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PolyModE Project: Update on progress during the second year
January 2012
The first half of the PolyModE project was mainly devoted to capacity building, both in terms of developing the scientific tools and biological resources and in terms of collaboration and team forming. During the first reporting period, we focused mainly on the identification and exploitation of suitable sources for novel polysaccharide modifying enzymes, while the second reporting period was mostly devoted to the heterologous expression of these genes and the purification of enzymatically active, recombinant proteins. These enzymes are now being used to generate and analyse novel, enzymatically modified polysaccharides. Eventually, during the second half of the PolyModE project, we will analyse in detail the physico-chemical properties and biological funtionalities of these novel polysaccharides. Our final goal is to integrate successful enzymatic modification steps into existing commercial processes to develop viable chemo-enzymatic strategies for the production of novel, beneficial products for the food and health market.
We began by screening a number of potentially interesting biological sources for the novel enzymes targeted, and identified the most promising ones. Each of the six hydrocolloid work packages follows two independent approaches to identify such sources. In some cases, one of these approaches is a metagenomic one, in which the total DNA present in an environmental sample is isolated and preserved in a large metagenomic library. As compared to a genomic library which contains the genome of one species only, a metagenomic library contains the genomes of all (microbial) species present in the environmental sample. This approach has the advantage that the metagenomic library will also contain the genes present in the vast majority of those microorganisms which do not readily grow on artificial media and which are, therefore, all but unknown and unexploited today. As an example, we have established a metagenomic library from selected soil samples taken from the site of a company isolating chitin from shrimp shells and processing it into chitosans. The rationale here is that in soil samples with a more than ten years’ history of exposure to chitin and chitosan, we can expect a microflora enriched in bacteria and fungi specialised on degrading these polysaccharides, and these microbes can be expected to have evolved all kinds of sophisticated enzymatic tools for the modification and breakdown of chitin and chitosan. In a first step, we collected soil samples from different sites in the company (and nearby, for controls) and established a protocol suitable for the isolation of metagenomic DNA from them. In parallel, we isolated bacteria and fungi from these soil samples which exhibited the ability to degrade chitin or chitosan (in which case, of course, we only look at the minority of culturable microorganisms). Two samples which gave good yields of metagenomic DNA and also displayed a rich biodiversity of chitin/chitosan degrading microbes were selected, mixed, and used for the establishment of the metagenomic library. The next task then is to find, in the more than 40,000 clones of the library, those clones containing a gene (or genes) for chitin/chitosan modifying enzymes. In principle, this screening of the library can be performed either by sequence-based or by function-based methods. While sequence-based methods are simple, they rely on at least some prior sequence knowledge of the gene family of interest - so that only genes related to already known genes can be found. In contrast, functional screenings are a lot more demanding as a suitable screening assay needs to be developed for each enzyme targeted, but they allow the identification of truly novel enzymes and their genes or even gene families. We therefore opted for the functional approach, and we have established a combined screening assay which allows us to search for chitinases, chitin deacetylases, and chitosanases at the same time. With these tools and resources at hand, we are now screening the libraries in the hope to identify novel enzymes.
In parallel, we have also followed a knowledge-based, genomic approach targeting the same class of enzymes. We isolated a number of bacteria and fungi from the chitin/chitosan exposed soil samples and identified them using morphological and molecular classification tools. We found one bacterial species to be especially prominent in our samples, and we then searched the available gene databases for genes of this species that contain tell-tale domains, such as chitin binding domains, chitin or chitosan hydrolase domains, or chitin deacetylase domains. Using this sequence information, the corresponding genes were then cloned from the bacteria we had isolated, the genes were heterologously expressed in E. coli and the recombinant enzymes were purified. The biochemical characterisation of the first set of such genes already yielded one enzyme with a novel combination of properties never described before. This is now under further investigation and will be patent-protected, if possible, prior to publication. Another gene identified thus has been converted using protein engineering into an analytical tool to reveal the presence of chitosan in natural samples, and we have developed a screening strategy for enzymes converting chitin into chitosan using this tool; this chitosan affinity protein and its uses as well as the generic strategy used have been submitted for IPR protection and for publication in an international scientific journal. A fungal gene thus identified turned out to be the prototype of a whole new class of genes, and we have been successful now in its difficult heterologous expression which succeeded only in an alternative yeast host available within the PolyModE consortium. This is a highly promising class of enzymes, potentially with properties that have been sought after in vain for decades. This is just one example where success was only possible through close collaborations between different PolyModE partners.
Other work packages followed a transcriptomic approach to identify novel polysaccharide modifying enzymes. The rationale here is to identify genes of a selected organism which are active under conditions where the enzymes targeted can be expected to be needed by this organism. The organism is grown under these specific conditions, and total mRNA is isolated, then transcribed into cDNA which can be cloned in a cDNA library. If the genome of the organism is fully sequenced and a DNA microarray of all genes identified is available, the identification of genes specifically up-regulated under the conditions specified is rather straight-forward. As an example, this approach has been followed for the identification of novel enzymes modifying algal polysaccharides such as alginate or carrageenan. A marine bacterium which had been isolated previously from algal samples and which is known to be able to grow on a wide range of algal polysaccharides as a sole carbon source – indicating that it must have evolved genes coding for enzymes capable of degrading such polysaccharides – has now been used in these experiments. Significantly up-regulated genes were further analysed using a range of bioinformatic tools to identify the most promising candidates. These have been cloned for heterologous expression in E. coli or, where this was not successful, in alternative hosts. Enzymatically active, recombinant proteins have been obtained in many cases, and a range of novel alginate and carrageenan modifying enzymes have been found.
