Identification



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PolyModE Project: Update on progress during the first year
The first year 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.
In particular, we screened 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 with 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 will now start the actual screening of the library.
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. We have started the biochemical characterisation of the first set of such genes, and we have already identified one 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.
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 are now being cloned for heterologous expression in E. coli and further analysis of their properties.
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 above discussed cases. First, full length cDNAs of the genes of interest need to be cloned 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 would be 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 will be available to others needing it.
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.
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. A second focus which will become more prominent in the future will be three-dimensional structure analysis of proteins using x-ray crystallography. Once higly pure recombinant enzymes are available in large enough quantities, these techniques will allow the development of protein engineering strategies to optimise their preformance under biotechnological production conditions, based on the known 3D structures. 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.
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. Already, close collaborations have started, materials are being exchanged and the first exchange visits of scientists - mostly young scientists working on their doctoral or post-doctoral projects within the framework of PolyModE - are being planned.
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 was 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 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 the first year.
Overall, we had a very successful and promising start of the project, and we are looking ahead with great expectations.