(138f) Production of Poly-L-Malic Acid By Engineered Aureobasidium Pullulans with Enhanced Reductive Tricarboxylic Acid Pathway

Aureobasidium pullulans is a versatile black yeast with a large genome containing genes encoding various hydrolases and enzymes in many secondary metabolic pathways. This study focused on using engineered A. pullulans with enhanced reductive tricarboxylic acid (rTCA) pathway for the production of poly(L-malic acid) (PLMA), which can be used as a novel drug carrier and its monomer (L-malic acid) has wide applications as food additive and C4 platform chemical. To achieve industrial-scale production, improvements in fermentation performance, including product titer, yield, and productivity, are required, which can be achieved via metabolic engineering and process optimization.

Among the known MA-biosynthesis pathways, the rTCA pathway has the highest theoretical malic acid (MA) yield of 2 mol/mol glucose. We introduced and overexpressed heterologous and native enzymes in the rTCA pathway, including pyruvate carboxylase (PYC), malate dehydrogenase (MDH), and PLMA synthetase (PMS), to evaluate their impacts on PLMA production. Episomal expression of MDH (mdhA) and PYC (pycA) from Aspergillus oryzae increased PLMA yield by 32% and 37%, respectively. Co-expressing pycA and mdhA led to a high PLMA titer of 58.6 ± 6.3 g/L and yield of 0.65 ± 0.07 g/g glucose. CRISPR-Cas genome engineering was then used to insert pycA and mdhA into A. pullulans’ genome to obtain genetically stable mutant strain suitable for use in industrial fermentation. A high-cell-density (HCD) fermentation operated at fed-batch and repeated batch modes produced 96.7 g/L PLMA at a high productivity of 0.90 g/L/h and yield of 0.73 g/g glucose. Techno-economic analysis of the HCD fermentation process showed that PLMA and MA can be produced from glucose at a cost of less than $1.5/kg, demonstrating its feasibility for industrial applications.