December 7-8, 2016: Nano3Bio at the NRW Nano-Conference
The Nano3Bio team had a well visited booth at the 7th NRW Nano-conference which was held in Münster for the first time. Achim Hennecke from the beemo company responsible for dissemination in our project, and Petra Tewes-Schwarzer from Care Sense Consulting responsible for exploitation of our results, as well as Cordula Kurth and Stefan Cord-Landwehr were present to answer questions and to convince people of the advantages of using well-defined functional biopolymers such as biotechnologically produced chitosans as building blocks for their nano-formulations. Sruthi Sreekumar presented her results on the production of chitosan-based nanoparticles using different methods and on the characterization of the particles, revealing the influence of production method and of the chitosan used on particle size and charge, solubility and stability in physiological media. Also, almost the whole group of Prof. Francisco Goycoolea, who also is a Nano3Bio partner, attended the conference and presented their results on posters.
November 30, 2016: Rita Weyer successfully defended her MSc thesis
Rita Weyer had worked for her Master thesis partly in our group and partly in the BioBase Europe Pilot Plant in Ghent, one of the industrial partners of our current European research project Nano3Bio. Her second referee had been Prof. Francisco Goycoolea who returned from his new position in Leeds, UK, to attend the defense. Rita had cloned a number of bacterial chitin synthase and chitin deacetylase genes in a Gram-positive bacterium with GRAS (Generally Regarded As Safe) status, with the goal to set up additional cell factories for the biotechnological production of fully defined, monoclonal partially acetylated chitosan oligosaccharides (paCOS). She also included in her work some mutants of these enzymes which we had previously developed in order to broaden the portfolio of available paCOS. After testing all her constructs on a laboratory scale here in Münster, she selected the most promising ones to grow them in larger fermenters in Ghent. Optimizing the scale-up to the 7.5 L-scale and the down-stream processing to purify the paCOS obtained was not an easy task, but it was finally successful and provided us i.a. with highly pure, fully acetylated chitin hexamer with a tremendous market value, which will last us for many experiments to come.
November 9, 2016: half-annual “F2F” consortium meeting in Münster
This year’s fall meeting of the ZIM project F2F was organized by the Münster members of the consortium, i.e. the biotech start-up company Cysal and the two groups from WWU, Prof. Bodo Philipp’s group from the Institute for Microbiology and Molecular Biotechnology and our own group. We welcomed Dr. Mareike Dirks-Hofmeister, the former doctoral candidate and post-doc from our group who is now head of research in the biotech company Weiss BioTech, the second industrial F2F partner, and Prof. Volker Wendisch and his doctoral candidate Elvira Sgobba from the University of Bielefeld. From our side, Bruno Moerschbacher, Marina Vortmann and Soofia Khan Ahmadi participated. Together, we aim to valorize fungal biomass that routinely accumulates as a waste product during large-scale fungal fermentation processes in technical enzyme production by developing strategies to convert waste fungal biomass to high-value fine chemicals, such as amino acids and dipeptides. We discussed the progress all partners made and the problems they encountered, and took decisions for the work to be focused on during the coming six months. As one milestone for the project, we defined a joint publication to which all three academic partners will contribute, providing proof-of-principle for our strategy.
November 2, 2016: new PhD student Soofia Khan Ahmadi arrived from Malaysia
Today, our new Iranian PhD student Soofia Khan Ahmadi who won a three-years doctoral fellowship from DAAD has started her doctoral project with us, after a two months German language class in Göttingen. Soofia will join our new European research project FunChi for the development of plant strengthening chitosans from fungal mycelium left over as a waste fraction of industrial fermentations for the production of technical enzymes. Soofia will focus on the identification and optimization of hydrolytic enzymes required for the extraction of high quality chitin from the fungal cell walls. To this end, she will closely collaborate with the group of Prof. Arthur Ram from the University of Leiden, who is an expert in fungal cell wall genetics and who is also a partner in the FunChi project. Besides Prof. Ram, Prof. Andreas Hensel from the Faculty of Chemistry and Pharmacy, an expert in carbohydrate chemistry, will be on her doctoral committee.
