Currently, piperazic acid is chemically synthesized using ecologically unfriendly processes. Microbial synthesis from glucose is an attractive alternative to chemical synthesis. In this study, we report the production of L-piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum; this strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring and constitutive genetic circuit reconstitution, to build a robust L-piperazic acid synthetic cascade. These genetic modifications enable A. melanogenum to directly convert glucose to L-piperazic acid without relying on the use of either chemically synthesized precursors or harsh conditions. This bio-based process overcomes the shortcomings of the conventional synthesis routes. The ultimately engineered strain is a very high-efficient cell factory that can excrete 1.12 ± 0.05 g l-1 of L-piperazic acid after a 120-h 10.0-l fed-batch fermentation; this is the highest titre of L-piperazic acid reported using a microbial cell factory.