Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

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, P _{rsp _ 7571} and P _{rsp _ 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 P _{rsp _ 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.

Keywords: Co-enzyme Q10; Promoter library; Red fluorescent protein; Rhodobacter sphaeroides; Transcriptomics.

Fig. 1

Screening and characterization of selected candidate native promoters from R. sphaeroides. A. Box plot of the FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) transcripts at 24 h for the two circular chromosomes in JDW-710 and WT. B. Transcription profile of chromosome I and the selected native promoters (highlighted in red) at 24 h in JDW-710 and WT. C. Evaluation of promoter strength by GusA enzymatic activity. D. Promoter strengths of the selected native and reference promoters in JDW-710. Control, JDW-710 containing the pBBR plasmid. Pkana, the kanamycin resistance gene promoter in the pBBR plasmid. The activity of tac was set to 100%. Data are expressed as the mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Characterization of selected native promoters in different culture media and time periods. A-B. Cell growth and GusA activity of the JDW-710 transformants in TSB medium in shake flasks. Because of variations in tac activity, the maximum activity of tac (at 32 h) was set to an expression level of 100%. C-D. Cell growth and GusA activity of JDW-710 transformants in Q₁₀ fermentation medium in shake flasks. JDW-710 containing the pBR322 plasmid served as the control. Because of the variation in tac activity, the maximum activity of tac (at 24 h) was set to an expression level of 100%. Control, JDW-710 containing the pBBR plasmid. Pkana, the kanamycin resistance gene promoter in the pBBR plasmid. Data are expressed as the mean ± SEM.

Fig. 3

Truncation and characterization of promoter P_{rsp_7571}. A. Truncation of the strong native promoter P_{rsp_7571}. T334 (334bp), T167 (167bp) and T84 (84bp) are three P_{rsp_7571}-derived promoters generated by sequence truncation. B. Relative GusA activity of truncated forms of P_{rsp_7571} in JDW-710 in TSB medium. The activity of P_{rsp_7571} at 24 h was set to an expression level of 100%.

Fig. 4

Construction of the P_{rsp_7571}-derived promoter library. A. Workflow of constitutive promoter library construction using the visualized RFP reporter system. B. The strength of 21 sequenced synthetic promoters. P_{rsp_7571} and the tac promoter is highlighted in red. The activity (RFU, relative fluorescence units) of each promoter was calibrated by cell growth (OD₇₀₀). T334-xx and T167-xx represent T334-and T167-derived promoters, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Enhanced Q₁₀ production achieved by engineering the synthetic pathway in JDW-710. A-C. Time course of cell growth, Q₁₀ production and yield in shake-flask fermentation. D. Biosynthetic pathway of Q₁₀ in R. sphaeroides. E-F. Transcriptional verification of dxs and ubiE expression in different strains in 24 h by q-PCR. The transcription of dxs and ubiE in JDW-710 (reference sample) was set to an expression level of 1.0, and the data for other strains are expressed as the fold increase of the mRNA level over the reference sample. JDW:pBBR (blank control), JDW-710 containing pBBR plasmid; DOE2 (JDW-710:pBBR-pT334*-dxs-ubiE), dxs and ubiE doubly overexpressed under the control of T334* in JDW-710; *P < 0.05.