Abstract
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.
Highlights
Microbial production of organic acid from renewable resources is a promising and sustainable approach due to its ecological and economical advantages (Kamzolova et al, 2014)
The maximum activity was 30.34 U/mg, which coincides well with the mRNA level and lactate production. These results indicated that the expression of pyruvate formate lyase in A. succinogenes does not depend on the activation of pyruvate formate lyase 1-activating protein, though its function notation in NCBI is to activate pyruvate formate lyase 1 under anaerobic conditions
We developed a markerless knockout method for A. succinogenes
Summary
Microbial production of organic acid from renewable resources is a promising and sustainable approach due to its ecological and economical advantages (Kamzolova et al, 2014). Actinobacillus succinogenes, a facultative anaerobe and natural succinate producer, is one of the most promising strains for succinic acid production due to its wide carbon utilization spectrum and robustness to environment (Mckinlay et al, 2005; Pateraki et al, 2016). A. succinogenes NJ113 has been reported to be a potential industrial application for the bioproduction of pyruvic acid under microaerobic fermentation condition, and dissolved oxygen environment was found to have a vital role in promoting pyruvic acid production (Wang et al, 2016). Initial oxygen aeration was necessary to enhance lactic acid biosynthetic capacity for A. succinogenes 130ZT in subsequent anaerobic cultivation (Li et al, 2010). Physiological changes during aerobic growth of engineered E. coli strain could significantly affect succinate production in the subsequent anaerobic phase (Vemuri et al, 2002)
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