In this study, we developed a green light-regulated T7 RNA polymerase expression system (T7 RNAP system), to provide a novel and versatile high-expression system for cyanobacteria without using any chemical inducer, realizing high expression levels comparable with previously reported for recombinant gene expression in cyanobacteria. The T7 RNAP system was constructed and introduced into Synechocystis sp. PCC6803. T7 RNAP was inserted downstream of the cpcG2 promoter, which is recognized and activated by the CcaS/CcaR two-component green-light-sensing system, to compose a vector plasmid, pKT-CS01, to achieve the induction of T7 RNAP expression only under green light illumination, with repression under red light illumination. The reporter gene, superfolder green fluorescent protein (sfGFP), was inserted downstream of the T7 promoter. Transcriptional analyses revealed that T7 RNAP was induced under green light but repressed under red light. Expression of the sfGFP protein derived from pKT-CS01 was observed under green light illumination and was approximately 10-fold higher than that in the control transformant, which expressed sfGFP directly under the cpcG2 promoter, which is directly regulated by CcaS/CcaR, under green light illumination. Comparison with the strong promoter expression systems Pcpc560 and PtrcΔlacO revealed that the expression of sfGFP by the T7 RNAP system was comparable with the levels obtained with strong promoters. These results demonstrated that the green light-regulated T7 RNAP gene expression system will be a versatile tool for future technological platform to regulate gene expression in cyanobacterial bioprocesses.
Keywords: CcaS/CcaR; Cyanobacteria; Green light-regulated gene expression system; Light-regulated bioprocesses; Synechocystis sp. PCC6803; T7 RNA polymerase.