(130c) CRISPR/Cas9 Genome Editing to Disrupt Pullulan Biosynthesis and Enhance Poly-L-Malic Acid (PMA) Production in Aureobasidium Pullulans

Aureobasidium pullulans is a black yeast naturally produces poly-L-malic acid (PMA) as an extracellular product. In the industrial production of PMA, downstream process was hindered by the presence of pullulan, a polysaccharide co-produced in A. pullulans fermentation that can co-precipitate with PMA with alcohol addition. The production of pullulan also increased fermentation broth viscosity and decreased PMA product yield from glucose. Therefore, we aimed to disrupt pullulan biosynthesis in A. pullulans by applying CRISPR/Cas9 optimized with homologous recombination for targeted gene disruption. Specifically, the amags2 gene encoding a multidomain α-glucan synthetase 2 (AmAgs2) was disrupted. Compared to the wild-type strain, the mutants with disrupted AmAgs2 showed a significantly decreased pullulan production, from 8.86 ± 1.97 g/L to 2.97 ± 1.36 g/L, with PMA yield increased from 0.58 g/g to 0.65 g/g in shake-flask batch fermentations. This study also introduced a reliable method for the quantification of pullulan in the PMA fermentation broth and streamlined genome editing in A. pullulans, paving the way for further engineering this robust non-model black yeast for industrial production of PMA and malic acid in a sustainable bioprocess.