(please scroll down for a more detailed account in English)
Im Arbeitskreis von Prof. Dr. Bruno Moerschbacher und seinem Akademischen Rat Dr. Nour Eddine El Gueddari arbeiten etwa zwei Dutzend Nachwuchs-Wissenschaftler/innen - Bachelor- und Master-Studierende, Doktorand/inn/en und Post-Doktorand/inn/en - zusammen mit und unterstützt von zur Zeit drei Technischen Assistentinnen gemeinsam an verschiedenen Forschungsprojekten. Diese dienen langfristig alle der Entwicklung einer „Wissensbasierten Bioökonomie“, und damit einer der zentralen Zukunftsfragen am Ende des Petrochemie-Zeitalters: das knapper werdende Erdöl macht eine Rückbesinnung auf nachwachsende Rohstoffe, insbesondere Biopolymere, nötig. Wenn das jedoch kein Schritt zurück werden soll sondern einer hin zu einer gerechten, nachhaltigen und modernen Lebensweise, dann muss die neue Bioökonomie eine wissensbasierte sein: Forschung kann zu den Erkenntnissen führen, mit deren Hilfe wir die unvergleichlichen und enorm vielfältigen Eigenschaften biologischer Rohstoffe optimal nutzen können.
Dabei interessieren wir uns weniger für die reinen Materialeigenschaften als vielmehr für die biologischen Aktivitäten der Biopolymere, insbesondere von komplexen Zuckern (Polysacchariden oder Oligosacchariden) wie Chitosanen aus Krabbenschalen, Ulvan aus Grünalgen, Pektin aus höheren Pflanzen oder bakteriellen Exopolysacchariden wie Xanthan. Wir wollen verstehen, wie die biologischen Funktionen der komplexen Zucker von ihren biochemischen Strukturen und ihren physikochemischen Eigenschaften bestimmt werden. Dazu identifizieren, charakterisieren und optimieren wir Polysaccharid-modifizierende Enzyme, also die biologischen Werkzeuge derjenigen Organismen, die diese Polysaccharide produzieren oder abbauen. Wir entwickeln Methoden, um die mit solchen Enzymen modifizierten Polysaccharide oder aus ihnen enzymatisch hergestellten Oligosaccharide bio- und physikochemisch zu analysieren und ihre biologischen Aktivitäten zu erkennen, zu messen und zu verbessern. Letztlich können wir so Produkte herstellen, die für bestimmte Aufgaben optimiert sind, z.B. für einen biologischen Pflanzenschutz oder für neuartige Wundverbände, unter denen auch chronische Wunden zuverlässig heilen können.
Neben diesen Arbeiten zu Polysaccharid-modifizierenden Enzymen und den biologischen Aktivitäten ihrer Produkte bearbeiten wir auch eine andere Gruppe von Enzymen, die Polyphenoloxidasen (PPO). Auch dieses Forschungsgebiet hat sich aus unseren Arbeiten zur Molekularen Phytopathologie entwickelt, also zu der Frage, wie Pflanzen sich gegen Krankheiten schützen. Im Rahmen dieser Arbeiten waren wir auf den Löwenzahn aufmerksam geworden, eine Pflanze, die zu den wenigen weltweit zählt, die praktisch nie krank werden. Wir hatten vermutet – und es hat sich partiell auch bestätigt – dass die Krankheitsresistenz des Löwenzahns etwas mit den PPOs zu tun haben könnte. Tatsächlich haben wir im Genom des Löwenzahns eine sehr große, aus elf Genen bestehende PPO-Genfamilie gefunden. Derzeit versuchen wir, diese Vielfalt zu verstehen, indem wir die PPO-Isoenzyme einzeln studieren und nach ihren natürlichen Substraten suchen, um letztlich die Eigenschaften ihrer Produkte analysieren zu können.
