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