Final Report Summary - NOVOSIDES (Novel Biocatalysts for the Production of Glycosides)
Executive Summary:
The aim of the NOVOSIDES-project is to develop NOVel biocatalysts for the production of glycOSIDES. Glycosylated compounds have a wide range of applications, but very few enzymes are able to glycosylate small organic molecules cost-efficiently at the industrial scale. Therefore, glycosylation reactions catalysed by transglycosidases (TG), glycoside phosphorylases (GP) and glycoside hydrolases (GH) have been explored in more detail. These biocatalysts are able to transfer a glycosyl group from a cheap and readily available donor substrate (e.g. sucrose) to a variety of acceptor substrates (polyphenols, flavonoids, terpenoids, etc). To maximize their performance, their specificity as well as stability was optimised by a combination of enzyme and process engineering. Furthermore, selected reactions were scaled-up to demonstrate their industrial relevance and to obtain enough product for application testing.
A large and diverse collection of enzymes was first established by the screening of culture collections and by the mining of public (meta)genome databases. A few of these represent totally new specificities (i.e. sucrose-6-phosphate phosphorylase and 4,6-alpha-glucanotransferase) but most are stable homologues of known enzymes. Nevertheless, the latter is absolutely crucial for their practical application, which has to be done at high temperatures (to avoid microbial contamination) and in the presence of organic solvents (to solubilize hydrophobic acceptors). This enzyme collection was then screened for activity on a variety of acceptor molecules from different chemical classes, to identify the most promising enzyme/acceptor combination for further optimization. For the high-throughput screening, new fluorescent probes have been developed that allow accurate and sensitive measurements of carbohydrate-active enzymes in a direct and continuous assay.
In a next step, the properties of the biocatalysts were optimized by means of protein engineering. On the one hand, their specificity towards alternative acceptor substrates has been significantly improved. For example, a single mutation in the active site of glucansucrase not only increased its transglycosylation activity three-fold but also shifted its product profile towards a single glucoside with uniform properties. On the other hand, improvements have also been realized in the enzymes’ stability under operational conditions. For example, the half-life of sucrose phosphorylase has been more than doubled by the introduction of six mutations that were designed in a semi-rational manner. In the course of the project, several of our best enzymes have been protected through the filing of patent applications.
Finally, the productivity of selected glycosylation reactions was optimized by means of intensive process engineering. To minimize the hydrolytic side reaction, the use of high concentrations of donor and acceptor substrates turned out to be absolutely crucial. For hydrophobic acceptors, however, this required the addition of organic solvents to the reaction medium. More specifically, a biphasic system was developed, which also enabled the convenient isolation of the glycosylated product through simple extraction. These procedures have recently been scaled-up at pilot plant facilities to generate more than 1 kg of highly pure and crystalline product. Interestingly, a Life Cycle Analysis (LCA) has revealed that the environmental impact of our technology is 9 times lower than the corresponding chemical process. These products can now be ordered through the catalogue of our commercial partner Carbosynth, and negotiations for custom manufacturing of other glycosides are currently on-going. Please visit our website (www.novosides.eu) for more information.
Project Context and Objectives:
The transfer of a glycosyl group represents one of the most important reactions in nature, and has a wide range of industrial applications (Davies et al., 1998). Indeed, glycosylation -i.e. the attachment of a carbohydrate moiety to an acceptor substrate- can drastically influence both the physicochemical and biological properties of an organic molecule (Kren, 2008). Prominent examples include the increased solubility of hydrophobic compounds, the improved pharmacokinetics of drugs, the optimised activity spectrum of antibiotics, and the modulation of flavours and fragrances. Consequently, the development of a cheap but efficient glycosylation technology that can be used on an industrial scale is highly desirable. That will be the major goal of the NOVOSIDES project.
Glycosylation reactions can be performed by means of conventional chemical catalysis but these suffer from a number of serious drawbacks (Nicolaou and Mitchell, 2001). First of all, the need for tedious activation and protection procedures results in long multi-step synthetic routes with a low overall yield. Furthermore, the enantioselectivity is difficult to control and a mixture of alpha- and beta-glycosides is therefore often produced. Finally, the chemical processes require the use of toxic catalysts and generate a considerable amount of waste. To overcome these limitations, the use of enzymes would be a major step forward. DeRoode et al. (2003) have calculated that enzymatic glycosylation reactions generate 5-fold less waste and have a 15-fold higher space-time yield, a tremendous improvement in eco-efficiency. However, enzymes are not yet routinely applied for glycoside synthesis at an industrial scale, in contrast to the opposite hydrolytic reaction (cleavage of glycosidic bonds with water as acceptor substrate).
Several types of carbohydrate-active enzymes (CAZymes) can be used for glycosylation reactions, each with specific characteristics (Seibel et al., 2006) (Fig. 1). Nature’s catalysts for glycosylation reactions are known as ‘Leloir’ glycosyl transferases (GT). Although very efficient, these enzymes require expensive nucleotide-activated sugars (e.g. UDP-glucose) as glycosyl donors, which hampers their industrial application. However, two special types of glycosyl transferases are the proverbial ‘exception to the rule’ and are active with low-cost donors. Glycoside phosphorylases (GP), on the one hand, only require glycosyl phosphates (e.g. glucose-1-phosphate) as donors, compounds that can be easily obtained in large quantities. Transglycosidases (TG), on the other hand, even employ non-activated carbohydrates (e.g. sucrose) for the transfer of a glycosyl group. Additionally, glycoside hydrolases (GH) can also be used for synthetic purposes, when applied under either kinetic (transglycosylation) or thermodynamic (reverse hydrolysis) control. In this project, the glycosylation reactions catalyzed by GH, GP and TG will be explored in more detail.
In general, one of the most crucial aspects for the industrial application of enzymes is their operational stability. Indeed, biocatalysts need to be sufficiently robust to withstand the harsh conditions of industrial processes. For glycosylation reactions in particular, enzymes are required that are both tolerant to high temperatures and to the presence of organic (co-)solvents. Nearly all carbohydrate conversions in the industry are performed at 60°C or higher, mainly to avoid microbial contamination which can be a very serious problem. Organic solvents, in turn, are needed to solubilise the hydrophobic compounds that are used as glycosyl acceptors. Fortunately, the stability of enzymes at high temperatures and in solvents usually coincide (Sellek and Chaudhuri, 1999). This means that improving one of these parameters is not accompanied by a trade-off with the other, but rather results in improvements of both parameters simultaneously.
Enzymes with high stability can be obtained by two complementary approaches: either by screening in extreme environments, or by the engineering of known enzymes (Vieille and Zeikus, 2001). Both approaches will be useful for this project, as two different scenarios can be encountered: either the identification of a thermostable enzyme whose specificity needs to be optimized, or the identification of an interesting novel specificity in an enzyme whose stability is inadequate. Some partners already have quite an extensive collection of enzymes available, and these will be screened first. Alternatively, the rapidly rising number of published (meta)genomes will be mined for new sequences that will complement our base collection. The candidate enzymes will then be engineered to obtain novel biocatalysts with improved performance under industrial conditions.
