One-Pot Sustainable Enzymatic Cascade for Production of High-Value Succinyl-CoA and 5-Aminolevulinic Acid

Succinyl-CoA, a vital metabolic intermediate, serves as a key cofactor in numerous biosynthetic pathways, including the synthesis of 5-aminolevulinic acid (5-ALA), which is extensively used in agriculture to improve crop growth and stress tolerance as well as in medicine for photodynamic therapy. This study demonstrates a sustainable biocatalytic approach for 5-ALA production from CoA using an in vitro enzymatic cascade relying on feedstocks of α-ketoglutarate and glycine. The green synthesis pathway integrates the 2-oxoglutarate dehydrogenase complex (SucA and SucB) with ALA synthase fromRhodobacter capsulatusin an atom-economical process. By coexpressing GroELS chaperone in the BD7G strain, we enhanced enzyme solubility over 90% and achieved 1.155 g of protein per liter. Process intensification through enzyme ratio optimization revealed that a 1:2 ratio of SucA to SucB maximized succinyl-CoA production of 0.5 mM, with NAD+ regeneration by a flavin reductase (FRE) and catalase (CAT) system under mild conditions. The integrated five-enzyme cascade achieved complete substrate conversion from α-ketoglutarate with 1 mM 5-ALA titer in one-pot. Notably, the process incorporates efficient CO2 capture strategies, with biocatalytic sequestration using carbonic anhydrase demonstrating superior sustainability metrics compared with chemical methods. This environmentally conscious enzymatic platform provides a novel strategy to produce high-value succinyl-CoA and an alternative to 5-ALA production.