Engineering the by-products pathway in Aureobasidium pullulans for highly purified polymalic acid fermentation with concurrent recovery of l-malic acid.

The fermentation of polymalic acid (PMA) by Aureobasidium pullulans, followed by acid hydrolysis to release the monomer l-malic acid (l-MA), has emerged as a promising process for the bio-based production of l-MA. However, the presence of specific by-products significantly affects the quality of the final products. In this study, chassis strains harboring an overexpressed endogenous malate dehydrogenase gene (ApMDH2) were engineered to delete key genes involved in the pullulan, melanin, and liamocin biosynthetic pathways. Furthermore, to enhance PMA synthesis productivity and prevent intracellular NADPH accumulation, an irreversible trans-hydrogenase transformation system was designed to efficiently convert NADPH to NADH. In fed-batch fermentation, the engineered strain produced the highest PMA titer (194.3 ± 1.1 g/L) and l-MA yield (0.89 ± 0.01 g/g) with an increased productivity (1.45 ± 0.06 g/L∙h). Moreover, a total of 86.19 % l-MA, with a purity of 99.7 %, was successfully extracted from fermentation broth.