Synechococcus elongatus UTEX 2973 was cultured in BG11 medium at 38°C, 250 ​μmol photons m−2 ​s−1 light, and 1% CO2 in an AlgaeTron growth chamber (Photon Systems Instruments, Czech Republic). For the mutant strains, the antibiotic kanamycin (10 ​μg mL−1) was used in BG11 agar plates or liquid medium. To test limonene production, mutant strains were grown in BG11 medium for 24 hours, and then diluted to an OD730 of 0.05 (0.06 ​g ​L−1 ​cell dry weight) to initiate the experiments. To compare the limonene production of cyanobacteria from previous studies, a higher initial OD730 of 0.4 (0.5 ​g ​L−1 ​cell dry weight) was used for the experiment. Strains were grown in 10 ​mL of BG11 medium in 50-mL flasks with 10% (v/v) of isopropyl myristate (Kato et al., 2017) covered as an organic overlay to trap limonene. IPTG (1 ​mM) was added to induce gene expression.

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