November 2, 2016: Paper accepted: “A chitin deacetylase from the endophytic fungus Pestalotiopsis sp. efficiently inactivates the elicitor activity of chitin oligomers in rice cells.” (http://rdcu.be/m77d)
Stefan Cord-Landwehr’s and his co-authors’ paper on the successful heterologous expression and characterization of the first chitin deacetylase gene from a plant endophytic fungus has been accepted for publication in Nature’s open access journal “Scientific Reports”. This is our second recombinant fungal chitin deacetylase, after the one from the wheat stem rust fungus we published earlier this year. Both enzymes yield defined partially acetylated chitosan oligomers (paCOS) when acting on fully acetylated chitin oligomers as a substrate but interestingly, the products of both enzymes differ in their pattern of acetylation. While the latter deacetylates all units except for the two units closest to the non-reducing end of the oligomer, the new enzyme also leaves the reducing end unit acetylated. Thus, when acting e.g. on the chitin tetra- to hexamers AAAA, AAAAA, and AAAAAA, PgtCDA produces AADD, AADDD, and AADDDD, while PestCDA yields AADA, AADDA, and AADDDA. In addition to thus offering a new tool for the biotechnological production of defined and novel paCOS, we were also able to show that this partial deacetylation converts the elicitor-active chitin hexamer into an elicitor-inactive paCOS. This observation is the first experimental evidence for one of the two postulated physiological roles of fungal chitin deacetylases, namely to prevent the triggering of defense reaction in infected plant tissues.
October 14, 2016: Paper accepted: “A broad pH range and processive chitinase from a metagenomic library.” (http://dx.doi.org/10.1590/1414-431x20165658)
Sarah Sacks Thimoteo’s and her co-authors’ first paper resulting from her sandwich year in our group has been accepted for publication in the “Brazilian Journal of Medical and Biological Research”. Sarah had identified a bacterial gene cluster containing three putative chitinase genes in a metagenomics library from a Brazilian industrial waste water sludge. She expressed all three genes in E. coli and proved that they indeed code for chitinases. In this paper, we describe the characterization of the first of these chitinases which is a multi-domain protein containing a catalytic domain of the Glycoside Hydrolase family GH18 and four additional domains, including two chitin binding domains. Careful analysis of substrate specificities and product patterns revealed that the enzyme is a processively acting endo-chitinase. The very broad pH range and unusually high salt tolerance of this enzyme make it a promising tool for biotechnological applications, e.g. as a bio-control agent protecting plants from insects and their larvae, as the enzyme would retain activity at the high pH of the insects’ midgut where it could attack the peritrophic membrane, decreasing food digestibility.
October 18-19, 2016: kick-off “FunChi” meeting in Münster
The European Research Area - Industrial Biotechnology research project FunChi coordinated by Bruno Moerschbacher had its belated kick-off meeting this week in Münster. We welcomed the CEO of our German industrial partner Weiss BioTech, Hans de Bie, and his research team consisting of Dr. Mareike Dirks-Hofmeister and Dr. Vasil Iliev, Prof. Arthur Ram and his PhD student Tim Leeuwe from our Dutch academic partner University of Leiden, Prof. Peter Punt from our Dutch industrial partner Dutch DNA Biotech, Prof. Antonio Molina and Dr. Sara Storres from our Spanish academic partner Polytechnical University of Madrid, as well as Prof. Marise Borja and Dr. Frederic Brunner from our Spanish industrial partner Plant Response Biotech. The goal of the FunChi project is to develop plant strengthening chitosans on the basis of chitin extracted from the cell walls of filamentous fungi used for the industrial production of technical enzymes. The Dutch partners will be responsible for optimizing the fungal strains for better chitin yields, the German partners will develop protocols for the extraction of high quality chitin from the waste mycelium, and the Spanish partners will convert the fungal chitin into plant strengthening chitosans.
