Table_1_Metabolic Regulation of Organic Acid Biosynthesis in Actinobacillus succinogenes.DOCX

Actinobacillus succinogenes is one of the most promising strains for succinic acid production; however, the lack of efficient genetic tools for strain modification development hinders its further application. In this study, a markerless knockout method for A. succinogenes using in-frame deletion was first developed. The resulting ΔpflA (encode pyruvate formate lyase 1-activating protein) strain displayed distinctive organic acid synthesis capacity under different cultivation modes. Additional acetate accumulation was observed in the ΔpflA strain relative to that of the wild type under aerobic conditions, indicating that acetate biosynthetic pathway was activated. Importantly, pyruvate was completely converted to lactate under anaerobic fermentation. The transcription analysis and enzyme assay revealed that the expression level and specific activity of lactate dehydrogenase (LDH) were significantly increased. In addition, the mRNA expression level of ldh was nearly increased 85-fold compared to that of the wild-type strain during aerobic–anaerobic dual-phase fermentation, resulting in 43.05 g/L lactate. These results demonstrate that pflA plays an important role in the regulation of C3 flux distribution. The deletion of pflA leads to the improvement of acetic acid production under aerobic conditions and activates lactic acid biosynthesis under anaerobic conditions. This study will help elaborate the mechanism governing organic acid biosynthesis in A. succinogenes.