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Periodic Report Summary 2 - SUSY (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions)

Project Context and Objectives:
Glycosylation or the process of fusing sugars to small pharmaceutical or food molecules can drastically improve their biological and physicochemical properties, offering the ability to reassure a high quality of drugs and nutrition. Access to this is a basic right for each European citizen. Such a modification can, for example, be used to improve solubility of healthy food or drug compounds, enhance activity of certain antibiotics or modulate the characteristics of flavours or odours. A famous example is the glycosylation of L-menthol, the ‘active’ or fresh compound of chewing gum, the slow hydrolytic degradation of the L-menthol glycoside in the mouth results in a slow release of L-menthol and thus a fresh sensation that will last over a longer period.

Many ways of performing glycosylation reactions do exist, either via the chemical way or by applying enzymes, but they all have an elevated cost price in common. The advantage of using enzymes for glycosylation instead of chemical synthesis are mainly ecologic as they produce less waste and no toxic intermediary compounds, and in addition use less energy due to their higher overall efficiency and specificity. Unfortunately, the enzymes that perform glycosylation reactions, known as glycosyl transferases (GTs), require nucleotide-activated sugars to properly do their job, which are way too expensive for industrial applications.

This European FP7 project, entitled SuSy, tries to address this problem by developing a more cost-effective multi-step process based on the unique properties of three types of sugar-modifying enzymes, i.e. levansucrase, sucrose synthase(s) and glycosyl transferase(s). Starting point of the process will be sucrose, a very cheap and abundant substrate. By coupling the three enzymes in a multi-step fashion, the expensive intermediary compounds can be created efficiently from cheap substrate sucrose and NDP can effectively be recycled throughout the process, which significantly reduces the production costs of glycosylated compounds. Unfortunately, the in nature available enzymes still suffer from a couple of drawbacks that need to be addressed before we can exploit them in our envisaged process, such as a narrow specificity and limited stability. Optimization of these enzyme parameters will be realized by searching for improved enzyme variants in either unexplored, natural habitats or in specifically for this project generated “smart” mutant libraries, enriched for the desired property.

To achieve the project’s ambitious goals, a tightly scheduled work plan will be followed that combines state-of-the-art enzyme and process engineering techniques. In a first step, a highly efficient system will be developed for cheap production of the expensive nucleotide-activated sugars and sucrose analogues, based on the action of the sugar-modifying enzymes. The availability of sucrose analogues is crucial to broaden the applicability of our glycosylation platform. Next, the production of the enzymes themselves needs to be optimized. Once a continuous supply of these sugar-modifying enzymes is guaranteed, their specificity and stability will be enhanced through enzyme engineering, generating the desired enzyme variants compatible with application in our multi-step process. Finally, the economic potential of our technology will be demonstrated by the development and scale-up of the process at pilot-plant facilities of the consortium partners. Both the finalized multi-step process and the produced glycosides will be actively marketed to potential end-users including enzyme-producing or enzyme-consuming companies to promote the valorisation of the project’s results. The tight collaboration between eight academic and industrial partners with complementary expertise should increase the chances of successful executing this project. Visit our project website ( for more background information.

Project Results:
During the first two years and a half of the project (M1-M30), considerable progress has been made towards the final goal to develop a cost-effective multi-enzyme process for glycosylation. Initial efforts were invested in establishing the BCA-assay as a fast, reliable and cheap assay for quantification of sucrose synthase (SuSy) activity, followed by characterization of a set of novel SuSy’s from plant and (cyano)bacterial origin with this optimized assay. The BCA assay specifically relies on the detection of fructose that is produced during the conversion of sucrose via sucrose synthase, and is compatible with high-throughput screening of mutant libraries. The SuSy from Acidiothiobacillus caldus (SuSyAc), a prokaryotic organism that lives under extreme temperatures, came out as most suitable candidate enzyme for engineering purposes and subsequent application in our process due to its natural activity at elevated temperatures, enhanced thermostability and promiscuity towards alternative substrates. Good bacterial overexpression of this enzyme and subsequent purification to almost 100% purity via affinity chromatography forms an additional benefit for its industrial application. In a next step, one of the partners immobilized SuSyAc on tailor-made carriers as one way to improve its operational stability and long-term storage, in addition to stability engineering.