Um die Ergebnisse unserer Forschung möglichst zeitnah in umwelt- und verbraucherfreundliche Produkte umsetzen zu können, arbeiten wir in unseren Projekten fast immer früh mit entsprechenden Firmen im In- und Ausland zusammen. So hat z.B. unser langjähriger Chitosanproduzent und –lieferant kürzlich auf der Grundlage unserer gemeinsamen Forschung drei neue, Chitosan-basierte Pflanzenschutzmittel entwickelt. Eines dieser Mittel hat bereits eine Zulassung in Europa, zwei weitere bisher nur in Indien, wo unser Partner Chitosan produziert und wo er auch gemeinsam mit Farmern vor Ort die neuen Mittel entwickelt hat.
Research in the group of Prof. Moerschbacher and Dr. El Gueddari aims to understand molecular structure-function relationships and cellular modes of action of functional biopolymers and their use in the knowledge-based transition towards a sustainable bio-economy. Our main focus is on chitin and chitosans, perhaps the most versatile and most promising functional biopolymers. Chitin is one of the most abundant biopolymers on Earth and, thus, an almost inexhaustible renewable resource. As a structural, fibre-forming polymer, it gives strength to fungal cell walls as well as to the exo- and endoskeletons of many invertebrate animals, such as squid, insects, shrimps and crabs, and it is even produced by some marine microalgae. Chitosans, partially deacetylated derivatives of chitin, are much less frequent in nature. They are only produced by a sub-group of fungi, the zygomycetes, and by some pathogenic fungi upon penetration into their host tissues. Commercially, chitosans are therefore produced by partial chemical deacetylation of chitin which can easily be extracted from waste materials such as shrimp or crab shells.
Our initial interest in chitosan arose from the observation that the wheat stem rust fungus, Puccinia graminis f.sp. tritici, one of the most devastating pathogens of wheat, converts the chitin in its cell walls into chitosan when it enters through stomates into the interior of what leaves, apparently in a clever attempt to evade the chitin-based immune system of its host plant. This had actually been the first description of natural chitosan appearance outside the Zygomycetes fungi, and it has since been shown for a range of other plant and even human pathogenic fungi which all appear to use the same pathogenicity strategy. Clearly then, this should be an interested target for an antifungal strategy. We also knew from ample literature that chitosan treatment can protect plants from disease but, as explained above, not consistently so. Thus, understanding molecular structure-function relationships of partially acetylated chitosans and their cellular modes of action in plants (and beyond) became the prime target of our research. However, we are still pursuing in parallel some other small projects related to molecular plant pathology (see below).
Chitosan as a Functional Biopolymer
Chitosans are the partially de-N-acetylated counterparts of chitin (poly-N-acetyl glucosamine) and as such, carry positive charges at slightly acidic, physiological pH. Chitosans are, thus, the only naturally occuring polycationic polymers, and they can easily interact with polyanions such as proteins, DNA, or phospholipid membranes. Consequently, many biological activities of chitosans have been reported, such as antimicrobial and plant strengthening activities, immuno-stimulatory and would haling activities. However, their detailed modes of action remained unresolved, and applications based on such bioactivities had not been successful due to a lack of reproducibility of the bioactivities: sometimes, plants treated with chitosan became resistant against disease, and sometimes, even large scale wounds treated with chitosan containing dressings healed without scars - but only sometimes, not always. We had suggested that this lack of reliability may be caused by subtle batch-to-batch differences in commercial chitosans. Chitosan is not a uniform material (that’s why we prefer to speak of chitosans, plural!), as chitosan molecules can differ e.g. in their degree of polymerisation (DP), degree of acetylation (DA), or pattern of acetylation (PA). Our observation that different chitosans differ in their biological activities - some e.g. are strongly antimicrobial while others not at all, while some others are inducing resistance reactions in plants while again others do not - was the starting point for a number of European research projects in which we focused on a detailed understanding of structure-function relationships of the physico-chemical properties and biological functionalities of partially acetylated chitosans.