Besides stability, the specificity of the enzymes will also have to be improved. Indeed, most of the enzymes used in this project have a strong preference for carbohydrate acceptors. Our enzyme collection will, therefore, be screened for activity on representative acceptors from various chemical classes. Interesting targets include benzenoids (e.g. resveratrol), flavonoids (e.g. quercetin), terpenoids (e.g. retinol), and steroids (e.g. digitoxigenin). This thorough screening program will allow the systematic evaluation of the acceptor promiscuity of the different enzymes, and consequently, the identification of the most promising candidates for further optimization through enzyme engineering.
In a final step, the target reactions will be optimized and developed into economical processes, in collaboration with our industrial partners. The product yield will be maximized by the optimization of the reaction conditions and the reactor configuration. An efficient downstream-processing procedure will also be developed, as this often contributes strongly to the price of the final product. Scale-up of the reaction will be performed at the pilot plant facilities of the industrial partner Bio Base Europe Pilot Plant. This will provide us with enough product to allow application testing and active marketing of the novel compounds, which will be the task of the industrial partner Carbosynth.
The identification of novel biocatalysts with improved properties, both from natural sources and from mutant libraries, requires the availability of a fast and reliable assay that can be used for high-throughput screening (HTS). Therefore, a novel type of fluorescent probe will be developed in this project that allows the detection of carbohydrate-active enzymes in a non-destructive, continuous assay. The system consists of diboronic acid-containing molecules, coupled to a fluorescent dye. The former specifically interact with the carbohydrate product while the latter serves as the reporter unit. This inexpensive sensing system is able to discriminate between substrate and product with high sensitivity and does not require the use of fluorogenic substrate-analogues. It can be used for real-time assays in microtiter plate format, enabling the processing of thousands of samples per day. The first generation of these fluorescent sensors has already been shown to work really well for sucrose phosphorylase, and optimised probes will now be applied to the screening of other types of CAZymes as well (Vilozny et al., 2009).
In summary, the major objectives of the NOVOSIDES project include:
- the development and exploitation of novel fluorescent probes that allow the monitoring of glycosylation reactions catalyzed by TG, GP and GH enzymes in a continuous and non-destructive assay.
- the identification of new TG, GP and GH enzymes that display activity on non-carbohydrate acceptors as substrate by screening in nature and in on-line databases
- the engineering of TG, GP and GH enzymes to increase their yields on non-carbohydrate acceptors to up to 70%
- the engineering of TG, GP and GH enzymes to increase their stability at 60˚C to more than 4 hours
- the development of a new database that lists all the glycosylation reactions that can be performed with our wild-type and variant enzymes
- the optimization of biocatalytic glycosylation reactions and of the downstream processing of glycosylated products to achieve a space-time yield of 500 g L-1 d-1
- the scale-up of biocatalytic glycosylation reactions at pilot-plant facilities to kg amounts
- the marketing of glycosylated products by their inclusion in a commercial catalogue
Project Results:
Since transglycosidases (TG), glycoside phosphorylases (GP) and glycoside hydrolases (GH) have not yet been thoroughly characterized with respect of their glycosylation potential, our first goal was to establish a large and diverse collection of representative enzymes. These could then be tested for activity on representative acceptor molecules, to reveal what kind of substrate promiscuity is already available in nature. In the course of the NOVOSIDES-project, more than 50 different enzymes have been obtained by the screening of culture collections and by the mining of online (meta)genome databases. All members of this base collection have been purified and thoroughly characterized. Although we mainly searched for more stable representatives, new specificities have also been discovered that could be useful for the glycosylation of alternative acceptor substrates. That is particularly true for sucrose-6’-phosphate phosphorylase and 4,6-alpha-glucanotransferase as new representatives of GP and TG enzymes, respectively.
For the GP enzymes, the group of Prof. Desmet at Ghent University (UGENT) has established a collection that contains at least one representative of all known phosphorylase specificities. All of these enzymes have been expressed recombinantly in E. coli, and have been decorated with a N-terminal (His)6-tag to enable their rapid purification. For their characterization, particular attention has been paid to sucrose phosphorylase (SP), as this enzyme is known to have the broadest acceptor specificity. In order to obtain thermostable homologues, the genomes of thermophilic organisms have been analysed but no new SP could be identified. However, the enzyme form the mesophile Bifidobacterium adolescentis was found to be remarkably stable under process conditions, both at high temperatures (needed to avoid microbial contamination) and at high solvent concentrations (needed to solubilize hydrophobic acceptors). Interestingly, the database mining also led to the discovery of a new GP specificity in the thermophile Thermoanaerobacter thermosaccharolyticum, i.e. sucrose-6’-phosphate phosphorylase (now designated as EC 2.4.1.329). This new enzyme seems to be involved in an alternative pathway for the degradation of sucrose that is more energy-efficient than classical pathways, which constitutes a major breakthrough in the scientific literature (Verhaeghe et al, 2014, Appl Microbiol Biotechnol). Meanwhile, the enzyme’s potential for glycosylation reactions at elevated temperatures (~60 °C) has been protected by a priority filing (WO 2014/060452), as this feature will be crucial for industrial applications.
For the GH enzymes, the group of Prof. Kren at the Academy of Sciences of the Czech Republic (IMIC) has established a very large collection of representatives of more than 10 glycosidase specificities. These enzymes mainly originate from fungal sources and are typically (thermo)stable because they are produced extracellularly. All enzymes have been purified by ammonium sulfate precipitation and can be stored in this form for a very long time. For their characterization, particular attention has been paid to alpha-L-rhamnosidase as this enzyme can be used for the production of the nutraceutical glycoside isoquercitrin. To increase protein yields, the enzyme from Aspergillus terreus has been expressed recombinantly in P. pastoris at a level of 100 U/ml (Weignerová et al, 2012, Bioresour Technol). Since the price of the enzyme has been a limiting factor, this new development has attracted serious interest from various end-users. In addition to rhamnosidase, diglycosidases have also been examined as a new class of GH enzymes. They selectively act on the bond between an aglycone and a disaccharide moiety, but have not yet been exploited for the synthesis of diglycosides. Several fungi were now screened for this rather special activity, and six of them were found to produce rutinosidase extracellularly. Finally, the enzyme from Aspergillus niger was recombinantly expressed in P. pastoris, resulting in a high yield (35 U/mL) and a high purity (> 99%). These results have just recently been published in the scientific literature (Šimcíková et al, 2015, Adv Synth Catal).
