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Periodic Report Summary 2 - NANO3BIO (NanoBioEngineering of BioInspired BioPolymers)

Project Context and Objectives:

The Nano3Bio project convenes a consortium of world renowned experts from eight EU universities, one large company, and 14 SME, to develop biotechnological production systems for nanoformulated chitosans. Chitosans, chitin-derived polysaccharides varying in their degree of polymerisation (DP), degree of acetylation (DA), and pattern of acetylation (PA), are among the most versatile and most promising biopolymers, with excellent physico-chemical and material properties, and a wide range of biological functionalities, but their economic potential is far from being exploited due to i) problems with reproducibility of biological activities as today’s chitosans are rather poorly defined mixtures, and ii) the threat of allergen contamination from their typical animal origin.
The Nano3Bio project will overcome these hurdles to market entry and penetration by producing in vitro and in vivo defined oligo- and polymers with controlled, tailor-made DP, DA, and PA. Genes for chitin synthases, chitin deacetylases, and transglycosylating chitinases/chitosanases will be mined from different (meta)genomic sources and heterologously expressed, the recombinant enzymes characterized and optimized by protein engineering through rational design and molecular evolution, e.g. targeting engineered glycosynthases. These enzymes and genes will be used for in vitro and in vivo biosynthesis in microbial and microalgal systems, focusing on bacteria and diatoms. The bioinspired chitosans will be formulated into biomineralised hydrogels, nanoparticles, nanoscaffolds, etc., to impart novel properties, including by surface nano-imprinting, and will be bench-marked against their conventional counterparts in a variety of cell based assays and routine industrial tests for e.g. cosmetics and pharma markets.
The process will be accompanied by comprehensive life cycle assessments including thorough legal landscaping, and by dissemination activities targeted to the scientific community and the general public.

Project Results:

During the second reporting period, the Nano3Bio project has made highly significant progress towards its final goals.
Work package 1 is devoted to identifying and characterizing suitable genes and enzymes for the biotechnological synthesis and modification of chitin and chitosan oligo- and polymers from a broad range of sources to harvest the potential of natural biodiversity. We have identified, cloned, and heterologously expressed a number of chitin synthases from viral, bacterial, fungal, diatom, and oomycete sources. We have for the first time achieved in vitro synthesis of chitin polymers, using oomycete chitin synthases. We have engineered a bacterial chitin synthase to increase the degree of polymerization of its product. We have identified chitinases with the highest transglycosylating activities so far reported, and we have increased this activity even further using site directed mutagenesis. We have used engineering to convert this transglycosylating chitinase into the first chito-synthase which, however, still has residual hydrolytic activity. We have heterologously expressed a range of viral, bacterial, fungal, oomycete, and microalgal chitin deacetylases and found that they produce partially acetylated chitosan oligomers and polymers with different, non-random patterns of acetylation. Based on crystal structures of a bacterial chitin deacetylase, we have developed the subsite capping model to understand substrate preferences of these enzymes, and we have used this model for the targeted engineering of deacetylases to generate muteins yielding products with changed patterns of acetylation. We have developed a new method to generate mutational libraries of an enzyme of interest through directed evolution for an affordable price; this method has been commercialized and it is being used now for the further engineering of a bacterial chitin deacetylase.
Work package 2 is devoted to using the enzymes identified and characterized in WP1 for the in vitro production of defined chitosan oligomers and polymers in a biorefinery approach. We have used the recombinant chitin deacetylases to produce a large library of fully defined, partially acetylated chitosan oligomers. As an example, we are able to produce the full set of 14 different partially acetylated chitosan tetramers. These oligomers are now being used in WP4 to assess their biological activities, and thus to get insight into the so far hypothetical influence of the pattern of acetylation on bioactivities of chitosans. Similarly, we are using these enzymes for the production of chitosan polymers with non-random patterns of acetylation, and these novel chitosans are now being tested for the physico-chemical properties and for their biological functionalities. Using the transglycosylation approach, we have generated small amounts of the highly precious chitin oligomers with a degree of polymerization of larger than six. While these oligomers are in principle available through conventional methods and have also been commercially available in the past, they are no longer available to the scientific community even though they are crucial to understand chitin perception in plant disease resistance on a molecular level, as a prerequisite for developing sustainable plant protection strategies based on the plants’ own immune system. We have developed protocols for the large scale production of activated chitin and chitosan oligomers which are needed for the biotechnological polymerisation into fully defined chitosan polymers using our chito-synthase, and we have first evidence for the production of small polymers using this approach.
