Malic acid production by Aspergillus oryzae
Doktorsavhandling, 2014
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)-1) 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.
C4 dicarboxylic acids
Aspergillus oryzae
systems biology
fermentation
malic acid
metabolic engineering