Enhancing γ-aminobutyric acid production by Enterococcus faecalis through atmospheric and room temperature plasma mutagenesis, adaptive evolution, and systematic fermentation optimization

Abstract

In this study, Enterococcus faecalis was employed as the target microorganism to enhance γ-aminobutyric acid (GABA) production. A high GABA-producing mutant strain, En1203, was obtained through atmospheric and room temperature plasma (ARTP) mutagenesis combined with gradient resistance screening. Subsequently, a microdroplet cultivation (MMC) based adaptive evolution strategy was applied to further improve the tolerance of the mutant strain to high concentrations of monosodium glutamate (MSG), leading to the selection of a genetically stable high-producing strain, EM05. On this basis, key medium components, including substrate composition, carbon sources, nitrogen sources, and sodium succinate, were systematically optimized. The results demonstrated that the use of a mixed substrate consisting of L-glutamic acid and MSG significantly enhanced GABA accumulation efficiency. Under the optimized medium conditions, the maximum GABA concentration reached 60.7 g/L in shake-flask fermentation. Furthermore, scale-up fermentation in a 3 L bioreactor revealed that strain EM05 maintained robust growth and metabolic stability under high substrate loading, resulting in a further increase in GABA production to 64.2 g/L. Overall, this study demonstrates that the combination of mutagenesis breeding and adaptive evolution, together with medium optimization, is an effective strategy for improving the yield and stability of GABA production by lactic acid bacteria, providing valuable theoretical insights and technical support for its application in food fermentation.

Keywords

Enterococcus faecalis / ARTP mutagenesis / Adaptive evolution / Fermentation medium optimization

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Funding

Natural Science Foundation of Anhui Province (No. 2508085QC105)

National College Student Innovation and Entrepreneurship Training Program (No. 201810363051)

Youth Fund of the National Natural Science Foundation of China (No. 32300059)

RIGHTS & PERMISSIONS

Jiangnan University