Redirecting formic acid flux and balancing redox for high-yield succinic acid production in Actinobacillus succinogenes

Succinic acid (SA) is an important organic dicarboxylic acid with broad applications in in chemical, pharmaceutical, and food industries. Actinobacillus succinogenes is a natural succinate producer considered a promising industrial strain. However, the formation of by-products such as formic acid (FA) and acetate acid (AA) during SA biosynthesis remains one of the major challenges. In this study, we focused on the role of FA metabolic branch in A. succinogenes. By inactivation of pyruvate formate-lyase using A. succinogenes base editors CBE, the strain that inhibited FA pathway named ΔpflB was obtained, and its growth, SA production, as well as intracellular nicotinamide adenine dinucleotide (NADH) were investigated. Subsequently, strategies were tried to restore NADH regeneration using overexpression of malate dehydrogenase(mdh), transhydrogenase(pntAB), and formate dehydrogenase(fdh) to regulate the intracellular NADH levels and NADH/NAD⁺ ratio of strain ΔpflB. We found that FA branch of A. succinogenes was critical for growth metabolic and redox balance in SA biosynthesis. Finally, through adaptive laboratory evolution to optimize the growth of strain ΔpflB, an evolved strain G100 with growth advantages was obtained after 100 generations. Compared with the parental strain, its SA yield increased by 37.2% and 39.2% in shake-flask and 3 L fermenter, respectively. Meanwhile, the intracellular NADH levels and the NADH/NAD⁺ ratio after evolution was significant adjusted during the fermentation process. The ΔpflB strain shows potential for industrial applications.