Fig. 1.

Identification of a high limonene-producing mutant of Synechococcus 2973. (A) The plasmid pSL3385 containing lims from M. spicata and gpps from A. grandis was introduced in Synechococcus 2973. A synthetic RBS for the gpps gene was obtained from the RBS calculator, with half of the TIR compared to the RBS in trc1O promoter. (B) Limonene production in Synechococcus 2973 expressing the plasmid pSL3385. One mutant produced 2.5-fold higher of limonene compared to the other strains. The experiment was conducted with the initial OD₇₃₀ of 0.05 in 1% CO₂ and 500 ​μmol photons m⁻² ​s⁻¹ light for 2 days (C) Schematics of testing the difference in genome background in the high and low limonene producers. The plasmid pSL3385 was cured and then re-introduced into the strains. (D) Limonene production of the re-constructed high and low limonene-producing strains. The experiment was conducted with the initial OD₇₃₀ of 0.05 in 1% CO₂ and 250 ​μmol photons m⁻² ​s⁻¹ light for 2 days. RBS, ribosome biding site; TIR, translation initiation rate.