For the TG enzymes, the group of Prof. Dijkhuizen at the University of Groningen (RUG) has established a collection of nearly 20 wild-type and mutant glucansucrases with different preferences for linkage types as well as for the degree and type of branching. Particular attention has been paid to enzymes that form alpha-1,2-linkages, since these are rather rare and have not yet been extensively characterized. All enzymes have been recombinantly expressed in E. coli and purified by affinity chromatography. The highest expression and yields of purified proteins were obtained with truncated gtf genes yielding highly active glucansucrase enzymes lacking the N-terminal regions with repeating units of unknown functions. Interestingly, a new subfamily in of TG enzymes could be identified, with GTFB of Lactobacillus reuteri as representative. This enzyme has no detectable activity with sucrose, but uses malto-oligosaccharides (MOS) as donor and acceptor substrates. Indeed, a transfer from alpha-1,4- to alpha-1,6-bonds can be observed in that case, meaning that enzyme should be named 4,6-alpha-glucanotransferase. The resulting products represent a totally new class of modified starches that are alpha-amylase-resistant and could thus have important application in the food industry. These important findings have been patented and meanwhile also been published in the scientific literature (Dobruchowska et al., 2012, Glycobiol).
Once a base collection of different enzymes was established, their acceptor promiscuity could be evaluated by means of high-throughput screening. To that end, a list of about 50 target compounds has been compiled in collaboration with our commercial partner Carbosynth (CBS). This list not only covers the wide diversity of chemical structures that can be tested, but also includes several acceptors with known industrial applications. The screening has been performed with the three enzyme classes, using sucrose phosphorylase, alpha-L-rhamnosidase and glucosyltransferase A as representatives. In general, it can be stated that these enzymes display a broad acceptor specificity, resulting in the formation of glycosides with the majority of compounds tested. However, the activity is typically rather low and hardly better than the contaminating hydrolytic activity. This means that either the enzymes or the processes will have to be engineered for our technology to become economically viable. Nevertheless, many of these reactions were not yet reported in the literature and have since been published by our consortium (De Winter et al, 2013, Green Chem and Bioresour Technol). Furthermore, an overview of the glycosylation potential of our base collection can also be found on your website in the form of a dedicated database (www.novosides.eu).
For the screening of glycosylation reactions, the use of a fluorescent probe has been evaluated by the group of Prof. Schiller at the University of Jena (FSU JENA). This probe consists of a receptor (BBV) that binds selectively to fructose, which is released from the donor substrate during the reaction. As a consequence, the receptor can no longer act as a quencher of a commercially available dye (HPTS), which results in a strong fluorescent signal. This fluorescent assay was already validated for sucrose phosphorylase and has now been tested with the other enzymes in our collection. Unfortunately, the probes were found to suffer from low sensitivity, selectivity and stability under screening conditions. Therefore, new probes with improved properties have been developed by changing the structure of the receptor as well as the dye. In that way, a 100-fold increase in sensitivity could be realized and the light sensitivity could be practically solved. Furthermore, introducing fluorine moieties in the receptor eliminates the need for a dye and allows to monitor the reactions directly by NMR, which is highly sensitive and selective. This has resulted in a new real-time and continuous assay for carbohydrate-active enzymes, which is a major breakthrough in the field. Interestingly, the new compounds even allowed to turn the sensing system into a logic gate that can be used for ‘sugar computing’. This remarkable achievement has attracted considerable attention from both the popular press and our scientific peers (Elstner et al, 2012, J. Am. Chem. Soc.. and 2014, Angew. Chem.).
In a next step, the properties of the biocatalysts were optimized by means of protein engineering. Only the results obtained with sucrose phosphorylase will be mentioned here, as the other enzyme variants still need to be protected through the filing of patent applications. For SP, the low activity on alternative acceptor substrates compared to the contaminating hydrolytic activity means that the product yields are rather low. To increase the ratio of transglycosylation over hydrolysis (T/H), the determinants of the enzyme’s specificity were first examined by so-called ‘alanine scanning’ of the active site. Replacing an amino acid with alanine amounts to the removal of its side chain without affecting the main chain conformation. However, none of the alanine mutants displayed a modified substrate specificity, which means that non-carbohydrate acceptors make no specific interactions with these residues. Improving the transglycosylation activity will thus probably require an extensive remodelling of the active site, which cannot be accomplished with simple aminoacid substitutions. Nevertheless, this mutational analysis has generated new insights into the enzyme’s mode of action and has meanwhile been published by our consortium (Verhaeghe et al, 2013, J Mol Catal B). In addition, no improved mutants could be identified by means of random mutagenesis of the complete gene sequence. These findings indicate that SP is already highly optimized to outcompete water as an acceptor substrate, and that further improvements will need to be realized by process engineering instead.
For the engineering of stability, we have used the SP from B. adolescentis as template since that is the most stable representative in our collection. At its most flexible positions (as reflected by B-factors in the crystal structure), so-called ‘consensus’ residues were then introduced, which are the aminoacids that occur most frequently at the corresponding position in all known SP enzymes. These residues have already been selected by nature and should thus certainly give rise to a functional enzyme with high stability. Combining the 6 best mutations into a single enzyme resulted in a half-life at 60°C that has more than doubled compared to the wild-type enzyme. Furthermore, the solvent concentration at which it retains half of its activity went up from 34% to 41%. These impressive results have meanwhile been published (Cerdobbel et al, 2012, PEDS) and patented (WO/2011/124538). In a next step, we have tried to extend the consensus concept to the complete sequence of SP, where at every position the most abundant residue from the multiple sequence alignment was introduced. In that way, a synthetic enzyme could be created that not only should have maximal stability but also maximal freedom-to-operate since it is not covered by any patents. However, we have found that the consensus is not necessarily the best construct but rather behaves like an ‘average’ that balances the properties of the individual sequences in the alignment. Indeed, the consensus was found to be better than the SP from L. mesenteroides but worse than the SP from B. adolescentis. These important new insights have just been accepted for publication (Aerts et al, 2013, Biotechnol Bioengin) and were even selected for a highlight feature by the editor.
Finally, the productivity of selected glycosylation reactions was optimized by means of intensive process engineering. To minimize the hydrolytic side reaction, the use of high concentrations of donor and acceptor substrates turned out to be absolutely crucial. For hydrophobic acceptors, however, this required the addition of organic solvents to the reaction medium. On the one hand, monophasic systems have been developed that make use of ionic liquids as environmentally friendly or ‘green’ co-solvents (De Winter et al, 2013, Green Chemistry). On the other hand, the use of ethylacetate as solvent resulted in a biphasic system from which the glycosylated product could be purified more easily (De Winter et al, 2014, Org. Proc. Res. Develop.). The latter has been used to develop a representative process for each enzyme class, i.e. the production of pyrogallol, catechol and quercetin glycoside with SP, GTFA and rhamnosidase, respectively. Product yields of more than 85% could be obtained in each case, which is more than enough for their commercial exploitation. To demonstrate the techno-economic feasibility of our technology, these reactions have been scaled-up at the facilities of our industrial partner Bio Base Europe Pilot Plant (BBEPP). More than 1 kg of highly pure and crystalline product was generated, which can now be ordered through the commercial catalogue of Carbosynth. Interestingly, a Life Cycle Analysis (LCA) has revealed that the environmental impact of our process is 9 times lower than that of the corresponding chemical process, which is a very strong argument for the uptake of our technology in an industrial sector that is constantly looking for innovative solutions to improve its eco-efficiency.