Work package 3 is devoted to using the genes identified and characterized in WP1 for the in vivo production of defined chitosan oligomers and polymers in a cell factory approach. We have succeeded in expressing various of the chitin synthase and chitin deacetylase genes identified, characterized, and optimized in WP1 in the gram-negative bacterium Escherichia coli, and some in the gram-positive bacterium Corynebacterium glutamicum, thus producing a range of different defined partially acetylated chitosan oligomers. Through systematic metabolic engineering and optimization of the fermentation conditions, we have increased the yield of monoclonal chitooligosaccharides by a factor of twenty. And systematic optimization of down stream processing protocols allows us to produce highly pure chitosan oligomers, already approaching scales sufficient for initial testing for industrial applications. While screening microalgae for their suitability as eukaryotic chitosan production hosts, we identified several species naturally producing chitin, and even - very unexpectedly - chitosan. Microalgae are already fermented at an industrial scale, and we are confident that this will offer a very rewarding opportunity to exploit a new natural source of chitin. We aim to develop it into the first commercial source of chitin from non-animal origin - certainly a boost for the development of chitosan-based applications in sensitive markets such as bio-cosmetics. Moreover, microalgae will be the first commercial source of natural chitosan not derived chemically from natural chitin. And the results of WP4 let us expect that biotechnologically produced, natural chitosans will differ significantly from conventionally produced chitosans.
Work package 4 is devoted to the chemical and biological analysis of biotechnologically produced chitosans delivered by WP2 and WP3, in comparison to the best conventional chitosans available on the markets today, and to a wide range of speciality chitosan produced at lab scale. We have developed new micro-scale techniques to analyse the degree and pattern of acetylation of partially acetylated chitosan oligomers and polymers. Quantitative mass spectrometric sequencing techniques allow to quantify the different oligomers even in complex mixtures, and enzymatic / mass spectrometric fingerprinting analyses allow analysis of polymers in unparalleled detail. This enabled us to verify that chitosans produced by the enzymatic action of chitin deacetylases have non-random patterns of acetylation, unlike chitosans chemically derived from chitin which invariably have random patterns of acetylation. We expect that these novel chitosans will also differ from conventional chitosans in their physico-chemical properties and in their biological functionalities which are currently under investigation. We have already established that the pattern of acetylation of chitosan oligomers crucially determines their ability to induce disease resistance reactions in plant cells, and we are currently analyzing its influence on the antimicrobial, plant growth promotion, and different pharmacological and biomedically relevant functionalities of chitosans.
Work package 5 is devoted to the evaluation of the application potential of the novel, biotechnologically produced chitosans delivered by WP2 and WP3, as compared to that of the best conventional chitosans available on the markets today, and to a wide range of speciality chitosan produced at lab scale in WP4. While the biotech chitosans were not yet available during the first half of the project, we have begun to develop new nanoformulations of well defined conventional chitosans, to set the benchmarks for the biotech chitosans to come. We have developed enzymatic methods for the biomineralization of chitosan hydrogels potentially useful for bone repair. We have found a way to stabilize chitosan nanofibers produced by electospinning in aqueous solvents so that they can be used as drug delivery agents in biomedical systems. We have started to prepare chitosan-based, surface-imprinted nanocapsules to interfere with bacterial quoarum sensing as a future alternative to antibiotic treatments. We have developed chitosan nanoparticles for the very efficient delivery of siRNA to mammalian cells and we have begun to assess their potential to influence wound healing. We have for the first time followed the uptake and intracellular transport of chitosan nanoparticles into and in mammalian cells. In the long run, perhaps the most influential discovery we made relates to a method to use chitosan particles for the diagnosis and possibly even treatment of cancer pre-metastases. A more immediate potential of chitosans we are developing is their dual use as thickener and preservative in hand cream and body wash formulations.
