L-piperazic acid (L-Piz) is one of components in many bioactive compounds synthesized by Streptomyces spp. Therefore, it is very important to develop a sustainable and eco-friendly bioprocess to produce L-Piz from renewable glucose. In this study, it was found that the engineered Aureobasidium melanogenum DFAK-ArgB6 strain overexpressing the ArgB gene encoding N-acetylglutamate kinase could produce 3.0 g/L L-Piz from glucose during the fed-batch fermentation. The yield was 55.6 mg L-Piz/g of glucose and the productivity was 24.8 mg/L/h. This was the highest amount of L-Piz produced by microorganisms so far. Finally, the partially purified L-Piz had purity of 86.4%. Display omitted •L-piperazic acid (L-Piz) is one of components in many bioactive compounds.•The DFAK-ArgB6 strain could produce the highest amount of L-Piz (3.0 g/L).•The partially purified L-Piz was obtained.