Potential Impact:
In various industries, enzymes are increasingly used as efficient biocatalysts to perform a wide range of chemical reactions. The use of biocatalysis can have significant benefits, both economical and ecological, compared to conventional chemical technology, such as increased conversion efficiency, higher product specificity, improved product purity, lowered energy consumption and a significant decrease in chemical waste. Biocatalytic reactions are typically performed at normal temperatures and pressures, whereby no hazardous intermediate products are formed or toxic waste is generated. Biocatalysis has thus developed into a main contributor to “green chemistry”.
One of the major objectives of NOVOSIDES was the development of biocatalytic glycosylation reactions into economical processes that can be run at an industrial scale. These new processes will illustrate the potential of enzymatic chemistry for the production of glycosides and stimulate further research in this field. It will also raise interest from the industry to collaborate with academic partners, which typically has been lacking for these particular targets. Interested parties can basically be divided into enzyme producers and enzyme users. Europe currently produces about 70% of the world’s enzymes, with Novozymes and Genencor (now DuPont Industrial Biosciences) as the market leaders. The NOVOSIDES project has significantly expanded the range of biocatalysts that can be used by the industry and thus helped to consolidate Europe’s position in this field.
Besides the enzyme market, a second sector that can be stimulated by the NOVOSIDES project is the chemical industry, a vital sector for Europe’s economy that accounts for 2.4% of its Gross Domestic Product (GDP) and 6% of its industrial workforce. It is a sector for which the European Union (EU) is famous worldwide and considered to be in a commanding position. Continuous innovations are required to hold on to this top spot and to adapt to the rapidly changing economical and ecological climate. Because of the complex nature of the synthetic procedures, a lot of glycosides are currently considered to be unprofitable. Providing the sector with a novel technology that is more efficient will allow them to expand their activities and produce a whole range of new compounds. This has already become apparent in recent months, as our first glycoside products have initiated interactions with different companies that want to incorporate our processes in their operations.
Besides the economical advantages, the ecological benefits are also apparent. On average, enzymatic glycosylation reactions generate 5-fold less waste than their chemical counterparts. Furthermore, the waste is not toxic, in contrast to that generated by the chemical industry. These advantages strongly contribute to the development of green chemistry. Because of the higher space-time yield of enzymatic reactions and the use of moderate temperatures, much less energy is also consumed during the biocatalytic production process. In that way, the NOVOSIDES project could also contribute to the reduction in carbon emissions, as stipulated in the Strategic Energy Technologies (SET) plan of the European Commission (EC). A complete Life Cycle Analysis (LCA) of our processes has confirmed that our total environmental impact is 9-fold lower when compared to the corresponding chemical processes.
These outcomes fit perfectly with recent policies that have been implemented by the EC, such as the Bioeconomy Strategy ("Innovating for Sustainable Growth: a Bioeconomy for Europe”). The goal of that ambitious plan is to enable the transition from a fossil- to a bio-based economy by stimulating further innovation in the field of biotechnology. The bioeconomy in the EU already has a turnover of nearly €2 trillion and employs more than 22 million people, accounting for 9% of total employment. In combination with the increased research funding for the bioeconomy under Horizon 2020, the new Bioeconomy Strategy should generate an added value of about €45 billion and a significant number of new jobs by 2025. Furthermore, the introduction of biobased products into the market is also supported by the EC’s updated Industrial Policy (building on the Lead Market Initiative), which should help to turn our scientific technology into industrial reality. It can, for example, be mentioned that the two staff members recruited for the NOVOSIDES-project by the Bio Base Europe Pilot Plant will remain in service after the project has ended. This nicely illustrates that our accomplishments already have a concrete impact, albeit at a modest scale for the time being.
To raise awareness about the benefits of our technology, results have been disseminated in various ways and forms. First of all, a newsletter has been regularly distributed to a very wide audience of academic and industrial scientists working in our field. In addition, our commercial partner Carbosynth has promoted our project at various exhibitions with a demo booth and leaflets, which were always very popular. In all those cases, interested parties were referred to our website (www.novosides.eu) for more information. In turn, the other partners have regularly presented their latest results at major conferences and in scientific publications. It is important to note that several of our papers featured on the cover of top-level journals, such as Nano Today (IF 19), Angewandte Chemie (IF 11), Journal of Materials Chemistry (IF 7), and Chemistry: A European Journal (IF 6). This is clear proof of the high-quality research being done in the framework of our project. Furthermore, one of our collaborators made it into the prestigious EYCA final of EuCheMS.
In addition, Lubbert Dijkhuizen has been invited by BASF to present his work on glucansucrases and Tom Desmet by Cargill to talk about the NOVOSIDES project in general. These invitations nicely illustrate that our line of work is very relevant for the industry. In addition to those informal contacts, a dedicated event has been organized to stimulate the exploitation of the NOVOSIDES-technology. On the 25th of November 2013, a Glycoside Application Event was held at the premises of Bio Base Europe Pilot Plant). More than twenty industrial representatives were present from companies as diverse as Procter & Gamble (personal care industry), Cosun (agricultural industry), Cargill (carbohydrate industry), Dupont (enzyme industry), Solvay (chemical industry) and Vesalius Biocapital (investors). This has resulted in the identification of new markets where glycosides could find applications. At the end of the meeting, 1-to-1 partnering was initiated, allowing private discussions with individual companies to take place.
At the end of the NOVOSIDES project, a dedicated workshop was also organized to present our results to interested parties from both academia and industry. To maximize the number of participants (120 in this case), the workshop was held as a dedicated session of the Fourth International Conference on Novel Enzymes, organized in Ghent, Belgium, on October 15th 2014. After the workshop, a demonstration session was organized to explain our technology to the general public. Invitations to this session were widely circulated by the project partners, and reached colleagues, family and friends as well as members of the press. In the end, about 70 people were present at the session. The different partners engaged the audience to explain their role in the project, and how this resulted in a glycosylation process that is environmentally friendly as well as economically attractive. As a visual aid, the properties of enzymes and glycosides were illustrated by means of an animated movie, which was compiled by a specialized firm and can be found at: https://www.youtube.com/watch?v=_jWwqiEfr2c.
Finally, it should also be mentioned that our results have attracted the interest of the popular press. That is particularly true for the fascinating properties of the new fluorescent probes. Under the title “The sweetest calculator in the world” they have featured in several online magazines, such Phys.org Science Daily, Scientific Computing, and Biotechnology.de. Furthermore, Prof. Schiller has also talked about their applications during radio interviews with MDR Figaro and Deutschlandfunk. In addition, the Novosides-project also made the national news in the Czech Republic, by means of an interview with Prof. Kren.
