P97: Improved Production of Malic Acid in Aspergillus oryzae NRRL 3488 through Metabolic Engineering

Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive TCA (rTCA) pathway. Malate produced by this route is then transported from the cytosol to the external environment by an unidentified transporter. An amino acid BLAST search of the A. oryzae RIB40 genome sequence using the sequence of a known transporter for malic acid, the Schizosaccharomyces pombe mae1 gene, identified 5 possible orthologues of the mae1 gene. Transcriptional analysis using the Affymetrix Aspergillus GeneChip indicates that only one is expressed in NRRL 3488 malic fermentations. Over expression of this dicarboxylate transporter in NRRL 3488 led to a 2-fold increase in malic acid production. When cytosolic variants of the native NRRL 3488 pyruvate carboxylase and malate dehydrogenase genes comprising the rTCA pathway were overexpressed in conjunction with the transporter, malic acid production increased by an additional 1.3-fold. These results illustrate the potential of this fungal strain as a new host for the production of malic acid.