October 17, 2016: new PhD student Naivy Yuvicel Nava Cruz arrived from Mexico
This weekend, our new PhD student Naivy Yuvicel Nava Cruz from Mexico has arrived in Münster to attend the “get-to-know weeks” organized by the Graduate Centre of WWU which had granted her a small fellowship to come here for two weeks. As Naivy had already successfully applied for a four-years doctoral fellowship from the Mexican funding agency CONACYT, she will start her doctoral research project already in November. Naivy plans to investigate how chitosans can protect plants from pathogenic nematodes. To this end, she will profit from our expertise on bioactivities of chitosans, in particular regarding their plant growth promoting and disease resistance inducing activities. Her project will be a collaborative project with Prof. Joachim Hallmann from the Julius Kühn Institute for Epidemiology and Pathogen Diagnostics in Münster, an expert in plant pathogenic nematodes. The third member of her doctoral committee will be Prof. Eva Liebau from our University, an expert for human pathogenic nematodes.
October 10, 2016: Rebecca Melcher successfully defended her PhD thesis
This Monday afternoon, Rebecca Melcher successfully defended her PhD thesis with a public presentation and discussion of her research results followed by a closed examination with her doctoral committee consisting of the professors Bruno Moerschbacher, Francisco Goycoolea, and Dirk Prüfer. Rebecca did her PhD in the framework of MCGS, the Indo-German graduate school for Molecular and Cellular Glyco-Sciences. She was part of the third and sadly last generation of MCGS doctoral candidates. Her project had involved i.a. the development of a medium-throughput screening assay for plant disease resistance inducing compounds which she used to identify elicitor- and priming active chitosan and ulvan oligomers. This had been essential for elucidating e.g. structure-function relationships of partially acetylated chitosan oligomers as plant resistance inducing compounds or the physiological role of a soluble chitin deacetylase in a plant endophytic fungus. These results will help us to develop novel knowledge-based plant disease protectants based on chitosans. After her successful defense, Rebecca will stay with us for a few more months to help us finalize some open ends of her work, trying to collect the last missing data for another manuscript or two to be submitted shortly.
October 4-7, 2016: half-annual “Nano3Bio” meeting in Mannheim
This year’s fall meeting of the European Nano3Bio project which is coordinated by Prof. Moerschbacher was organized by Dr. Christian Gorzelanny from the Institute for Experimental Dermatology in Mannheim. The consortium meeting itself was preceded by a “Patenting Workshop” given by Johan Brants from the Belgian company Brantsandpatents, particularly attended by young researchers, including seven people from our group. During the meeting itself, all partners presented their results of the past six months and their plans for the coming six months. From our group, the PhD students Stefan Cord-Landwehr, Maha Attjioui, Jasper Wattjes, and Lea Hembach as well as the master student Frauke Lankamp presented their results, and Bruno Moerschbacher reported the work of Nour Eddine El Gueddari who could not attend himself. After the meeting, Dr. Dominique Gillet from the French company Gillet Chitosan, our long-standing provider of high quality chitosans, and Derek Latil from the Spanish company Greenaltech who recently discovered the first non-fungal natural chitosan producers, namely a special group of green algae, accompanied us to Münster to discuss future joint research and business options.
September 23, 2016: Service unit online: “ChitoProf - Professional ChitosanS for the Life Sciences”
Today, we finally went online with our service unit “ChitoProf” to deepen the impact of our research. We want to reach out to scientists in academia and industry who are interested in elucidating structure-function relationships of chitosans as a knowledge-base for the development of reliable chitosan-based applications and products. We want to help bridge the gap between academic research and industrial development by supplying well-defined chitosan polymers and oligomers as well as well-defined chitosan modifying enzymes on a laboratory scale. If demand develops for larger quantities, we will be able to satisfy it in collaboration with our partners, such as Gillet Chitosan or BBEPP. We are also offering our expertise in chitosan characterization to help reduce the number of papers in which people report on using “chitosan” without giving any specifications of the material they used, and to help reduce the frequency of failures in developing applications caused by the use of ill-defined chitosans.