List of Websites:
Prof. dr. Tom Desmet, Coordinator
Ghent University, Center for Industrial Biotechnology and Biocatalysis
TEL: +32 9264 9920, EMAIL: tom.desmet@ugent.be
www.novosides.eu
The aim of the NOVOSIDES-project is to develop NOVel biocatalysts for the production of glycOSIDES. Glycosylated compounds have a wide range of applications, but very few enzymes are able to glycosylate small organic molecules cost-efficiently at the industrial scale. Therefore, glycosylation reactions catalysed by transglycosidases (TG), glycoside phosphorylases (GP) and glycoside hydrolases (GH) have been explored in more detail. These biocatalysts are able to transfer a glycosyl group from a cheap and readily available donor substrate (e.g. sucrose) to a variety of acceptor substrates (polyphenols, flavonoids, terpenoids, etc). To maximize their performance, their specificity as well as stability was optimised by a combination of enzyme and process engineering. Furthermore, selected reactions were scaled-up to demonstrate their industrial relevance and to obtain enough product for application testing.
A large and diverse collection of enzymes was first established by the screening of culture collections and by the mining of public (meta)genome databases. A few of these represent totally new specificities (i.e. sucrose-6-phosphate phosphorylase and 4,6-alpha-glucanotransferase) but most are stable homologues of known enzymes. Nevertheless, the latter is absolutely crucial for their practical application, which has to be done at high temperatures (to avoid microbial contamination) and in the presence of organic solvents (to solubilize hydrophobic acceptors). This enzyme collection was then screened for activity on a variety of acceptor molecules from different chemical classes, to identify the most promising enzyme/acceptor combination for further optimization. For the high-throughput screening, new fluorescent probes have been developed that allow accurate and sensitive measurements of carbohydrate-active enzymes in a direct and continuous assay.
In a next step, the properties of the biocatalysts were optimized by means of protein engineering. On the one hand, their specificity towards alternative acceptor substrates has been significantly improved. For example, a single mutation in the active site of glucansucrase not only increased its transglycosylation activity three-fold but also shifted its product profile towards a single glucoside with uniform properties. On the other hand, improvements have also been realized in the enzymes’ stability under operational conditions. For example, the half-life of sucrose phosphorylase has been more than doubled by the introduction of six mutations that were designed in a semi-rational manner. In the course of the project, several of our best enzymes have been protected through the filing of patent applications.
Finally, the productivity of selected glycosylation reactions was optimized by means of intensive process engineering. To minimize the hydrolytic side reaction, the use of high concentrations of donor and acceptor substrates turned out to be absolutely crucial. For hydrophobic acceptors, however, this required the addition of organic solvents to the reaction medium. More specifically, a biphasic system was developed, which also enabled the convenient isolation of the glycosylated product through simple extraction. These procedures have recently been scaled-up at pilot plant facilities to generate more than 1 kg of highly pure and crystalline product. Interestingly, a Life Cycle Analysis (LCA) has revealed that the environmental impact of our technology is 9 times lower than the corresponding chemical process. These products can now be ordered through the catalogue of our commercial partner Carbosynth, and negotiations for custom manufacturing of other glycosides are currently on-going. Please visit our website (www.novosides.eu) for more information.
Project Context and Objectives:
The transfer of a glycosyl group represents one of the most important reactions in nature, and has a wide range of industrial applications (Davies et al., 1998). Indeed, glycosylation -i.e. the attachment of a carbohydrate moiety to an acceptor substrate- can drastically influence both the physicochemical and biological properties of an organic molecule (Kren, 2008). Prominent examples include the increased solubility of hydrophobic compounds, the improved pharmacokinetics of drugs, the optimised activity spectrum of antibiotics, and the modulation of flavours and fragrances. Consequently, the development of a cheap but efficient glycosylation technology that can be used on an industrial scale is highly desirable. That will be the major goal of the NOVOSIDES project.
Glycosylation reactions can be performed by means of conventional chemical catalysis but these suffer from a number of serious drawbacks (Nicolaou and Mitchell, 2001). First of all, the need for tedious activation and protection procedures results in long multi-step synthetic routes with a low overall yield. Furthermore, the enantioselectivity is difficult to control and a mixture of alpha- and beta-glycosides is therefore often produced. Finally, the chemical processes require the use of toxic catalysts and generate a considerable amount of waste. To overcome these limitations, the use of enzymes would be a major step forward. DeRoode et al. (2003) have calculated that enzymatic glycosylation reactions generate 5-fold less waste and have a 15-fold higher space-time yield, a tremendous improvement in eco-efficiency. However, enzymes are not yet routinely applied for glycoside synthesis at an industrial scale, in contrast to the opposite hydrolytic reaction (cleavage of glycosidic bonds with water as acceptor substrate).
Several types of carbohydrate-active enzymes (CAZymes) can be used for glycosylation reactions, each with specific characteristics (Seibel et al., 2006) (Fig. 1). Nature’s catalysts for glycosylation reactions are known as ‘Leloir’ glycosyl transferases (GT). Although very efficient, these enzymes require expensive nucleotide-activated sugars (e.g. UDP-glucose) as glycosyl donors, which hampers their industrial application. However, two special types of glycosyl transferases are the proverbial ‘exception to the rule’ and are active with low-cost donors. Glycoside phosphorylases (GP), on the one hand, only require glycosyl phosphates (e.g. glucose-1-phosphate) as donors, compounds that can be easily obtained in large quantities. Transglycosidases (TG), on the other hand, even employ non-activated carbohydrates (e.g. sucrose) for the transfer of a glycosyl group. Additionally, glycoside hydrolases (GH) can also be used for synthetic purposes, when applied under either kinetic (transglycosylation) or thermodynamic (reverse hydrolysis) control. In this project, the glycosylation reactions catalyzed by GH, GP and TG will be explored in more detail.
In general, one of the most crucial aspects for the industrial application of enzymes is their operational stability. Indeed, biocatalysts need to be sufficiently robust to withstand the harsh conditions of industrial processes. For glycosylation reactions in particular, enzymes are required that are both tolerant to high temperatures and to the presence of organic (co-)solvents. Nearly all carbohydrate conversions in the industry are performed at 60°C or higher, mainly to avoid microbial contamination which can be a very serious problem. Organic solvents, in turn, are needed to solubilise the hydrophobic compounds that are used as glycosyl acceptors. Fortunately, the stability of enzymes at high temperatures and in solvents usually coincide (Sellek and Chaudhuri, 1999). This means that improving one of these parameters is not accompanied by a trade-off with the other, but rather results in improvements of both parameters simultaneously.
Enzymes with high stability can be obtained by two complementary approaches: either by screening in extreme environments, or by the engineering of known enzymes (Vieille and Zeikus, 2001). Both approaches will be useful for this project, as two different scenarios can be encountered: either the identification of a thermostable enzyme whose specificity needs to be optimized, or the identification of an interesting novel specificity in an enzyme whose stability is inadequate. Some partners already have quite an extensive collection of enzymes available, and these will be screened first. Alternatively, the rapidly rising number of published (meta)genomes will be mined for new sequences that will complement our base collection. The candidate enzymes will then be engineered to obtain novel biocatalysts with improved performance under industrial conditions.
