Metabolic engineering of Ustilago trichophora: an isotope-assisted metabolomics approach for the improvement of malate production from glycerol

Periodic Reporting for period 1 - Meta-Origami (Metabolic engineering of Ustilago trichophora: an isotope-assisted metabolomics approach for the improvement of malate production from glycerol)

Okres sprawozdawczy: 2019-02-01 do 2021-01-31

Now more than ever is the era for affordable, reliable, and sustainable energy. A significant contribution from modern biotechnology into the efforts in reducing the dependence on fossil fuels is biodiesel. A report from the World Bioenergy Association showed that 160 billion liters of biodiesel were produced worldwide in 2018. However, biodiesel cannot compete with fossil-based diesel due to the high-cost production. 10% of the products in a common biodiesel production process is crude-glycerol, which is the main low-value by-product. Therefore, the development of glycerol conversion technologies is encouraged to take advantage of the whole biodiesel production process.
Recently, the fungal family Ustilaginaceae with more than 600 species has attracted increasing attention due to their capability in using non-food biomass or bio-industrial waste streams as substrate to produce organic acids, glycolipids, sugar alcohols and other molecules of industrial interest. Among them, Ustilago trichophora was found to have the highest natural production of malate, one of the top value-added building block compounds from biomass (Department of Energy, USA), from glycerol. Further optimizations on this strain increased the malate titer to over 200 g/L with a maximum production rate of 1.8 g/L/h, which is the highest reported titer for microbial production. Of notes, even though a high titer of malate was achieved with U. trichophora, the yield was just approximately 30% of the theoretical maximum. These data suggest that if we can overcome the carbon lost during cultivation, U. trichophora will be a novel candidate for industrial production of malate, contributing directly to the valorization of crude glycerol.
Therefore, the objective of project “Meta-Origami” is applying modern biotechnology methodologies to understand the metabolism of U. trichophora while using glycerol to produce malic acid. The results will guide ongoing efforts in metabolic engineering to maximize the performance of U. trichophora with respect to titer, productivity, and importantly yield of malic acid.
A parallel goal of the MSCA Individual Fellowship is to foster the development of the individual researcher and the host institute.

Project “Meta-Origami” was conducted via 6 work packages (WPs). Two high through-put and sensitive methods were developed to analyze intracellular (WP1) and extracellular metabolites (WP2) in U. trichophora TZ1. With the information from the metabolic profiles, the major by-products while U. trichophora TZ1 used glycerol to produce malic acid were identified. As a result, we could eliminate one major extracellular by-product and significantly improve malic acid production both in small scale and large scale cultivation (WP3). Beside the benefits from more malic acid production, we could also save cost from product purification in post processing steps. WP4 covered training activities. Throughout the whole project, the individual researcher received necessary training from the host to fulfil the work in project “Meta-Origami”. On the other hand, the methods developed in WP1 and WP2 were also transferred to the host institute and contributed to co-author publications. Additional communication activities have been done via attending international conferences, symposia, and university visits. Moreover, the individual researcher was the co-editor for a special issue “Metabolic Engineering and Synthetic Biology Volume 2” for Metabolites Journal, MDPI. She also took part in student supervision including 3 bachelor theses, 2 internship and 3 master theses. The scientific outcomes were published on open access journals and updated on Google Scholar, RWTH library, Research Gate, LinkedIn and ORCID (WP5). The project was managed under WP6.
Together, these works contributed to six publications:
1. Phan, A.N.T.; Blank, L.M. GC-MS-Based Metabolomics for the Smut Fungus Ustilago maydis: A Comprehensive Method Optimization to Quantify Intracellular Metabolites. Frontiers in Molecular Biosciences 2020, 7.
2. Liebal, U.W.; Phan, A.N.T.; Sudhakar, M.; Raman, K.; Blank, L.M. Machine Learning Applications for Mass Spectrometry-Based Metabolomics. Metabolites 2020, 10, 243.
3. Nies, S.C.; Alter, T.B.; Nölting, S.; Thiery, S.; Phan, A.N.T.; Drummen, N.; Keasling, J.D.; Blank, L.M.; Ebert, B.E. High titer methyl ketone production with tailored Pseudomonas taiwanensis VLB120. Metabolic Engineering 2020, 62, 84-94.
4. Ullmann, L.; Phan, A.N.T.; Kaplan, D.K.P.; Blank, L.M. Ustilaginaceae Biocatalyst for Co-Metabolism of CO2-Derived Substrates toward Carbon-Neutral Itaconate Production. Journal of Fungi 2021, 7, 98.
5. Agostino, V.; Lenic, A.; Bardl, B.; Rizzotto, V.; Phan, A.N.T.; Blank, L.M.; Rosenbaum, M.A. Electrophysiology of the Facultative Autotrophic Bacterium Desulfosporosinus orientis. Frontiers in Bioengineering and Biotechnology 2020, 8.
6. Phan, A.N.T.; Blank, L.M. Special Issue “Metabolic Engineering and Synthetic Biology Volume 2”. Metabolites 2021, 11, 35.

One of the major motivations of this project is the potential of Ustilaginaceae in using non-food biomass or bio-industrial waste streams as substrate to produce industrially relevant compounds. Unlike other filamentous fungi that tend to form pellets in the cultures, Ustilaginaceae can keep yeast-like morphology in axenic culture. It is an important feature for strain improvement of fungi using metabolic engineering for industrial production because this morphology will allow similar oxygen supply to all cells even at large scale. Therefore, when a general framework for U. trichophora strain improvement using metabolic engineering was generated, it can be easily used to exploit the full potential of the Ustilaginaceae. By using state-of-the-art metabolic engineering technologies, this project resulted in a better understanding on the metabolic pathways involved in the uptake and degradation of glycerol of U. trichophora. First, this fundamental biochemical information was successfully used for the improvement of malate production using U. trichophora. Furthermore, it also shed light on the applications of U. trichophora to produce other valuable chemicals from glycerol. Results emerging from the project attracted more attention of scientists in the biotechnological utilization of Ustilaginaceae, which will also promote transferring of knowledge among European countries and throughout the world.
Greater impacts come from the fact that biotechnology is one of the key enabling technologies for the development of industry in Europe generally and in Germany particularly. From the report of the World Bioenergy Association (WBA), around 15.7 billion liters of biodiesel, accounted for 37.5% of the world biodiesel production, were produced in Europe in 2018. Therefore, successful in improving malic acid production in this study can add value to the biodiesel industry and make the overall biodiesel refinery ecologically as well as economically more sound. The ideas of using non-fossil non-food sources will positively influence the societal acceptance of the concept and encourage the development of the envisaged bioeconomy.
Altogether, these scientific, social, environmental, and economic benefits will contribute towards a more sustainable society, with great opportunities for the preservation and creation of jobs, and a reduced dependence on fossil fuels.

The Institute of Applied Microbiology, RWTH Aachen

"Overview of project ""Meta-Origami"""

Periodic Reporting for period 1 - Meta-Origami (Metabolic engineering of Ustilago trichophora: an isotope-assisted metabolomics approach for the improvement of malate production from glycerol)

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