PolyModE: The Magic Ingredients of Molecular Chefs.
Or: The Language of Sugars
The European Commission supported PolyModE as a European research project for the development of novel enzymes for the optimised use of plant, bacterial, and human hydrocolloids based on complex sugar molecules in the food sciences and for technical and medical applications. During the coming four years, the EU will provide six million Euro for the project involving 15 participants: universities, research centres, multinationals and small biotech companies from Germany, France, Denmark, the Netherlands, Sweden and Bulgaria. The co-ordinator of the project named „PolyModE - POLYsaccharide MODifying Enzymes" aiming at the deciphering of the molecular language of complex sugar polymers is Prof. Bruno Moerschbacher from the Department of Plant Biochemistry and Biotechnology at the University of Münster in Germany.
Polymeric sugars - so-called polysaccharides - are by far the most abundant biomolecules; they include such ordinary substances as starch made from potato or maize, and cellulose in cotton. As renewable resources, they are, thus, of high priority to man in many areas, from food sciences to textiles and medical products. Apart from simple sugar polymers like starch and cellulose which are basically long chains consisting of a single type of sugar, there are also highly complex sugar polymers made up of many different types of sugar, such as pectin used for gelifying jams. Such complex sugars are highly important additives in the food industries where they are thickening sauces, giving texture to cheeses and ice cream, or increase the viscosity of ketchup still allowing it to flow out of the bottle when shaken.
These sugars which are also termed hydrocolloids are typically obtained from plants and algae. Pectin, e.g., is mostly extracted from citrus peel, alginate from brown algae, and carrageenan and agar from red algae. Some hydrocolloids are also produced by bacteria, such as xanthan, or by fungi, such as chitosan; some are animal derived products such as gelatine (which, however, is not a sugar but a protein) from bones, hyaluronan from cartilage or heparin from mucosa.
In many cases, however, those hydrocolloids with the best properties are produced only from some very specific organisms and are, thus, available only in limited quantities. As an example, three quarters of the annually produced ca. 50.000 tons of carrageenan are extracted from only two species of red algae, and these have begun to become rare due to over-utilisation and global climate change. The researchers of the PolyModE project, therefore, want to isolate the enzymes - i.e. the tools used by these red algae to produce their superior quality carrageenan - and optimise them using state-of-the-art molecular genetic methods. These enzymes should then be suitable to be used in a biotechnological process to convert the inferior quality carrageenan from other, widespread red algal species into a superior quality product. A different goal of the PolyModE project will be the optimisation of pectin modifying enzymes. Today, pectin is extracted commercially mostly from citrus peel. Recently, however, the rising prices for energy and the development of a novel technique to burn orange peel have prompted the orange juice producing companies to use their orange peel for the generation of energy needed for juice production, rather than selling them to the pectin producing companies. This is why e.g. the PolyModE partner Danisco, one of the largest producers of food ingredients world wide, is now in search of alternative pectin raw material sources. The PolyModE project will, therefore, search for novel enzymes that can modify low quality plant material to provide highly functional pectin. Thus, PolyModE will create an enlarged platform for future functional developments in these and other hydrocolloids for food, health, and nutritional as well as technical applications.
Concomitantly, the PolyModE researchers are trying to understand the subtle molecular differences between the different sugar polymers from different organisms. In fact, complex sugar polymers have a lot more in store. Sugars are masters of diversity. They can build an enormous diversity of different structures, and information is contained in these structures. Cells recognise each other by the sugar structures on their surfaces, our immune system often recognises pathogens by their typical sugars, and the different blood groups as well as rejection of transplanted organs are based on different sugar structures. The language of sugars, however, is a lot more complex than the language of genes or proteins and so far, we can hardly read it, much less understand it.