Besides stability, the specificity of the enzymes will also have to be improved. Indeed, most of the enzymes used in this project have a strong preference for carbohydrate acceptors. Our enzyme collection will, therefore, be screened for activity on representative acceptors from various chemical classes. Interesting targets include benzenoids (e.g. resveratrol), flavonoids (e.g. quercetin), terpenoids (e.g. retinol), and steroids (e.g. digitoxigenin). This thorough screening program will allow the systematic evaluation of the acceptor promiscuity of the different enzymes, and consequently, the identification of the most promising candidates for further optimization through enzyme engineering.
In a final step, the target reactions will be optimized and developed into economical processes, in collaboration with our industrial partners. The product yield will be maximized by the optimization of the reaction conditions and the reactor configuration. An efficient downstream-processing procedure will also be developed, as this often contributes strongly to the price of the final product. Scale-up of the reaction will be performed at the pilot plant facilities of the industrial partner Bio Base Europe Pilot Plant. This will provide us with enough product to allow application testing and active marketing of the novel compounds, which will be the task of the industrial partner Carbosynth.
The identification of novel biocatalysts with improved properties, both from natural sources and from mutant libraries, requires the availability of a fast and reliable assay that can be used for high-throughput screening (HTS). Therefore, a novel type of fluorescent probe will be developed in this project that allows the detection of carbohydrate-active enzymes in a non-destructive, continuous assay. The system consists of diboronic acid-containing molecules, coupled to a fluorescent dye. The former specifically interact with the carbohydrate product while the latter serves as the reporter unit. This inexpensive sensing system is able to discriminate between substrate and product with high sensitivity and does not require the use of fluorogenic substrate-analogues. It can be used for real-time assays in microtiter plate format, enabling the processing of thousands of samples per day. The first generation of these fluorescent sensors has already been shown to work really well for sucrose phosphorylase, and optimised probes will now be applied to the screening of other types of CAZymes as well (Vilozny et al., 2009).
In summary, the major objectives of the NOVOSIDES project include:
- the development and exploitation of novel fluorescent probes that allow the monitoring of glycosylation reactions catalyzed by TG, GP and GH enzymes in a continuous and non-destructive assay.
- the identification of new TG, GP and GH enzymes that display activity on non-carbohydrate acceptors as substrate by screening in nature and in on-line databases
- the engineering of TG, GP and GH enzymes to increase their yields on non-carbohydrate acceptors to up to 70%
- the engineering of TG, GP and GH enzymes to increase their stability at 60˚C to more than 4 hours
- the development of a new database that lists all the glycosylation reactions that can be performed with our wild-type and variant enzymes
- the optimization of biocatalytic glycosylation reactions and of the downstream processing of glycosylated products to achieve a space-time yield of 500 g L-1 d-1
- the scale-up of biocatalytic glycosylation reactions at pilot-plant facilities to kg amounts
- the marketing of glycosylated products by their inclusion in a commercial catalogue
Project Results:
Since transglycosidases (TG), glycoside phosphorylases (GP) and glycoside hydrolases (GH) have not yet been thoroughly characterized with respect of their glycosylation potential, our first goal was to establish a large and diverse collection of representative enzymes. These could then be tested for activity on representative acceptor molecules, to reveal what kind of substrate promiscuity is already available in nature. In the course of the NOVOSIDES-project, more than 50 different enzymes have been obtained by the screening of culture collections and by the mining of online (meta)genome databases. All members of this base collection have been purified and thoroughly characterized. Although we mainly searched for more stable representatives, new specificities have also been discovered that could be useful for the glycosylation of alternative acceptor substrates. That is particularly true for sucrose-6’-phosphate phosphorylase and 4,6-alpha-glucanotransferase as new representatives of GP and TG enzymes, respectively.
For the GP enzymes, the group of Prof. Desmet at Ghent University (UGENT) has established a collection that contains at least one representative of all known phosphorylase specificities. All of these enzymes have been expressed recombinantly in E. coli, and have been decorated with a N-terminal (His)6-tag to enable their rapid purification. For their characterization, particular attention has been paid to sucrose phosphorylase (SP), as this enzyme is known to have the broadest acceptor specificity. In order to obtain thermostable homologues, the genomes of thermophilic organisms have been analysed but no new SP could be identified. However, the enzyme form the mesophile Bifidobacterium adolescentis was found to be remarkably stable under process conditions, both at high temperatures (needed to avoid microbial contamination) and at high solvent concentrations (needed to solubilize hydrophobic acceptors). Interestingly, the database mining also led to the discovery of a new GP specificity in the thermophile Thermoanaerobacter thermosaccharolyticum, i.e. sucrose-6’-phosphate phosphorylase (now designated as EC 2.4.1.329). This new enzyme seems to be involved in an alternative pathway for the degradation of sucrose that is more energy-efficient than classical pathways, which constitutes a major breakthrough in the scientific literature (Verhaeghe et al, 2014, Appl Microbiol Biotechnol). Meanwhile, the enzyme’s potential for glycosylation reactions at elevated temperatures (~60 °C) has been protected by a priority filing (WO 2014/060452), as this feature will be crucial for industrial applications.
For the GH enzymes, the group of Prof. Kren at the Academy of Sciences of the Czech Republic (IMIC) has established a very large collection of representatives of more than 10 glycosidase specificities. These enzymes mainly originate from fungal sources and are typically (thermo)stable because they are produced extracellularly. All enzymes have been purified by ammonium sulfate precipitation and can be stored in this form for a very long time. For their characterization, particular attention has been paid to alpha-L-rhamnosidase as this enzyme can be used for the production of the nutraceutical glycoside isoquercitrin. To increase protein yields, the enzyme from Aspergillus terreus has been expressed recombinantly in P. pastoris at a level of 100 U/ml (Weignerová et al, 2012, Bioresour Technol). Since the price of the enzyme has been a limiting factor, this new development has attracted serious interest from various end-users. In addition to rhamnosidase, diglycosidases have also been examined as a new class of GH enzymes. They selectively act on the bond between an aglycone and a disaccharide moiety, but have not yet been exploited for the synthesis of diglycosides. Several fungi were now screened for this rather special activity, and six of them were found to produce rutinosidase extracellularly. Finally, the enzyme from Aspergillus niger was recombinantly expressed in P. pastoris, resulting in a high yield (35 U/mL) and a high purity (> 99%). These results have just recently been published in the scientific literature (Šimcíková et al, 2015, Adv Synth Catal).