The CARAPAX Project: Antimicrobial and Plant Resistance Inducing Activities of Chitosans
In a first European research project, CARAPAX, we analyzed the influence of DP (degree of polymerization) and DA (degree of acetylation) on the antimicrobial and plant resistance inducing activities of chitosans. Partners from France and Norway produced series of well-defined chitosans for us, and partners from Greece and Aachen tried to modify them using specific enzymes. We found that both bioactivities greatly depend on these parameters: chitosan polymers with intermediate DP and low DA had the highest antimicrobial activity, while those with high DP and intermediate DA were best in inducing resistance reactions in plants. Based on these results, one of our industrial partners, the chitosan producer Gillet Chitosan, later developed his first three chitosan based plant protectants. Depending on the application area, different chitosans are used, e.g. for soil amendment, leaf spraying, or seed coating. As an example, a single seed treatment with 40 g of the appropriate chitosan suffices to protect a whole hectare of e.g. groundnut from disease for a full growing season, leading to a ca. 25 % increase in yield.
Small CARAPAX Follow-Up Projects
Our work in the CARAPAX project was supported and extended by two smaller research projects focusing on the biological activities of partially acetylated chitosan oligosaccharides (paCOS), both of them financially supported by DAAD. These were the projects Chito-Oligosaccharides together with colleagues from Brazil, and Chitosan-Based Plant Protection together with colleagues from India. The first project dealt with the enzymatic production of paCOS and the determination of their resistance inducing capacity in plants. In the second project, we aimed to combine resistance inducing chitosans with plant growth promoting rhizobacteria (PGRP) producing chitosan degrading enzymes. These small projects mainly served to intensify our international relations through the exchange of doctoral students. In a parallel project which was financially supported by the Arnold-Hueck Stiftung, we found that the plant strengthening bacterium Pantoea agglomerans secrets an exopolysaccharide which induces a state of alert in plant cells, making them more attentive to the presence of potential pathogens which they can then ward off more efficiently. Even then, we hypothesized that a similar mechanism of cellular priming might be responsible for the plant strengthening effect of chitosans.
The NanoBioSaccharides Project: Nanoformulation and Biomedical Activities of Chitosans
In a second large European research project, NanoBioSaccharides, and its international satellite project NBS-TTC, we found that the biological activities of chitosans towards human cells appears to be even more complex, possibly due to the presence of a chitosan hydrolysing enzyme, chitotriosidase, in human tissues. This enzyme degrades chitosans in a sequence specific way so that the PA (pattern of acetylation) of a chitosan determines both its rate of degradation (turn-over rate) and the quantity and quality of chitosan oligomers produced. In collaboration with the Department of Dermatology, we found that chitosan oligomers can inhibit human matrix-metalloproteases and induce an inflammatory response, both of these activities are potentially relevant for wound healing. We therefore began to explore the use of enzymes for the production of chitosans with different, defined, non-random PA. In order to improve the solubility of chitosan at physiological, neutral pH and its bioavailability in human tissues, we collaborated with partners in France and Spain who produced and characterized chitosan nanoparticles. One Mexican post-doctoral researcher in the Spanish lab, Dr. Goycoolea, later became an independent young research leader in our institute in Münster, in the framework of the Indo-German MCGS project, and we are collaborating with him to further explore the many options offered by chitosan nanoformulations, such as in drug and gene delivery.
The PolyModE Project: Chitosan Modifying Enzymes to Produce Chitosans with Non-Random PA
Current chemical means of producing chitosans invariably lead to polymers with random PA (pattern of acetylation). Therefore, in our third large European research project, PolyModE, we began exploring the possibility to use enzymes to generate chitosans with non-random PA. Our focus was on processive chitin de-N-acetylases to generate chitosan polymers with a more block-wise rather than random PA, and on sequence specific chitosan hydrolases to generate chitosan oligomers (paCOS) with at least partially defined rather than random PA. We have either purified such enzymes from the culture media of bacteria and fungi producing them, or we have cloned and heterologously expressed the genes coding for such enzymes from different bacteria and fungi, and we started to characterize the purified proteins. In the framework of the PolyModE project, we obtained proof of principle that such enzymes do exist and can be used to produce chitosans with non-random PA, but the analytical tools available to characterize the structure of chitosans were not sensitive enough to allow detailed analyses of the PA. In this project, we also collaborated with partners from The Netherlands on pectin and xanthan deacetylases and with partners from France on alginate epimerases and carrageenan and glycosaminoglycan sulfatases, to synergistically profit from our varied expertises.
