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Directed Evolution and Rational Design of Mechanosensitive Channel MscCG2 for Improved Glutamate Excretion Efficiency

Zhihua Nie,Pi Liu,Yu Wang,Xuan Guo,Zijian Tan,Jie Shen,Zijing Tang,Jianping Lin,Jibin Sun,Ping Zheng,Leilei Zhu

Mechanosensitive amino acid exporters have drawn increasing attention due to their important roles in extracellular accumulation of the target amino acids. Protein engineering is a powerful approach to tailor the properties of amino acid exporters and illustrate structure–function relationships. Here we report the first protein engineering effort on the mechanosensitive glutamate exporter MscCG2 from Corynebacterium glutamicum for improved excretion efficiency of glutamate and understanding of the structure–function relationship. MscCG2 was engineered through directed evolution and computer-assisted design with a coupled assay in microtiter plate format. Improved MscCG2 variants were identified with up to 2.5-fold increase in the level of glutamate excretion in the early stage of fermentation and 1.5-fold in the late stage of fermentation under experimental conditions. Furthermore, the identified variants exhibited enhanced efflux of 4-fluoroglutamate (4-FG), an analog of glutamate. Structure analysis employing homology modeling and molecular dynamics (MD) simulation reveal that identified amino acid substitutions enlarge the size of the seven portals on the equator of MscCG2 and expand the narrowest rim of its inner channel, respectively. This study demonstrates the great potential of protein engineering in improving the secretion efficiency of exporters for enhanced bioproduction.