Poly-malic acid (PMA) is a water-soluble polymer consist of repeated malic acid linked by ester bonds, which can be produced by A. pullulans. PMA has attracted significant attentions due to its biocompatibility and degradability with promising applications in medicine, agriculture, and pharmaceutical industry. PMA must be produced with high yield and high purity for commercial applications. However, the production of PMA in A. pullulans strains is limited by the co-production of pullulan polysaccharide, which presents a challenge in downstream product separation and purification. The removal of pullulan production via metabolic engineering should increase product purity for applications as well as direct the substrate to generate more main products and thus enhance PMA production. Three enzymes, α-glucan synthetase (Ags2), pullulan synthetase (PUL1), and UDP-glucosyltransferase (UGT1), in the pullulan biosynthesis pathway were identified and knocked out in A. pullulans by using CRISPR-Cas genome editing technique to inhibit pullulan production. Compared to the wild type, the mutant A. pullulans-ΔAgs2 produced significantly less pullulan with a 9.3% increase in PMA production, achieving a PMA yield of 0.59 g/g glucose in batch fermentation. Overexpressing the malate dehydrogenase (MDH) from A. oryzae in A. pullulans-ΔAgs2::mdhA further increased PMA production to reach the highest yield of 0.66 g/g glucose. Fed-batch fermentation in a bioreactor produced PMA at a high titer of greater than 50 g/L that could be readily recovered by alcohol precipitation.
