THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Malic acid production by Aspergillus oryzae

Malic acid is a C4 dicarboxylic acid which is used as an acidulant in food and beverages. It is also considered as a bio-building block to replace petrochemically derived compounds in the post oil era. This organic acid can be biotechnologically derived from fermentation using renewable feedstocks as carbon source. Aspergilli are among the best producers of organic acid and A. flavus/oryzae is the best natural producer of malic acid. The mechanism of malic acid production in A. oryzae was first assessed by transcriptome analysis. A nitrogen starvation response, probably regulated by a transcription factor related to the S. cerevisiae Msn2/4 transcriptional activator of stress related genes, was found to result in high malic acid production. Furthermore the pyruvate carboxylase reaction was identified as a metabolic engineering target. This gene, together with the malate dehydrogenase and a malic acid exporter was overexpressed in the strain 2103a-68, which was characterized in a second project. The overexpression led to an 80% increase in yield during the starvation phase (1.49 mol (mol gluc)) and a triplication of the specific production rate. The increase in citric acid production in the engineered strain and its evaluation through model simulations led to the curation of the A. oryzae GEM. The existing model was curated with special emphasis on the mitochondrial transport reactions and let to a more defined network around the production of organic acids. Furthermore, the performance of the strain 2103a-68 on xylose as carbon source was evaluated as well and the good results led to the final project of manipulating the carbon source utilization by deleting the carbon catabolite repressor CreA. This work contributed to the understanding of the regulation of malic acid production. This knowledge was used for the development of A. oryzae as an organic acid producer through metabolic engineering. Furthermore, the evaluation of xylose as an alternative carbon source paved the way towards the use of lignucellulosic feedstocks and showed the suitability of A. oryzae for the biorefinery of the future.