A Green Light-Regulated T7 RNA Polymerase Gene Expression System for Cyanobacteria
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ORIGINAL ARTICLE
A Green Light-Regulated T7 RNA Polymerase Gene Expression System for Cyanobacteria Chika Shono 1 & Dwi Ariyanti 1,2 & Koichi Abe 1 & Yuta Sakai 1 & Ippei Sakamoto 1 & Kaori Tsukakoshi 1 & Koji Sode 1,3 Kazunori Ikebukuro 1
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Received: 14 July 2020 / Accepted: 10 September 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract 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, pKTCS01, 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 PtrcĪlacO 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 T7 RNA polymerase . Green light-regulated gene expression system . Light-regulated bioprocesses . CcaS/CcaR . Synechocystis sp. PCC6803 . Cyanobacteria
Introduction
Chika Shono and Dwi Ariyanti contributed equally to this work. * Koji Sode [email protected] * Kazunori Ikebukuro [email protected] 1
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan
2
Faculty of Biotechnology, Sumbawa University of Technology, Olat Maras, Moyo Hulu, Sumbawa, West Nusa Tenggara 84371, Indonesia
3
Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA
The design of bioprocesses using cyanobacteria as host microorganisms is a challenging but is also attractive considering the potential of cyanobacteria to produce various valuable compounds, due to their photoautotrophic properties, utilizing CO2 and a simple nutrient com
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