Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803

Abstract

In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated using GFPuv as a reporter gene inserted in a broad-host-range vector. A clear induction of protein expression from native PcpcG 2 under green-light illumination was observed; however, the expression level was very low compared with Ptrc , which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine-Dalgarno-like sequence derived from the cpcB gene was inserted in the 5' untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green-light sensing system resulted in about 40-fold higher protein expression than with the wild-type promoter with a high ON/OFF ratio under green-light illumination. The engineered green-light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses.

Figure 1

Control of gene expression by green light in Synechocystis sp. PCC6803 harbouring pKT230-P_cpcG2-GFPuv.A. Growth curve under red (20 μmol m⁻² s⁻¹), green (20 μmol m⁻² s⁻¹), or both green and red light (green/red). Growth was determined by measuring the optical density at 730 nm.B. Fluorescence intensity of cells was normalized by their respective optical density at 730 nm after 39 h of incubation under each light illumination. After each culture was washed by phosphate buffered saline, fluorescence intensity was measured by a plate reader (λex. 395 nm/λem. 520 nm) (Thermofisher Scientific, Waltham, MA)

Figure 2

Evaluation of engineered green-light sensing system in Synechocystis sp. PCC6803 under red or green/red light.A. Schematic representation of each construct introduced into pKT230: P_cpcG2-GFPuv (PcpcG2), P_cpcG2-GFPuv-P_ccaS-ccaS (PcpcG2+ccaS), P_cpcG2-GFPuv-P_ccaR-ccaR (PcpcG2+ccaR).B. Relative normalized fluorescence intensity of Synechocystis sp. PCC6803 harbouring PcpcG2, PcpcG2+ccaS, or PcpcG2+ccaR under red or green/red light. Each value is normalized to cell optical density and provided relative to the normalized value of PcpcG2 under green light (set as 1).C. Relative normalized fluorescence intensity of Synechocystis sp. PCC6803 harbouring PcpcG2, pKT230-P_cpcG2-SD-GFPuv (PcpcG2-SD), P_cpcG2-SD-GFPuv+ccaR (PcpcG2-SD+ccaR) under red or green/red light. Each value is normalized to cell optical density and provided relative to the normalized value of PcpcG2 under green light (set as 1).D. Relative normalized fluorescence intensity of Synechocystis sp. PCC6803 harbouring pKT230-P_trc-SD-GFPuv (Ptrc-SD), whose SD-like sequence was replaced by the SD-like sequence of cpcB, and P_cpcG2-SD+ccaR. Each value is normalized to cell optical density.