September 22, 2016: Paper accepted: “Physical properties and biological stability of soft gelled chitosan-based nanoparticles” (DOI link)
Francisco Goycoolea’s and Nour Eddine El Gueddari’s and their co-authors’ paper on structure-function relationships of partially acetylated chitosan polymers concerning their ability to form soft nanoparticles, i.e. the influence of structural parameters of the chitosans used on the functional characteristics of the nanoparticles obtained, has been accepted for publication in the Journal “Macromolecular Bioscience”. The work we report here was performed in the context of our European research project NanoBioSaccharides almost ten years ago but their publication failed at the time - perhaps we were too much ahead of times. Back then, it was generally assumed that once chitosans (or other biopolymers) are formulated as nanoparticles, the details of their chemical structure - such as their degree of polymerization (DP) or their degree of acetylation (DA) - become irrelevant. However, as we found, this is not the case. Both the size of the particles and their stability in physiological media strongly depend on the DA and the DP of the chitosan used. And as no such systematic study has appeared to this day, our data are still new and relevant. In fact, they formed the basis for a number of papers we published over the past few years on the generation of tailored nanoparticles for different applications, such as copper formulation for plant protection or gene delivery into human cells.
August 28, 2016: Paper accepted: “A recombinant fungal chitin deacetylase produces fully defined chitosan oligomers with novel patterns of acetylation” (DOI link)
Shoa Naqvi’s and her co-authors’ paper on a recombinant chitin deacetylase from the wheat stem rust fungus and its unique ability to produce partially acetylated chitosan oligomers with fully defined sequence has been accepted for publication in the Journal “Applied and Environmental Microbiology”. In this paper, we describe the heterologous expression of the fungal gene in the bacterial host E. coli which required the systematic development of a sophisticated protocol. The enzyme can convert fully acetylated chitin oligomers into partially acetylated chitosan oligomers with a defined pattern of acetylation, namely leaving the two GlcNAc residues closest to the non-reducing end of the oligomer acetylated while deacetylating all other units. With this paper, chitin deacetylases emerge as a powerful new tool to produce fully defined chitosan oligomers which cannot currently be produced in any other way. We are using these oligomers to gain a detailed molecular and cellular understanding of structure-function relationships and modes of action of chitosans, as a prerequisite for the knowledge-based development of reliable applications of this promising functional biopolymer.
July 30, 2016: Paper accepted: “Corynebacterium glutamicum possesses β-N-acetylglucosaminidase” (http://rdcu.be/nnN3)
This paper is the first in what hopefully be a series of papers resulting from our collaboration with Prof. Volker Wendisch’s group at the University of Bielefeld aiming to allow C. glutamicum to grow on chitin. C. glutamicum is a Gram-positive bacterium used industrially on a large scale for the production of amino acids mainly as animal feed additive. The Wendisch group is interested in broadening the spectrum of C- and N-sources for this bacterium, and chitin as a waste material from the shrimp peeling industry would be an interesting candidate. Therefore, they teamed up with us to profit from our expertise in chitin degradation, and we are since providing them with potentially suitable enzymes such as chitinases. The here described discovery of an N-acetylglucosaminidase - which can cleave the dimer chitobiose, the typical end product of chitinase activity, into two GlcNAc monomers - was a side result of that project. However, we found that even the presence of these two enzymes does not suffice to allow C. glutamicum to grow on chitin, probably because crystalline chitin is not an easy substrate for the chitinase. We are currently continuing this work in a small CiM-project, F2F, supported by BMWi, with Prof. Bodo Philipp’s group from our Institute for Microbiology and Molecular Biotechnology as a third partner.