Polymalate (PMA), a promising drug delivery system, can be produced by Aureobasidium pullulans NRRL 62031. The key genetic determinant for PMA polymerization and the primary pathway for malate synthesis were identified. Four gene knockout targets and one overexpression target were found to enhance PMA titer. Genetic insights into PMA hydrolysis were also obtained through bioinformatics. This study lays the foundation for the large-scale production of PMA and its medical applications.