Metabolic engineering and metabolomics based profiling of cyanobacteria for enhanced succinate production

Succinate, a versatile four‑carbon dicarboxylic acid, is pivotal in synthesizing industrial chemicals, pharmaceuticals, and biodegradable polymers. Traditional production methods are constrained by intensive energy requirements, complex procedures, and reliance on non-renewable fossil resources, impeding sustainable manufacturing. This study introduces a robust and renewable production platform by integrating metabolic engineering with untargeted metabolomics in the fast-growing cyanobacterium Synechococcus elongatus PCC 11801. Specifically, we engineered heterologous expression of glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase (MS), under native cyanobacterial promoters to circumvent carbon loss typically encountered during decarboxylation in the TCA cycle. Concurrently, CRISPR-Cpf1 genome editing was utilized to knock out succinate dehydrogenase (SDH), which ordinarily diverts succinate toward fumarate, thus reinforcing succinate accumulation. High cell density cultivation in enriched 5× BG11 medium under optimized culture conditions with low light intensity and elevated CO₂ gave up to 350 mg/L extracellular succinate titer. Comprehensive metabolome profiling revealed that increased succinate production was associated with extensive reprogramming within central carbon metabolism, marked by enhanced glycolytic throughput, accumulation of TCA cycle intermediates, and pronounced changes in amino acid profiles and redox balance, but also imposed metabolic stress. These findings emphasize the effectiveness and promise of integrating genetic engineering with advanced metabolomic profiling and optimization of cultivation conditions to facilitate the sustainable photosynthetic production of succinate and other value-added chemicals.