Introducing extra bicarbonate transporters bicA and sbtA in the cyanobacterium synechocystis sp. PCC 6803 for enhanced ethanol production

The cyanobacterium Synechocystis sp. PCC 6803 is a model organism for genetic manipulation. Utilising genetically engineered cyanobacteria to produce ethanol offers significant potential as a renewable and sustainable source of biofuel, as waste carbon dioxide (CO2) can be utilised to sustain the organism, decreasing greenhouse gas emissions. In addition, use of cyanobacteria as a biofuel producer may inroad into dwindling fossil fuel resources. Synechocystis has multiple carbon transport systems that allow it to transport CO2 or bicarbonate into the cell and convert it to essential nutrients by photosynthesis. In an attempt to achieve an increased ethanol yield in a genetically engineered strain of Synechocystis sp. PCC 6803, initially phenotypic differences between six sub-strains of Synechocystis were studied to select an optimum strain for transformation and prevent future micro-evolution. The transformation efficiency rate was calculated and the AA314 (Berkeley) wild-type (WT) strain was chosen as a suitable strain. Following this, cassettes with either an additional inducible copy of the endogenous bicarbonate (HCO3-) transporter gene, bicA, or the HCO3- transporter gene sbtA, were created. Inducibility was engineered by coupling the light inducible PSBAII promoter to the cassettes. These cassette constructs were transformed into Synechocystis PCC 6803 wild-type at the PSBAII neutral site. A strain with increased biomass levels, when grown at 0.5% CO2, was produced (strain UL061 containing bicA). However strains expressing sbtA did not show biomass enhancement under the construct conditions utilised. Two further cassette constructs were then created, a bicA construct and sbtA construct with Zeocin™ resistance. These constructs were transformed into the genetically engineered ethanol-producing Synechocystis PCC 6803 (UL004) at the slr0397 neutral site. An unstable ethanol-producing strain containing bicA was produced, but a stable ethanol-producing strain containing sbtA was created (strain UL066), although ethanol levels remained unaltered in strain UL066.