Small PolyModE Follow-Up Projects
In extension of the PolyModE project, we initiated a strong discovery program for novel chitin and chitosan modifying enzymes (CCME), with partners from India, financially supported by DAAD. We followed two parallel strategies, a knowledge-based approach in the Endophytes project and an un-biased metagenomics approach in the Soil Metagenomics project. In the former one, we used fungi which had been isolated from the interior of leaves from tropical trees in the Indian Western Ghats mountains by our partner. We found that these endophytic fungi secret massive amounts of CCME including chitin de-N-acetylases (CDA), and we have since cloned one very interesting CDA gene from the fungus Pestalotiopsis and characterized the recombinant enzyme which has become an important tool for us to produce partially acetylated chitosan oligosaccharides (paCOS) with full defined PA (pattern of acetylation). When we sequenced the metagenomic DNA isolated from the soil of our commercial chitosan producer in India which had been exposed to chitosans for more than ten years, we identified a large number of genes potentially coding for CCME, and using a chitinase gene, we proved that it is possible to obtain functional enzyme from this approach. We were also granted a small Prototypes project from BMBF to produce a few defined paCOS in larger amounts sufficient for bioactivity. Another small project which was associated to the PolyModE project was the Ulvan project together with a partner from Brazil which was also supported by DAAD. Here, we found that the plant strengthening, sulfated heteropolysaccharide ulvan from green algae has strong priming activity towards plant cells, and we are currently pursuing an enzymatic modification approach to reveal the role of (DP) degree of polymerization and DS (degree of sulfation) on this bioactivity.
The ChitoBioEngineering Project: Towards Biotechnological Production of Defined paCOS
The European Industrial Biotechnology project ChitoBioEngineering which on the German side was supported by BMBF was a direct and logical follow-up to the PolyModE project. In the latter, we had found that CDA (chitin deacetylases) can be used to convert fully acetylated chitin oligomers into paCOS (partially acetyated chitosan oligosaccharides) with fully defined PA (patterns of acetylation). In the ChitoBioEngineering project, we combined different CDA genes with different bacterial chitin oligosaccharide synthase (oCS) genes in bacterial production strains. Depending on the choice of oCS, we obtained chitin oligomers of different DP (degree of polymerization), and depending on the choice of CDA, we can convert them to paCOS with different DA (degree of acetylation) and PA. Partners in Belgium optimized the production strains for higher yield and fermented them in pilot scale, yielding kg amounts of paCOS. And while we engineered the oCS to broaden the spectrum of DP we can produce, a Spanish partner engineered the CDA to broaden our portfolio in terms of DA and PA. The s produced monoclonal paCOS are precious tools for the detailed analysis of structure-function relationships of chitosans and their cellular modes of action.
Small ChitoBioEngineering Follow-Up Projects
The availability of large amounts of well-defined paCOS as obtained using the well-defined chitosan hydrolases of the PolyModE and its follow-up projects and the fully defined monoclonal paCOS from the ChitoBioEngineering project prompted us to focus on the biological activities of these paCOS. In the small ChitoGrow project financially supported by BMWi, we teamed up with a German chitosan producer who supplied a high quality mixture of paCOS with plant growth promoting activities. We first had difficulties reproducing the growth promoting effect under controlled plant growth conditions even though the effect had been clear in the field, until we found that it is only observed under abiotic stress conditions, such as drought stress. Currently, we are aiming at a cellular understanding of this effect. Another small project supported by BMWi is the F2F project in which we collaborate with the microbiologists from Münster and Bielefeld as well as with a biotech start-up and a fermentation company. Here, we aim to convert fungal mycelia obtained as a waste fraction from industrial fermentations for the production of technical enzymes into fine chemicals. Our interest in the synthetic biotechnology project is to use our knowledge on chitin and chitosan modifying enzymes to engineer a bacterial substrate converted which can feed on the chitin in the cell walls of the fungal fermentation wastes and concomitantly support the growth of a bacterial fine chemicals producer, in a bacterial consortium. F2F, thus, aims to contribute to a transition in chemical industry from petroleum-based chemical feedstock to renewable resources.
