Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein...

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q₁₀ (Q₁₀) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, P and P , showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a P -derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q₁₀ was modulated by the selected promoter T334* in JDW-710; the Q₁₀ yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.