Directed evolution of Actinobacillus succinogenes for high succinic acid production

Abstract:

Succinic acid, an important high-value platform compound, is widely used in chemical, food, and medicine fields. The chemical production of succinic acid has the defects of high pollution and high carbon emissions. Therefore, the fermentation method has become the main direction of succinic acid production at present. Actinobacillus succinogenes, a naturally succinic acid-producing strain, is praised for the high titer, high osmotic pressure tolerance, and short fermentation time, thus serving as an ideal strain for industrial production. Improving the succinic acid titer of A. succinogenes and thus reducing the production cost is an urgent need for industrial production. In this study, we developed a simple high-throughput screening method by combining bromothymol blue with atmospheric room-temperature plasma mutagenesis to direct the evolution of wild type A. succinogenes. A high-yielding mutant strain, A4-K74, was screened out. After 72 hours of anaerobic fermentation in a shake flask, this strain accumulated a maximum of 56.3 g/L of succinic acid, which represented a 40.8% increase compared with that of the wild type strain. Moreover, A4-K74 demonstrated good genetic stability. The transcriptome analysis revealed that the enhancement of sulfur metabolic pathway and amino acid (such as cysteine) synthesis pathway was the potential reason for the increase in succinic acid titer, which provided a reference for the future metabolic engineering of A. succinogenes and was conducive to the further promotion of the industrial production of succinic acid by bioprocessing.