Engineered cyanobacteria with additional overexpression of selected Calvin-Benson-Bassham enzymes show further increased ethanol production.

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in PCC 6803. The ethanol production of the strains overexpressing two CBB enzymes (FBA ​+ ​TK, FBP/SBPase ​+ ​FBA or FBP/SBPase ​+ ​TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.