Enhanced fumarate production using high-density cultivation of Synechocystis sp. PCC 6803

Cyanobacteria are photosynthetic microorganisms that can fix CO2 via the Calvin–Benson–Bassham cycle and store carbon as glycogen, which is subsequently catabolized through multiple pathways. Unicellular cyanobacteria such as Synechocystis sp. PCC 6803 possess a unique tricarboxylic acid (TCA) cycle, lacking 2-oxoglutarate dehydrogenase, and excrete various low-molecular-weight carboxylic acids under dark, anaerobic conditions. Fumarate, which is a TCA cycle metabolite widely used in food additives and bioplastics, has been produced at titers below 120 mg/L in cyanobacteria, thereby limiting its industrial potential. Here, high-density cultivation of genetically engineered Synechocystis 6803 was established, and a fumarate-producing mutant (ΔfumC) lacking fumarase (fumC) was characterized. The ΔfumC mutant exhibited increased photosynthetic activity and grew comparably to the wild-type strain under high-density conditions. Furthermore, the combination of fumC knockout with ppc overexpression (encoding phosphoenolpyruvate carboxylase) enabled fumarate production at the highest titer reported to date (>2 g/L) under photoautotrophic conditions. This study highlights the effectiveness of metabolic engineering combined with cultivation optimization to overcome the intrinsic bottlenecks in cyanobacterial carboxylic acid production, providing a promising platform for sustainable bioproduction from CO ₂ .