Stamatina Roussou: Metabolic engineering of Synechocystis PCC 6803 for acetate production

Abstract

The increasing need for sustainable production has driven interests towards photosynthetic microorganisms as cell factories. Their ability of capture and utilization of CO2 makes them excellent candidates for production. In this thesis, the cyanobacterium Synechocystis PCC 6803 was engineered to enhance acetate production, under photoautotrophic conditions. Native acetate formation in this organism as a side product, is low and was quantified for the first time. Introduction of a heterologous phosphoketolase established a direct link between the Calvin–Benson–Bassham cycle and acetyl-P, resulting in a 40-fold increase in acetate production. Further pathway optimization, including overexpression of phosphotransacetylase, increased production up to 120-fold compared to the control. High-density cultivation of a strain overexpressing also acetate kinase, enabled cumulative acetate titer of 7.1 g/L over 12 days, among the highest reported in cyanobacteria.

To further improve yields, strategies targeting carbon fixation were explored. Overexpression of key CBB-cycle enzymes, revealed product-dependent effects. While combination of the enzymes enhanced ethanol production; acetate production benefited primarily from single gene overexpression (aldolase). The contrasting responses are attributed to differences in pathway precursors, with phosphoketolase competing directly for CBB intermediates while ethanol’s precursor is pyruvate. Deletion of CP12 protein improved acetate production in specific backgrounds, highlighting the importance of carbon flux regulation.

Proteomic and metabolomic analyses demonstrated that the engineered pathway creates a strong metabolic sink, increasing carbon fixation while imposing metabolic stress. Further acetate production was enhanced through overexpression of bicarbonate transporters while less stress was noticed, emphasizing the importance of balancing carbon acquisition and allocation.

Finally, the engineered strains were successfully applied in synthetic consortia, where photosynthetically produced acetate supported the formation of value-added products such as hydrogen and 1-butanol.

Overall, this work establishes Synechocystis as a promising platform for sustainable acetate production and highlights the importance of integrating metabolic engineering with systems-level analysis.