The CuChi-BCA Project: Towards Chitosan-Based Plant Protection
The CuChi-BCA project was the first Indo-German Public Private Partnership “2+2” research project. It was an extremely rewarding project as it built on our cumulative expertise gained in the previous European projects CARAPAX, NanoBioSaccharides, and PolyModE. From the CARAPAX project, we knew which chitosans are best suited for plant strengthening and disease protection. But we had also learned that the efficacy of chitosan treatments was not sufficient for successful applications in intensive European agriculture. We therefore wanted to combine chitosan with well-known copper-based fungicides aiming for a synergistic interaction which would at the same time allow a reduction in the copper application rate required for reliable plant protection. However, copper and chitosan form insoluble complexes so that we decided for nano-formulated chitosans, resorting to our knowledge on chitosan nanoformulations from the NanoBioSaccharides project. Together with Indian partners, we decided to extend this chitosan-copper nano-formulation towards a triple combination with the plant growth stimulating fungus Triochoderma as a biocontrol agent. In order to allow a co-formulation of Trichoderma with fungicidal copper, the Indian partners identified copper-tolerant Trichoderma strains, and we added our expertise on chitosan hydrolases from the PolyModE project to select for Trichoderma strains which can convert the chitosan into plant strengthening paCOS. The results of this project were stunning: we achieved a reduction in copper dosage of more than one third in Germany and more than two thirds in India, while maintaining or even improving quantity and quality of harvested grapes in Germany and potatoes in India.
The Nano3Bio project: Towards Biotechnological Production of Defined Chitosans
Our fourth large European research project, Nano3Bio, is a direct consequence of the highly successful ChitoBioEngineering project. When we had then developed biotechnological production processes for defined paCOS (partially acetylated chitosan oligosaccharides), we are now aiming at the biotechnological production of chitosan polymers. To this end, we pursue two parallel strategies. Firstly, we aim to use CCME (chitin and chitosan modifying enzymes) such as CDA (chitin deacetylases) and chitinase-derived glyco-synthases (GS) in an in vitro biorefinery approach. CDA can be used to convert fully acetylated chitin oligomers into defined paCOS, and our Spanish partner aims to engineer transglycosylating chitinases from our Indian partner into GS with the goal to polymerise these paCOS into fully defined, repetitive chitosan polymers. Secondly, we aim to use fungal chitin polymer synthases (pCS) of our Swedish partner in suitable expression hosts metabolically optimized by our Belgian partner, in combination with different CDA, to produce chitosan polymers in a cell factory approach in vivo. We are convinced that such biotechnologically produced “third generation” chitosans will be even more tightly defined in terms of their structure and functions than todays “second generation” chitosans which are well defined in terms of their DP (degree of polymerization) and DA (degree of acetylation) but always have random PA (pattern of acetylation). These second generation chitosans which, as a result of the CARAPAX and similar projects, are available now in industrial scale from some few chitosans producers worldwide with reliable qualities, are currently responsible for a renaissance of industrial interest in this promising biopolymer. Third generation chitosans will hopefully support this development and allow the development of medical products based on them, to finally exploit the phenomenal biomedical activities of chitosans.
The FunChi Project: Towards Chitosan Production from Fungal Cell Walls
Our second European Industrial Biotechnology project FunChi which on the German side is financially supported by FNR and BMEL builds on the F2F project. In FunChi, we aim to develop a process for the isolation of polymeric chitin or chitosan from fungal cell walls, while the F2F project targets the chitin monomer N-acetylglucosamine. In both cases, the source of fungal cell walls is the spent mycelium of Aspergillus niger left over as a waste fraction of biotechnological enzyme production. As this production is done in huge fermenters of several hundred thousands of litres volume, the source is abundantly available in constant high quality. Together with partners in the Netherlands, we will engineer the cell wall of the fungus for higher chitin content and better chitin extractability, and together with our Spanish partners we aim to develop a plant protection product based on the fungal chitin. Our own focus, in close collaboration with the German industrial partner, will be on the development of the chemo-enzymatic process for the extraction and purification of high quality chitin or chitosan from the fungal mycelium.