Biocontainment of Engineered Synechococcus elongatus PCC 7942 for Photosynthetic Production of α-Farnesene from CO2

Biocontainment systems have been developed to mitigate the concerns regarding biosafety and environmental risk because of the possible escape of genetically modified organisms into the environment following large-scale outdoor cultivation. Here, we present a biocontainment system entailing genetically modified Synechococcus elongatus PCC 7942, also engineered for alpha-farnesene production using a de-evolutionary strategy. In this approach, the gene cluster encoding the beta-carboxysome and the associated carbon concentrating mechanism (CCM) were deleted in the alpha-farnesene-producing cyanobacteria, resulting in no cell growth and no alpha-farnesene production at ambient CO2 concentrations (100% air bubbling). However, cell growth and alpha-farnesene production were detected in the CCM-deficient strains at high CO2 concentrations (5% CO2 [v/v], 10% CO2 [v/v]), albeit at levels lower than those of the parental control. To overcome this limitation, the overexpression of carbonic anhydrase and bicarbonate transporter genes in the CCM-deficient strains restored cell growth and the production level of alpha-farnesene (5.0 +/- 0.6 mg/L) to that of the parental control. The production of alpha-farnesene in the later strains strictly depended on CO2 concentration in the photobioreactor and did not rely on a chemical induction process. Thus, next generation bio-solar cell factories could be promoted with the suggested biocontainment system.