Rapid RFP Screening in Microbial Transformants

In a study published in Synthetic and Systems Biotechnology (Shi et al., 2021), researchers from East China University of Science and Technology developed a promoter library for Rhodobacter sphaeroides, an industrially important photosynthetic bacterium used for coenzyme Q10 production. To rapidly screen transformants with varying promoter strengths, the team employed the LUYOR-3260GR portable fluorescent protein excitation lamp with a red filter (LUV-50A).

Abstract

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, Prsp_7571 and Prsp_6124, 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 Prsp_7571-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.

Red Fluorescence Analysis

During the promoter library construction, a portable fluorescent protein excitation lamp LUYOR-3260GR and red filter LUV-50A (LUYOR Instrument Co., Ltd), were used to screen R. sphaeroides transformants on agar plates. For 48-well microplate culture, the red fluorescence was measured with a BMG CLARIOstar microplate reader with excitation at 570 nm and emission at 620 nm.

Photo below: RFP fluorescence captured by fluorescent protein excitation lamp LUYOR-3260GR and red filter LUV-50A

Reference

Shi, T., Zhang, L., Liang, M., Wang, W., Wang, K., Jiang, Y., Liu, J., He, X., Yang, Z., Chen, H., Li, C., Lv, D., Zhou, L., Chen, B., Li, D., Zhang, L.-X., & Tan, G.-Y. Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides. Synth. Syst. Biotechnol. 7, 15-25 (2022).