Engineering of riboregulators for cyanobacteria

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Koichi Abe, Yuta Sakai, Saki Nakashima, Koji Sode, Kazunori Ikebukuro

1) Tokyo University of Agriculture and Technology 2) JST, Japan

Direct conversion of CO2 to fuel or drug using the ability of carbon fixation of photoautotrophic organisms, such as cyanobacteria, has received increasing attention to solve the energy or environmental problem. We aim to construct a synthetic cyanobacterial host designated as “The CyanofactoryTM”, in which we can control biofuel production, aggregation, and cell lysis by various light signals. Among the many biological tools for cell optimization, RNA has great potential because it can be conveniently designed based on its sequence and predicted structure. In this study, we designed two coupled riboregulators (crRNA and taRNA) to regulate gene expression in cyanobacteria. Located in the 5 untranslated region of a target gene, the crRNA forms a stem-loop structure that includes the ribosome-binding site (RBS), thus preventing the binding of the ribosome to the RBS, resulting in repression of translation of the target gene. The taRNA, which includes a complementary sequence to a region of the crRNA, disrupts the crRNA stem-loop structure, resulting in activation of translation of the target gene. The strict control of gene expression that can be achieved with riboregulators allows us to better control cell activity with various external signals. We designed riboregulators that has RBS designated RBS* showed higher expression level in Synechocystis sp. PCC6803. Fist of all, we evaluated the function of designed riboregulators using GFP as reporter protein in E. coli. By adjusting intramolecular or intermolecular hybridization energy of crRNA or between crRNA and taRNA, respectively, we have succeeded in construction of crRNA, which has RBS* and taRNA. Designed riboregulators also worked well in Synechocystis sp. PCC6803. Since riboregulators can adjust proper gene expression level, they would be useful tools for biofuel production in cyanobacteria.