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Poly-γ-glutamic acid (γ-PGA) is a biopolymer with various industrial applications. Despite its diverse applications, γ-PGA of defined molecular structure is costly. Sustainable synthesis of designer-γ-PGA was achieved with a B. subtilis 168 derivative as host. In-depth investigations of the metabolism provided new targets for enhanced γ-PGA synthesis. The developed strains and cultivation conditions demonstrate the huge potential of microbial γ-PGA for the use in industrial applications.