While genes make use of only four ‚letters' and proteins use twenty, where these letters are nicely arranged in a continuous sequence, there are more than twenty letters in the alphabet of sugars, and these can firstly be modified by different types of ‚accents', they can secondly be linked in different ways, and they can thirdly even be arranged in branched patterns. This is what makes ‚reading' (chemical analysis) of the information present in complex sugars so incredibly demanding. And even if using state-of-the-art mass spectrometry may allow us to read the information, we are still far from understanding it. Today, we only know very few of the ‚words' of the language of sugars, such as one that inhibits coagulation of human blood and one that alerts plants to the presence of pathogenic fungi.
But if we want to use this language e.g. for medical purposes, we will not only have to be able to read and understand it, we will also need to learn writing it. Thus, researchers of the PolyModE partner Sanofi-Aventis have, in a demanding chemical process, synthesised the small but highly complex five-letter sugar which inhibits blood coagulation, producing a therapeutic agent for e.g. thrombosis or dialysis patients that is almost free of side effects. Chemical synthesis of this heparin fragment, however, is extremely difficult, time consuming, and expensive, its synthesis in fact requires more than fifty steps. This is why the PolyModE researchers want to investigate and use the cells' own writing and reading tools. In cells, both processes are performed by enzymes. It is these polysaccharide modifying enzymes which are in the focus of the PolyModE project. These, the researchers want to find, to produce them in large quantities and in high purity using molecular genetic methods, to study their properties, and to optimise the conditions for their use in the synthesis or modification of complex sugar structures in cell free systems.
To this end, biologists and chemists, microbiologists and biochemists, molecular geneticists and biotechnologists from three European universities - Münster in Germany, Wageningen in the Netherlands, and Uppsala in Sweden - and three research centres - INRA Jouy-en-Josas close to Paris, CNRS Roscoff in Brittany, and BAS Sofia in Bulgaria - have teamed up with the two multinationals Danisco and Sanofi-Aventis, with a chitosan producer (Gillet Chitosan from Nancy) as well as with half a dozen young biotech companies in the PolyModE project.
The biotech companies involved were selected based on their proven record of driving the development of modern biotechnologies in areas such as metagenomics and directed gene evolution (LibraGen from Toulouse and Geneart from Regensburg), or novel expression and fermentation systems (Artes from Langenfeld and GTP from Toulouse). The scientific team is complemented by professional management and consultancy teams (LIP from Lyon and CSC from Senden). Together, these partners possess a very broad spectrum of expertise and experience in the development and use of state-of-the-art methodology in sugar biology and chemistry, perfectly complementing each other and allowing the direct transfer of results from the lab to the industry scale. At the same time, the project offers ideal opportunities for young scientists to perform excellent basic research with a tangible potential for application, in a stimulating, interdisciplinary and international context.
The research of the PolyMode project will then not only open up new resources for known hydrocolloids, but it may also generate novel complex sugars with further improved properties. "If we understand how e.g. human cells produce certain types of heparin, or why they react with a stimulation of the immune system when confronted with certain types of chitosan, then we might be able to use optimised enzymes for the generation of designer polysaccharides that specifically support wound healing or suppress blood coagulation" says Prof. Moerschbacher. He is convinced: "Such specifically acting, complex sugar polymers which are produced in a completely biological process, will have an enormous potential in many fields. They are well compatible with the human body, and they are easily degraded in the environment."
This will be music to the ears of aficionados of the trendy molecular gastronomy, as many of the magic tricks of the molecular chefs rely on the detailed knowledge and skilful use of complex sugar hydrocolloids. Three stars can be earned by those alone who understand the language of sugars.
