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, we established high-density cultivation of genetically engineered Synechocystis sp. PCC 6803 and characterized a mutant lacking fumarase (ΔfumC), an enzyme that catalyzes the reversible conversion of fumarate to malate in the TCA cycle. The ΔfumC mutant exhibited increased photosynthetic activity and grew comparably to the wild-type strain under high-density conditions. The ΔfumC strains were cultivated at high density in nutrient-enriched BG-11 media (5 × , 10 × , and 15 ×) under 1% CO2 and 300 μmol photons/m2/s, resulting in fumarate production of up to 1.7 g/L fumarate under these 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 CO2.