. © 2013 Elsevier GmbH.