PolyModE project results
Peak oil and peak soil, peak water and peak grain – the beginning of the 21st century marks a crucial transition, from an oil-based economy to a bio-based economy, from exploiting fossil resources to using renewable resources. In many areas such as material sciences or energy, this transition to a knowledge-based bio-economy will heavily rely on the large and diverse group of biopolymers. Bio-based renewable energies mostly rely on the degradation of biopolymers, essentially starch and lignocelluloses, and the consumption of their monomeric building blocks to release the energy stored in them during their photosynthetic biosynthesis. Renewable biomaterials, in contrast, typically rely on the production of biopolymers, either by extraction from biological sources or by biotechnological production means, sometimes combining both. Most biopolymer-based biomaterials such as many polysaccharides, lignin, or rubber are most prominently used for their superior structural properties only. Some biopolymers, however, and in particular polysaccharides, also possess functional properties, and these can be at the basis of a broad spectrum of applications, ranging from food sciences and agriculture over cosmetics and pharmaceutics to biomedical sciences. The potential of such functional biopolymers is evident, as they combine superior material properties with excellent biocompatibility and often highly versatile biological activities, promising advanced applications in many life-science related market fields. However, the potential of polysaccharides is still largely unfulfilled today, mostly as polysaccharides are very demanding both in terms of their chemistry and in terms of their biology. In particular, the typical microheterogeneity, which is a hallmark of natural polysaccharides, is a severe hurdle in establishing reliable structure/function relationships and, eventually, in developing successful applications based on them.
Natural polysaccharides have evolved to fulfill a plethora of functions in their natural biological context, and as each individual polysaccharide typically fulfills multiple roles, their structures represent a balanced compromise to achieve the best overall performance. This, however, is different in biotechnological applications where the polysaccharide used typically has to fulfill a special singe purpose, so that there is room for improvement over the natural compound. Also, the roles attributed to polysaccharides in biotechnological applications may differ from their natural roles, so that modifications may be required to change their structural and functional properties. Today, these modifications aiming at improving the performance of a polysaccharide are typically attempted using chemical means, such as acid or alkaline treatment, or the introduction of additional substituents. These chemical methods, while typically well established and easily upscaled to industrial dimensions, often have severe limitations, in particular regarding their specificity. Also, their environmental burden in terms of energy or water consumption, or in terms of toxic waste production, is sometimes high. The PolyModE project, therefore, aimed at developing enzymatic tools to perform such modifications specifically and in an environmentally benign form.
The central assumption of the PolyModE project was that it is the pattern of substitution of complex functional polysaccharides that fine-tune their physico-chemical properties and/or their biological activities. These could be patterns of e.g. acetylation, sulfation, or methyl-esterification, but they could also be patterns in the sequence of monosaccharide building blocks or their glycosidic linkage type, or even patterns in the distribution of different side chains. We predicted that nature uses enzymes to ‘ write’ these patterns, but also to ‘ read’ them, i.e. to specifically and partially degrade the complex polysaccharides to generate specific oligosaccharides as the individual ‘ words’ of the language of sugars. The PolyModE project targeted writing and reading enzymes for the guluronic acid distribution in alginates from red algae (C5-epimerases, lyases), for the pattern of sulfation in carrageenans from brown algae (sulfatases and sulfurylases), for the pattern of acetylation in chitosans from shrimp and fungi (deacetylases and hydrolases) as well as in the pattern of sulfation in human glycosaminoglycans (sulfotransferases and sulfatases), in the patterns of methyl- and acetyl-esterification in pectins from higher plants (acetyl- and methyl-esterases) and in the distribution patterns of acetylated and/or pyruvylated side chains in bacterial xanthan gums (acetyl-esterases, lyases, and hydrolases). These six polysaccharides represent the most important or most promising functional polysaccharides today, with diverse applications of alginates, carrageenans, pectins, and xanthans as functional food ingredients due to their superior material properties, in particular gelling abilities, and with highly promising applications of glycosaminoglycans and chitosans in biomedical fields due to their versatile and highly specific biological activities.
Six work packages were devoted to these polysaccharides and connected through a central work package focusing on the development of generic techniques in bioinformatics and molecular genetics, heterologous expression and fermentation, enzyme characterization and optimization, as well as structural and functional characterization of enzymatically modified polysaccharides. All seven work packages were highly successful and reached at least one of the two major goals so that in all cases, significant progress beyond the state of the art was achieved. Unfortunately, we were eventually denied a cost-neutral six-month extension of the project which would have allowed us, at no extra cost, to advance these results to the point where they would have been ready to be taken up by industry for further development and integration into their large scale production processes. We now aim at securing alternative funding so that these results will not be lost to European industry and society.