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Towards a sustainable bioeconomy

Research • Educate • Connect
Towards a sustainable bioeconomy

Ustilago maydis as biosurfactant producer

The eukaryotic microorganism Ustilago maydis is a natural producer of two biosurfactants, the cellobiose lipid ustilagic acid and mannosylerythritol lipids (MEL). The two glycolipids are produced as a mixture of natural variants under nitrogen depletion conditions. In case of MEL four forms exist, MEL-A to MEL-D. These variants differ in their degree of acetylation, with MEL-D constituting the completely deacetylated form. The underlying biosynthetic pathway is known (Fig. 1) and the corresponding five enzymes are organised at genome level in a gene cluster.

Fig. 1: Biosynthetic pathway of MEL in Ustilago maydis (Müntjes et al., 2020)[1].

The aim of the FocusLab Bio2 was to establish processes that enable MEL production based on sustainable raw materials. A new synthetic regulation of the gene cluster was intended to reduce the dependence of MEL production on severe nitrogen deficiency in order to ensure the constitutive production of MEL from pectin-rich side streams from the sugar industry. Due to their longstanding expertise in the genetic engineering of U. maydis the Core Group (CG) Feldbrügge addressed this aspect. For the novel synthetic regulation of the MEL gene cluster, a 2A peptide strategy was applied. Cluster genes, which are otherwise regulated and transcribed separately in eukaryotes, were placed under the control of a strong constitutive promoter and expressed together, resulting in a type of "polycistronic mRNA". It was demonstrated that separate proteins are formed from this mRNA during translation. Applying this strategy, three cluster genes were successfully co-regulated, leading to the production of a modified pattern of MEL variants even under the presence of nitrogen in the medium.

For MEL production on pectin-rich side streams, investigations initiated in the SeedFund UstiLyse and the BoostFund PectiLyse were continued. In these previous projects, CG Feldbrügge successfully established a genetic strategy in which hydrolytic enzymes can be activated in the yeast form of the fungus. This strategy was extended by complementing and improving the fungal enzyme repertoire with potent heterologous pectinolytic enzymes. In this regard, the possibility to use an unconventional secretion pathway recently described in U. maydis for the export of bacterial enzymes presented a unique feature.

A fruitful collaboration between microbiologists and biochemical engineers enabled new possibilities for strain characterisation and optimisation using online analytics. To this end, the CG Büchs used the RAMOS (respiration activity monitoring system) to monitor the cultivation process of the strains that secreted hydrolytic enzymes (Fig. 2). With the RAMOS, the oxygen and carbon dioxide transfer rate of eight parallel cultures can be determined continuously. This enabled predictions on the metabolism of U. maydis strains on different substrates, such as glucose, xylose and arabinose, and allowed for calculations of enzyme activities and substrate turnover.

Fig. 2: Respiration Activity Monitoring System. Biochemical Engineering, RWTH Aachen University.

In order to extend the substrate spectrum of U. maydis and to facilitate the degradation of the main component of pectin, polygalacturonic acid, U. maydis strains that secrete different combinations of endo- and exopolygalacturonases were examined (Fig. 3). With this approach, the growth of U. maydis on polygalacturonic acid was successfully established. The combination of two heterologous enzymes from other fungi proved to be the most effective approach. The RAMOS technique supported strain characterisation by establishing an indirect, non-invasive determination of the residual substrate concentration of polygalacturonic acid based on the total oxygen transfer rate. Subsequently, mixed cultures of overexpression strains were monitored with the RAMOS technology in different inoculation ratios to determine the optimal strain ratio for polygalacturonic acid degradation (Fig. 4). It was shown, that a higher ratio of the U. maydis strain overexpressing an exo-polygalacturonase led to the fastest degradation of polygalacturonic acid (Fig. 4 C).

Fig. 3: Interaction of endo- and exopolygalacturonases during degradation of polygalacturonic acid (Stoffels & Müller et al., 2020)2.
Fig. 4: Co-cultivation of U. maydis strains on polygalacturonic acid with varying inoculation ratios. The used strains AtPgaX and AaPgu[1] overexpress a fungal exo- and endopolygalacturonase, respectively. Medium: modified Verduyn mineral medium containing glucose (4 g/L) and polygalacturonic acid (20 g/L, purity 85 %) as carbon sources. (Stoffels & Müller et al., 2020)[2].

In summary, the results achieved in the FocusLab Bio2 represent a first, essential step towards the use of pectin-rich side streams from the sugar industry for the production of biosurfactants in U. maydis.


[1] Reprinted as a part of figure 5 from Frontiers in Microbiology, 11 /1384, Müntjes, K.,
Philipp, M., Hüsemann, L., Heucken, N., Weidtkamp-Peters, S., Schipper, K., Zurbriggen, MD., Feldbrügge, M. Establishing Polycistronic Expression in the Model Microorganism Ustilago maydis. Copyright (2020).
[2] Reprinted from Journal of Biotechnology, 10 /307, Stoffels P., Müller MJ., Stachurski S., Terfrüchte M., Schröder S., Ihling N., Wierckx N., Feldbrügge M., Schipper K., Büchs J., Complementing the intrinsic repertoire of Ustilago maydis for degradation of the pectin backbone polygalacturonic acid, 148-163, Copyright (2020), with permission from Elsevier.

Involved Core Groups

Prof. Michael Feldbrügge
Dr. Kerstin Schipper
Dr. Silke Jankowski
Magnus Philipp
Institute for Microbiology
Heinrich-Heine University Düsseldorf

Prof. Jochen Büchs
Dr. Nina Ihling
Maximilian Schelden
Chair of Biochemical Engineering
RWTH Aachen University