Work package 6 is devoted firstly to the first ever comprehensive Life Cycle Assessment of chitosan production. We have painstakingly collected all pertinent data for two different conventional chitin and chitosan polymer production pathways and deposited them in public databases. This allowed us to identify hotspots for environmental impact of these processes, allowing the commercial chitin and chitosan producers who provided the data to improve their productions. We have also compiled the data for the biotechnological production of chitosan oligomers using the cell factory approach, and we will benchmark it against the conventional production of the same oligomer mixture. Secondly, work package 6 is devoted to analyse the legal constraints for functional biopolymers such as chitosans to be developed into marketable products, particular relating to the fields of medicines, cosmetics, plant protection products, and functional feed ingredients. We have compiled a comprehensive list of the applicable law as well as an interpretation for the distinct phases relative to chitosan research and development. Currently, a summary report is being drafted which will include suggestions for possible areas of future policy decisions.
Work package 7 is devoted to the administrative, legal, and financial management of the large project which comprises 24 partners from 21 European and one Indian institution. Thanks to the involvement of a professional partner (LIP) with a strong record of successfully supporting the coordinator WWU, this task is handled efficiently. LIP also serves as a helpdesk whenever any of the partners needs administrative support. We have so far organized seven half-annual consortium meetings at partner sites, one of them in conjunction with the globally largest and most important chitin/chitosan meeting, EUCHIS/ICCC 2015, organized by P1 (WWU). Twice, these meetings were accompanied by training workshops, on bioinformatics and IPR protection, particularly for the young Nano3Bio researchers. They also organize Young Researcher Symposia as satellite to the consortium meetings, to improve communication and technology transfer. As a consequence, a large number of Researcher Exchange Grants have been granted from the central budget to allow young Nano3Bio researchers to visit a different partner lab, for a few days up to several months, to learn techniques and profit from the expertise and infrastructure of their hosts. The Paris consortium meeting saw the first convention of the Scientific and Exploitation Advisory Panels, and members of both panels have since joint us at all meetings, to support the strategic orientation of the project. A few patent applications already submitted or in the process of being prepared and a growing number of scientific manuscripts submitted or already published in renowned international scientific journals are proofs of the success of the project.
Work package 8 is devoted to dissemination and exploitation of the goals, strategies, and results of the Nano3Bio project, supported by two professional partners (beemo, CSC). We have identified the dissemination goals of all partners and their most important target audiences, and we have designed a dissemination strategy based on these. The Nano3Bio website as well as its social media channels are continuously updated and growing, and they are followed by a satisfyingly high number of users. We had an excellent visibility during the EUCHIS/ICCC 2015 conference in Münster, both by high quality scientific contributions and by the Nano3Bio conference app. We are planning one other large dissemination meeting during the last year of the project, most likely in conjunction with the EUROCARB 2016 which will be organized in Barcelona by one of the Nano3Bio partners (IQS). Two more Nano3Bio apps are in the process of being realized, one of them a wiki-style chitin/chitosan webpage which will shortly go public. The most important tool for planning dissemination and exploitation is the Plan for the Use and Dissemination of Foreground, PUDF, which is continuously being updated. We regularly devote ample time during the half-annual consortium meetings to identify the results with the greatest scientific and commercial potential, strategically planning for their long-term use to the benefit of the Nano3Bio partners, both from Academia and from Industry, and to European and global society at large.

Potential Impact:
By developing biotechnological ways of producing well defined polysaccharides and nanotechnological tools for their formulation and functionalization, yielding products with known physico-chemical properties and reliable biological activities, Nano3Bio aims to offer a fresh approach towards realising the ecological and economical potential of functional bio-polymers, opening new opportunities for the knowledge-based development of high added-value products.
By far the most promising and most advanced functional bio-polymer is the polysaccharide chitosan. The term chitosan really refers to a family of polysaccharides derived from one of the most abundant renewable resources on earth, chitin. Chitin is found in the exoskeletons of insects and crustaceans such as shrimp and crab, in the endoskeletons of mollusks such as squid, in many invertebrates as in egg shells of nematodes, as well as in the cell walls of fungi and some diatom algae.