In parallel, a genomic approach was also followed to target modifying enzymes for algal polysaccharides. Here, we made use of the recently deciphered genome of a brown alga which underwent a thorough bioinformatic analysis yielding more than thirty potentially interesting genes, and hints for the presence of novel pathways involved in alginate synthesis, modification, or degradation. These being eucaryotic genes, the heterologous expression is less straight-forward than in the case of bacterial genes. First, full length cDNAs of the genes of interest need to be isolated which are currently being cloned into suitable hosts, firstly E. coli again, but then possibly also eucaryotic expression systems available in the PolyModE consortium. In some cases, highly specialised expression systems are being required for the production of functional recombinant enzymes. One example are polysaccharide sulfatases which require a specific post-transcriptional sulfatase maturation step. A bacterial expression system able to perform this maturation step has been established by one of the partners and has successfully been made available to another partner needing it. We have also teamed up to develop a novel broad host range shuttle vector which gives us access to an even wider variety of (bacterial) exppression systems which we are only beginning to exploit.
Similarly, metagenomic, transcriptomic, genomic, and bioinformatic approaches for gene identification followed by heterologous gene expression and characterisation of the potentially novel enzymes encoded are being followed for all the six hydrocolloids targeted by the PolyModE project. An additional approach which is becoming more important in several work packages is the generation of cDNA expression libraries allowing the functional screening of eucaryotic genomes, a difficult tool which we have established through close collaborations between different PolyModE partners. In future, dedicated libraries for the comprehensive screening of engineered protein mutants will advance to the front of the PolyModE project, and we are already developing new tools to this end.
In parallel, the large work package devoted to the development of generic techniques brings together the resources and expertises of all academic and commercial PolyModE partners. Three interest groups have been formed, focusing on gene identification, heterologous expression/fermentation, and analytical tools. Regular teleconferences are held to coordinate the activities of the interest groups, and to focus the efforts of different partners on crucial problems. As an example, the analytical tools group decided to first focus on the development and use of NMR techniques to fully characterise selected oligosaccharides needed as reference compounds for the analysis of e.g. the products of polysaccharide hydrolytic enzymes. This is supported by the available and expanding expertise in organic synthesis of oligosaccharides with perfectly controlled architecture. This approach has been especially successful with three alginate lyases whose complementing substrate specificities have been elucidated using NMR. These enzymes are now being used to develop a fingerprinting analysis for sequencing alginates, a prerequisite for the ongoing development of enzymatic modification protocols to yield novel alginates with unusual patterns of guluronic acid distributions. A second focus which has become more prominent in the second year is the three-dimensional structure analysis of proteins using x-ray crystallography. As soon as a higly pure recombinant enzyme becomes available in large enough quantities, attempts at crystallisation are initiated with the goal to obtain structural information needed for the development of protein engineering strategies to optimise their preformance under biotechnological production conditions. Similarly, the gene identification group has focused on the further development of methods for the establishment and screening of metagenomic, genomic, and cDNA libraries, while the heterologous expression/fermentation group is focusing on further improving the existing range of pro- and eucaryotic expression systems which include gram-positive and gram-negative bacteria as well as different yeasts. A particular focus is on the the further development of the bacterial system Lactococcus lactis and on the yeast system Hansenula polymorpha.
Perhaps the most crucial point for success in the beginning of a large collaborative project bringing together teams from different disciplines, from different countries, from universities, research centres, SMEs, and multinational companies, is team building. This was achieved through the regular half-annual meetings of all consortium members which are not only well attended, but which are always conducted in an atmosphere of mutual trust and interest. All groups freely bring in their experiences, offer their advice and suggestions, and make available their infrastructure and biological or chemical samples to the benefit of the whole project. Close bilateral and multilateral collaborations have been initiated, ideas and materials are being exchanged freely, and the exchange visits of scientists - mostly young scientists working on their doctoral or post-doctoral projects within the framework of PolyModE - are becoming an integral instrument of success. These close interactions were greatly supported by the first PolyModE Technical Workshop organised jointly by three of the PolyModE partners. But the discussions between the young researchers are by no means limited to technical issues. In fact, they have started their own Young Researcher’s Round Table Discussions during the half-annual PolyModE meetings, in parallel to the management meeting of the principal investigators.
One important aspect in team building is also the professional support in all questions of scientific and organisational, e.g. financial or legal management, which is smooth in the PolyModE project due to the experience of the partners specifically and professionally involved in management. This includes the generation and regular use of the PolyModE website and the building of a corporate identity by the development of a project logo and corporate style. From the start of the project, we have taken active steps in the form of regular workshops during the consortium meetings to focus the attention of all partners on the needs of a research project devoted not only to the discovery of novel enzymes, but also to their development into tools suitable for improving the production and performance of hydrocolloid polysaccharides in food and life science applications. The continuous development of truly common targets and strategies will be an ongoing process throughout the lifetime of the PolyModE project, and it had a very successful start during its first half.
Overall, we had a very successful and promising first half of the project, and we are looking ahead to its second half with great expectations.