Development of tools for quantitative intracellular metabolomics of chemostat cultures.

In view of the high citric acid production capacity of , it should be well suited as a cell factory for the production of other relevant acids as succinic, fumaric, itaconic and malic. Quantitative metabolomics is an important tool in a synthetic biology approach to develop for the production of these acids. Such studies require well defined and tightly controlled cultivation conditions and proper rapid sampling, sample processing and analysis methods. In this study we present the development of a chemostat for homogeneous steady state cultivation of , equipped with a new dedicated rapid sampling device. A quenching method for quantitative metabolomics in based on cold methanol was evaluated using balances and optimized with the aim of avoiding metabolite leakage during sample processing. The optimization was based on measurements of the intermediates of the glycolysis, TCA and PPP pathways and amino acids, using a balance approach. Leakage was found to be absent at -20 °C for a 40 % (v/v) methanol concentration in water. Under these conditions the average metabolite recovery was close to 100 %. When comparing and metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both fungi, except for the intracellular citrate level which is higher for .