For the TG enzymes, the group of Prof. Dijkhuizen at the University of Groningen (RUG) has established a collection of nearly 20 wild-type and mutant glucansucrases with different preferences for linkage types as well as for the degree and type of branching. Particular attention has been paid to enzymes that form alpha-1,2-linkages, since these are rather rare and have not yet been extensively characterized. All enzymes have been recombinantly expressed in E. coli and purified by affinity chromatography. The highest expression and yields of purified proteins were obtained with truncated gtf genes yielding highly active glucansucrase enzymes lacking the N-terminal regions with repeating units of unknown functions. Interestingly, a new subfamily in of TG enzymes could be identified, with GTFB of Lactobacillus reuteri as representative. This enzyme has no detectable activity with sucrose, but uses malto-oligosaccharides (MOS) as donor and acceptor substrates. Indeed, a transfer from alpha-1,4- to alpha-1,6-bonds can be observed in that case, meaning that enzyme should be named 4,6-alpha-glucanotransferase. The resulting products represent a totally new class of modified starches that are alpha-amylase-resistant and could thus have important application in the food industry. These important findings have been patented and meanwhile also been published in the scientific literature (Dobruchowska et al., 2012, Glycobiol).
Once a base collection of different enzymes was established, their acceptor promiscuity could be evaluated by means of high-throughput screening. To that end, a list of about 50 target compounds has been compiled in collaboration with our commercial partner Carbosynth (CBS). This list not only covers the wide diversity of chemical structures that can be tested, but also includes several acceptors with known industrial applications. The screening has been performed with the three enzyme classes, using sucrose phosphorylase, alpha-L-rhamnosidase and glucosyltransferase A as representatives. In general, it can be stated that these enzymes display a broad acceptor specificity, resulting in the formation of glycosides with the majority of compounds tested. However, the activity is typically rather low and hardly better than the contaminating hydrolytic activity. This means that either the enzymes or the processes will have to be engineered for our technology to become economically viable. Nevertheless, many of these reactions were not yet reported in the literature and have since been published by our consortium (De Winter et al, 2013, Green Chem and Bioresour Technol). Furthermore, an overview of the glycosylation potential of our base collection can also be found on your website in the form of a dedicated database (www.novosides.eu).
For the screening of glycosylation reactions, the use of a fluorescent probe has been evaluated by the group of Prof. Schiller at the University of Jena (FSU JENA). This probe consists of a receptor (BBV) that binds selectively to fructose, which is released from the donor substrate during the reaction. As a consequence, the receptor can no longer act as a quencher of a commercially available dye (HPTS), which results in a strong fluorescent signal. This fluorescent assay was already validated for sucrose phosphorylase and has now been tested with the other enzymes in our collection. Unfortunately, the probes were found to suffer from low sensitivity, selectivity and stability under screening conditions. Therefore, new probes with improved properties have been developed by changing the structure of the receptor as well as the dye. In that way, a 100-fold increase in sensitivity could be realized and the light sensitivity could be practically solved. Furthermore, introducing fluorine moieties in the receptor eliminates the need for a dye and allows to monitor the reactions directly by NMR, which is highly sensitive and selective. This has resulted in a new real-time and continuous assay for carbohydrate-active enzymes, which is a major breakthrough in the field. Interestingly, the new compounds even allowed to turn the sensing system into a logic gate that can be used for ‘sugar computing’. This remarkable achievement has attracted considerable attention from both the popular press and our scientific peers (Elstner et al, 2012, J. Am. Chem. Soc.. and 2014, Angew. Chem.).
In a next step, the properties of the biocatalysts were optimized by means of protein engineering. Only the results obtained with sucrose phosphorylase will be mentioned here, as the other enzyme variants still need to be protected through the filing of patent applications. For SP, the low activity on alternative acceptor substrates compared to the contaminating hydrolytic activity means that the product yields are rather low. To increase the ratio of transglycosylation over hydrolysis (T/H), the determinants of the enzyme’s specificity were first examined by so-called ‘alanine scanning’ of the active site. Replacing an amino acid with alanine amounts to the removal of its side chain without affecting the main chain conformation. However, none of the alanine mutants displayed a modified substrate specificity, which means that non-carbohydrate acceptors make no specific interactions with these residues. Improving the transglycosylation activity will thus probably require an extensive remodelling of the active site, which cannot be accomplished with simple aminoacid substitutions. Nevertheless, this mutational analysis has generated new insights into the enzyme’s mode of action and has meanwhile been published by our consortium (Verhaeghe et al, 2013, J Mol Catal B). In addition, no improved mutants could be identified by means of random mutagenesis of the complete gene sequence. These findings indicate that SP is already highly optimized to outcompete water as an acceptor substrate, and that further improvements will need to be realized by process engineering instead.
For the engineering of stability, we have used the SP from B. adolescentis as template since that is the most stable representative in our collection. At its most flexible positions (as reflected by B-factors in the crystal structure), so-called ‘consensus’ residues were then introduced, which are the aminoacids that occur most frequently at the corresponding position in all known SP enzymes. These residues have already been selected by nature and should thus certainly give rise to a functional enzyme with high stability. Combining the 6 best mutations into a single enzyme resulted in a half-life at 60°C that has more than doubled compared to the wild-type enzyme. Furthermore, the solvent concentration at which it retains half of its activity went up from 34% to 41%. These impressive results have meanwhile been published (Cerdobbel et al, 2012, PEDS) and patented (WO/2011/124538). In a next step, we have tried to extend the consensus concept to the complete sequence of SP, where at every position the most abundant residue from the multiple sequence alignment was introduced. In that way, a synthetic enzyme could be created that not only should have maximal stability but also maximal freedom-to-operate since it is not covered by any patents. However, we have found that the consensus is not necessarily the best construct but rather behaves like an ‘average’ that balances the properties of the individual sequences in the alignment. Indeed, the consensus was found to be better than the SP from L. mesenteroides but worse than the SP from B. adolescentis. These important new insights have just been accepted for publication (Aerts et al, 2013, Biotechnol Bioengin) and were even selected for a highlight feature by the editor.
Finally, the productivity of selected glycosylation reactions was optimized by means of intensive process engineering. To minimize the hydrolytic side reaction, the use of high concentrations of donor and acceptor substrates turned out to be absolutely crucial. For hydrophobic acceptors, however, this required the addition of organic solvents to the reaction medium. On the one hand, monophasic systems have been developed that make use of ionic liquids as environmentally friendly or ‘green’ co-solvents (De Winter et al, 2013, Green Chemistry). On the other hand, the use of ethylacetate as solvent resulted in a biphasic system from which the glycosylated product could be purified more easily (De Winter et al, 2014, Org. Proc. Res. Develop.). The latter has been used to develop a representative process for each enzyme class, i.e. the production of pyrogallol, catechol and quercetin glycoside with SP, GTFA and rhamnosidase, respectively. Product yields of more than 85% could be obtained in each case, which is more than enough for their commercial exploitation. To demonstrate the techno-economic feasibility of our technology, these reactions have been scaled-up at the facilities of our industrial partner Bio Base Europe Pilot Plant (BBEPP). More than 1 kg of highly pure and crystalline product was generated, which can now be ordered through the commercial catalogue of Carbosynth. Interestingly, a Life Cycle Analysis (LCA) has revealed that the environmental impact of our process is 9 times lower than that of the corresponding chemical process, which is a very strong argument for the uptake of our technology in an industrial sector that is constantly looking for innovative solutions to improve its eco-efficiency.
