Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes

Abstract

We developed a synthetic promoter library for actinomycetes based on the -10 and -35 consensus sequences of the constitutive and widely used ermEp1 promoter. The sequences located upstream, in between and downstream of these consensus sequences were randomised using degenerate primers and cloned into an integrative plasmid upstream of the gusA reporter gene. Using this system, we created promoters with strengths ranging from 2% to 319% compared with ermEp1. The strongest synthetic promoter was used in a proof-of-principle approach to achieve the overexpression of a natural type III polyketide synthase. We observed high correlation between the number of gusA reporter gene RNA-Seq reads and the GusA reporter protein activity, indicating that GusA is indeed a transcription-level reporter system.

Keywords: Actinomycetes; Erme promoter; GusA; RNA-Seq; RppA; Synthetic promoter; Transcriptomic analysis.