(554c) Engineering Aureobasidium Pullulans to Overexpress Malate Dehydrogenase in the Reductive Tricarboxylic Acid Pathway for Poly (L-malic acid) Production from Glucose

Poly (L-malic acid) (PMA) can be produced by the yeast-like fungus Aureobaisidum pullulans. The high water-solubility, non-toxicity, and biocompatibility of PMA make it an ideal drug carrier for the pharmaceutical industry. The monomer of PMA, L-malic acid (MA), is an important C4 platform chemical and food additive. A. pullulans belongs to the order Dothideales of the family Dothideaceae, which has been applied in biotechnology for decades. However, PMA fermentation by A. pullulans requires further improvement to realize industrial production. Fermentation yield from the substrate, PMA titer, and productivity can be enhanced through metabolic engineering and process optimization. Among the metabolic pathways for PMA biosynthesis, the reductive tricarboxylic acid (rTCA) pathway that converts pyruvate to oxaloacetate and transfers to malate by a malate dehydrogenase (MDH) has the highest theoretical yield of 2 mol MA/mol glucose, which is the target for metabolic engineering of A. pullulans to enhance PMA production from glucose.

A PMA producing strain A. pullulans ZX-10 previously isolated in our lab. was used in this study. A mutant strain A. pullulan-mdh overexpressing a cytosolic malate dehydrogenase (mdh) in A. pullulans ZX-10 was obtained. The effects of mdh overexpression on PMA production was evaluated in shake-flask fermentation, which showed that A. pullulan-mdh gave a 16% higher PMA yield from glucose or 0.50 g/g compared to 0.43 g/g for the wild-type strain. The fermentation process was scaled up in a 1.5-L bioreactor and further optimized by fed-batch fermentation. Techono-economic analysis showed that MA can be produced from glucose at $1.83/kg, which is significantly lower than the current market price of >$2/kg for MA produced from current petrochemical-based processes.