定向进化产琥珀酸放线杆菌高产丁二酸

Directed evolution of Actinobacillus succinogenes for high succinic acid production

摘要:

丁二酸是一种重要的高值平台化合物，广泛应用于化工、食品、医药等多个领域。化学法生产丁二酸具有高污染、高碳排放等缺陷，因此发酵法成为目前丁二酸生产的主要方法。天然可产丁二酸的产琥珀酸放线杆菌(Actinobacillus succinogenes)具有丁二酸产量高、耐高渗、发酵时间短等优势，是一种重要的工业生产菌株。为了进一步提高A. succinogenes的丁二酸产量，降低生产成本，本研究建立了一种基于溴百里酚蓝的简便高通量筛选方法，结合常压室温等离子体诱变的方法对野生菌A. succinogenes进行了定向进化，筛选得到了高产诱变菌株A4-K74。此菌株在摇瓶中厌氧发酵72 h后最高积累56.3 g/L丁二酸，比野生菌株提高了40.8%，且具有良好的遗传稳定性。转录组分析揭示了硫代谢途径以及半胱氨酸等氨基酸合成途径的增强是丁二酸产量提高的潜在原因，为未来代谢工程改造A. succinogenes提供了参考，有利于进一步推进丁二酸的生物法工业化生产。

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.