Potential Impact:
In various industries, enzymes are increasingly used as efficient biocatalysts to perform a wide range of chemical reactions. The use of biocatalysis can have significant benefits, both economical and ecological, compared to conventional chemical technology, such as increased conversion efficiency, higher product specificity, improved product purity, lowered energy consumption and a significant decrease in chemical waste. Biocatalytic reactions are typically performed at normal temperatures and pressures, whereby no hazardous intermediate products are formed or toxic waste is generated. Biocatalysis has thus developed into a main contributor to “green chemistry”.
One of the major objectives of NOVOSIDES was the development of biocatalytic glycosylation reactions into economical processes that can be run at an industrial scale. These new processes will illustrate the potential of enzymatic chemistry for the production of glycosides and stimulate further research in this field. It will also raise interest from the industry to collaborate with academic partners, which typically has been lacking for these particular targets. Interested parties can basically be divided into enzyme producers and enzyme users. Europe currently produces about 70% of the world’s enzymes, with Novozymes and Genencor (now DuPont Industrial Biosciences) as the market leaders. The NOVOSIDES project has significantly expanded the range of biocatalysts that can be used by the industry and thus helped to consolidate Europe’s position in this field.
Besides the enzyme market, a second sector that can be stimulated by the NOVOSIDES project is the chemical industry, a vital sector for Europe’s economy that accounts for 2.4% of its Gross Domestic Product (GDP) and 6% of its industrial workforce. It is a sector for which the European Union (EU) is famous worldwide and considered to be in a commanding position. Continuous innovations are required to hold on to this top spot and to adapt to the rapidly changing economical and ecological climate. Because of the complex nature of the synthetic procedures, a lot of glycosides are currently considered to be unprofitable. Providing the sector with a novel technology that is more efficient will allow them to expand their activities and produce a whole range of new compounds. This has already become apparent in recent months, as our first glycoside products have initiated interactions with different companies that want to incorporate our processes in their operations.
Besides the economical advantages, the ecological benefits are also apparent. On average, enzymatic glycosylation reactions generate 5-fold less waste than their chemical counterparts. Furthermore, the waste is not toxic, in contrast to that generated by the chemical industry. These advantages strongly contribute to the development of green chemistry. Because of the higher space-time yield of enzymatic reactions and the use of moderate temperatures, much less energy is also consumed during the biocatalytic production process. In that way, the NOVOSIDES project could also contribute to the reduction in carbon emissions, as stipulated in the Strategic Energy Technologies (SET) plan of the European Commission (EC). A complete Life Cycle Analysis (LCA) of our processes has confirmed that our total environmental impact is 9-fold lower when compared to the corresponding chemical processes.
These outcomes fit perfectly with recent policies that have been implemented by the EC, such as the Bioeconomy Strategy ("Innovating for Sustainable Growth: a Bioeconomy for Europe”). The goal of that ambitious plan is to enable the transition from a fossil- to a bio-based economy by stimulating further innovation in the field of biotechnology. The bioeconomy in the EU already has a turnover of nearly €2 trillion and employs more than 22 million people, accounting for 9% of total employment. In combination with the increased research funding for the bioeconomy under Horizon 2020, the new Bioeconomy Strategy should generate an added value of about €45 billion and a significant number of new jobs by 2025. Furthermore, the introduction of biobased products into the market is also supported by the EC’s updated Industrial Policy (building on the Lead Market Initiative), which should help to turn our scientific technology into industrial reality. It can, for example, be mentioned that the two staff members recruited for the NOVOSIDES-project by the Bio Base Europe Pilot Plant will remain in service after the project has ended. This nicely illustrates that our accomplishments already have a concrete impact, albeit at a modest scale for the time being.
To raise awareness about the benefits of our technology, results have been disseminated in various ways and forms. First of all, a newsletter has been regularly distributed to a very wide audience of academic and industrial scientists working in our field. In addition, our commercial partner Carbosynth has promoted our project at various exhibitions with a demo booth and leaflets, which were always very popular. In all those cases, interested parties were referred to our website (www.novosides.eu) for more information. In turn, the other partners have regularly presented their latest results at major conferences and in scientific publications. It is important to note that several of our papers featured on the cover of top-level journals, such as Nano Today (IF 19), Angewandte Chemie (IF 11), Journal of Materials Chemistry (IF 7), and Chemistry: A European Journal (IF 6). This is clear proof of the high-quality research being done in the framework of our project. Furthermore, one of our collaborators made it into the prestigious EYCA final of EuCheMS.
In addition, Lubbert Dijkhuizen has been invited by BASF to present his work on glucansucrases and Tom Desmet by Cargill to talk about the NOVOSIDES project in general. These invitations nicely illustrate that our line of work is very relevant for the industry. In addition to those informal contacts, a dedicated event has been organized to stimulate the exploitation of the NOVOSIDES-technology. On the 25th of November 2013, a Glycoside Application Event was held at the premises of Bio Base Europe Pilot Plant). More than twenty industrial representatives were present from companies as diverse as Procter & Gamble (personal care industry), Cosun (agricultural industry), Cargill (carbohydrate industry), Dupont (enzyme industry), Solvay (chemical industry) and Vesalius Biocapital (investors). This has resulted in the identification of new markets where glycosides could find applications. At the end of the meeting, 1-to-1 partnering was initiated, allowing private discussions with individual companies to take place.
At the end of the NOVOSIDES project, a dedicated workshop was also organized to present our results to interested parties from both academia and industry. To maximize the number of participants (120 in this case), the workshop was held as a dedicated session of the Fourth International Conference on Novel Enzymes, organized in Ghent, Belgium, on October 15th 2014. After the workshop, a demonstration session was organized to explain our technology to the general public. Invitations to this session were widely circulated by the project partners, and reached colleagues, family and friends as well as members of the press. In the end, about 70 people were present at the session. The different partners engaged the audience to explain their role in the project, and how this resulted in a glycosylation process that is environmentally friendly as well as economically attractive. As a visual aid, the properties of enzymes and glycosides were illustrated by means of an animated movie, which was compiled by a specialized firm and can be found at: https://www.youtube.com/watch?v=_jWwqiEfr2c.
Finally, it should also be mentioned that our results have attracted the interest of the popular press. That is particularly true for the fascinating properties of the new fluorescent probes. Under the title “The sweetest calculator in the world” they have featured in several online magazines, such Phys.org Science Daily, Scientific Computing, and Biotechnology.de. Furthermore, Prof. Schiller has also talked about their applications during radio interviews with MDR Figaro and Deutschlandfunk. In addition, the Novosides-project also made the national news in the Czech Republic, by means of an interview with Prof. Kren.
List of Websites:
Prof. dr. Tom Desmet, Coordinator
Ghent University, Center for Industrial Biotechnology and Biocatalysis
TEL: +32 9264 9920, EMAIL: tom.desmet@ugent.be
www.novosides.eu