Development of tools for quantitative intracellular metabolomics of Aspergillus niger chemostat cultures.

Francisca Lameiras,Joseph J Heijnen,Walter M Van Gulik

doi:10.1007/s11306-015-0781-z

Francisca Lameiras, Joseph J Heijnen + Show 1 more

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https://doi.org/10.1007/s11306-015-0781-z

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Abstract

In view of the high citric acid production capacity of Aspergillus niger, 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 omics tool in a synthetic biology approach to develop A. niger 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 A. niger, equipped with a new dedicated rapid sampling device. A quenching method for quantitative metabolomics in A. niger 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 A. niger and Penicillium chrysogenum metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both fungi, except for the intracellular citrate level which is higher for A. niger.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0781-z) contains supplementary material, which is available to authorized users.

Highlights

Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Filamentous fungi like Aspergillus niger, form a key step in the global carbon cycle by their capacity to degrade plant cell wall wastes efficiently, as they have one essential advantage: the massive secretion of enzymes that are capable of degrading plant cell wall constituents into available sugars which can subsequently be taken up by the cells
Aspergillus niger was grown in a 7 L turbine stirred bioreactor, constructed for chemostat cultivation of filamentous fungi
A homogeneous steady state chemostat culture could not be achieved in this reactor, due to massive wall growth and accumulation of biomass in the headspace of the reactor (Fig. S1), as experienced in other studies (Schrickx et al 1993; Larsen et al 2004)

Summary

Introduction

Filamentous fungi like Aspergillus niger, form a key step in the global carbon cycle by their capacity to degrade plant cell wall wastes efficiently, as they have one essential advantage: the massive secretion of enzymes that are capable of degrading plant cell wall constituents into available sugars which can subsequently be taken up by the cells. This fungal characteristic is already exploited industrially in the production of enzymes such as glucoamylases and hemicellulases. Overproduction of dicarboxylic acids in A. niger requires metabolic engineering of the fungus, with respect to the product pathway and with respect to the import of lignocellulosic sugars (glucose, xylose, arabinose, etc.) and the export of the produced acids

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