While chitin is a linear polysaccharide built from just one monomeric sugar unit, namely acetylated glucosamine, chitosans are linear co-polymers of acetylated and non-acetylated residues. This partial deacetylation yields free amino groups which at slightly acidic pH values convey positive charges to chitosan, making it the only polycationic polysaccharide. As such, chitosans can easily interact with polyanionic biomolecules such as most proteins and nucleic acids, but also polyanionic phospholipidic membranes and sulphated polysaccharides such as e.g. human glycosaminoglycans at cell surfaces. Such purely electrostatic interactions are partly responsible for the many biological activities reported for chitosans.
Among these biological activities are:
• Amply demonstrated antimicrobial activities
• Plant growth promoting and disease resistance inducing activities
• Mucoadhesive and immunostimulatory activities
• Anti-proliferative and wound healing promotion activities
• The ability to complex and subsequently deliver genetic material in vivo
• Opening of cellular tight junctions in a reversible manner
The crux with all of these, however, so far has been their poor reproducibility. The development of marketable chitosan-based products has lagged far behind expectations due to repeated failures to achieve reliable and predictable biological functionalities. This failure to achieve reproducible bioactivities was at least partly due to the rather poor characterisation and the resulting batch-to-batch differences in commercially available chitosans.
Based on extensive experience, the Nano3Bio partners are addressing the objective of developing biotechnological biosynthesis strategies for partially acetylated chitosan oligomers and polymers with narrowly defined quality. Two approaches are being followed in parallel: an in vitro biorefinery approach and an in vivo cell factory approach. In the biorefinery approach, we are using bioengineered and optimized chitin synthesizing. modifying, and degrading enzymes on conventionally produced chitin and chitosans or their chemically activated, monomeric building blocks, to yield novel chitosans with more defined, non-random patterns of acetylation not achievable using chemical protocols. In the cell factory approach, we are using genes coding for chitin synthases and chitin deacetylases towards the production of monoclonal oligo-chitosans. Both approaches are being successful, partly beyond expectation. As an example, we have identified several natural chitosan producing organisms outside the kingdom of fungi to which natural chitosan production was thought to be limited. This offers unprecedented opportunities for the biotechnological production of pure chitosans in their native forms for which we already have strong reasons to believe that they differ from today’s conventional chemically produced chitosans. Moreover, the non-animal origin of these novel chitosans will allow rather swift entry into sensitive, high-price markets such as bio-cosmetics. The novel biotech-chitosans are not only different from conventional chitosans produced chemically, e.g. in having different, non-random patterns of acetylation, they are also more defined in terms of their chemical properties and - most likely, as this remains to be proven - biological functionalities, and the ones produced in cell factories will in addition equally avoid the problems of animal origin. Within the remaining time of the Nano3Bio project, we will characterize these novel chitosans and their nano-formulations more thoroughly, and assess their performance in a range of benchmarking application tests, in particular for cosmetic products, but also for other markets.
In the past, our series of highly successful European research projects CARAPAX, NANOBIOSACHARIDES, and PolyModE has paved the way from yesterday’s ill-defined and poorly reproducible “first generation chitosan” to today’s well-defined “second generation chitosanS” which begin to appear on the markets in sufficient quantities for the development of reliable chitosan-based products, firstly in the agricultural market sector and increasingly also in the biomedical sector. We are confident that the ongoing Nano3Bio project, directly building on the successful ERA-IB project ChitoBioEngineering, will guide the way towards tomorrow’s “third generation chitosans” which due to their biotechnological production processes will be different and more natural than the conventional chitosans derived by chemical processes from shrimp and crab shell wastes. The comprehensive Life Cycle Assessment which we perform in the Nano3Bio project will allow to further optimize the conventional production processes, lowering their environmental impact, and will guide the way to the development of sustainable biotechnological production processes. In parallel, the thorough analysis of the legal constraints hindering the commercial success of bio-based products, in particular products based on functional biopolymers such as chitosans, will point towards hot spots of required future legal activities to foster the transition to a sustainable, circular bio-economy.

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