(253c) Metabolic Engineering of Auroebasidium Pullulans for Polymalic Acid and Malic Acid Production from Food Processing Wastes

Aureobasidium pullulans is a versatile yeast-like fungus with a large genome and can secrete many hydrolases capable of degrading plant materials. It can also produce many secondary metabolites including poly(L-malic acid) (PMA). The goal of this project is to develop a fermentation process with metabolically engineered strains of A. pullulans for PMA production from low-cost food processing wastes such as soy molasses. PMA, a homopolymer of L-malic acid (MA), has unique properties and many applications such as carriers for drugs and flavor compounds. PMA fermentation also provides a novel production route for L-malic acid, a specialty chemical with wide applications in foods and pharmaceuticals and a potential C4 platform chemical with a large market. PMA fermentation followed with acid hydrolysis of PMA can provide a novel process for the production of MA, which currently is produced by chemical synthesis using petroleum-derived feedstocks. We aimed to develop a robust strain capable of producing PMA at high titer, purity, yield and productivity by rational metabolic engineering. In this study, we focused on strategies for strengthening the glyoxylate shunt in A. pullulans. First, genes encoding the rate-limiting enzymes in the pathway were overexpressed. A novel transcriptional activator, Cat8, was identified and co-expressed to regulate the glyoxylate shunt. To rewire the carbon flow towards glyoxylate shunt, a CRISPR genome-editing system was used to knock out the node gene in the TCA cycle. Furthermore, ethanol stress, as an operable process parameter, was used to regulate stress-responsive genes in glyoxylate shunt. Compared to the parental strain, the engineered strain developed through these strategies showed a 40% increase in the PMA yield (0.70 g/g) from glucose. PMA production in a fed-batch fermentation carried out in a stirred-tank bioreactor reached the highest titer (>155 g/L MA) ever reported. Techno-economic and life cycle analyses showed that PMA and MA can be produced from food processing wastes or by-products from corn, sugarcane, and soybean refinery industries at an economically competitive cost with significant reduction in emissions and energy consumption compared to current chemical synthesis and corn-based fermentation processes.