Furthermore, one of the partners was able to establish a highly efficient production system for the nucleotide-activated sugar UDP-glucose, starting from simple sucrose and the aid of SuSy from soybean (Glycine max). Under optimized conditions, 100 to 200 mM of UDP-glucose could be obtained via this system with an overall yield of more than 80 %. Isolation of this nucleotide sugar from the reaction mixture could be easily performed by a combination of anion exchange chromatography and desalting steps. The partner’s UDP-Glc is now available in a commercial catalogue. SuSyAc has also been engineered to towards an improved biocatalyst, providing an alternative to the soybean biocatalyst in this established UDP-glucose production/isolation system.

Also the other enzymes (glycosyl-transferase and levansucrase) have been engineered successfully. Superfamily databases containing sequential and structural data for the project enzymes were created and form a solid base for our enzyme engineering efforts. They were used to design mutant libraries, which are focussed on optimization of two enzyme characteristics, namely improving stability and changing the selectivity of the selected enzymes. By mutating specific positions, enzyme variants with reduced side-product formation have been generated, which significantly improves the enzyme’s efficiency. In addition, stabilized variants have been created hereby improving their industrial applicability.

The research efforts and results obtained so far will be transferred amongst the consortium partners to enable their implementation in Process development and scale-up, which have recently been initiated and will be continued in the upcoming period, allowing us to reach our final goal; the development of a cost-effective multi-enzyme process for glycosylation of small molecules.

Potential Impact:
Enzymes are of huge importance as efficient biocatalysts in European industry to perform a wide range of chemical reactions. Compared to conventional chemistry, choosing for biocatalyst implies a significant amount of benefits, both economic and ecological. The main advantages are increased conversion efficiency, broader product specificity, improved product purity and a five-fold decrease in chemical waste. In addition, enzymatic reactions are typically performed at environmental temperatures and pressures, whereby no hazardous intermediate products are formed or toxic waste is generated. Less energy is consumed during enzymatic conversions, contributing to the reduction of carbon dioxide emission. Taken all together, biocatalysis could be considered as a major pillar of the “green chemistry”.

A decade ago, a vision paper by ESAB and EuropaBio gave the offset of exploiting biocatalysts in industrial biotechnology by describing how this can contribute to a sustainable development of European industry, together with an increase in competitiveness with world-wide markets. Recently, the EC’s Joint Research Council (JRC) defined industrial biotechnology as one the six key enabling technologies for future industrial development. Nevertheless, the European Technology Platform for Sustainable Chemistry (SusChem) has warned us that novel enzymes with unique properties will be required in the upcoming decade to provide fuel for future business opportunities in the European enzyme industry. This idea fits nicely with this call’s topic, i.e. “Optimal and cost-effective industrial biocatalysts”.

One of the major impacts of SuSy is the development of a technological platform that will enclose the availability of cost-effective applications for GT’s, and subsequently increase their world-wide demand. GT’s could be exploited in a wide range of applications, emphasizing the potential impact on the enzyme market that could be achieved through broadening the range of available biocatalysts via our technology. In addition, as Europe currently is the lead producer of enzymes world-wide, the current project could help to consolidate this position. A crucial factor in the development of cost-effective GT’s is the collaboration between world-class scientists with complementary expertise that cover all required aspects. This could be nicely reflected by the successful execution of this project.

A second sector that will be stimulated 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. Continuous innovations in this field are crucial to adapt to rapid fluctuations in the economic and ecological climate. Providing the sector with a cost-effective technology to produce glycosides will allow them to expand activities and produce a whole range of new compounds. The products that were initially selected as targets form a powerful case study that should attract the main interest of potential investors. The resveratrol market, for example, has a total value of 40 billion dollar according to a report of Frost & Sullivan (2012). At this moment, improvement of solubility and stability of this compound has only been pursued by the multinational Omnichem-Ajinomoto through chemical phosphorylation, but this conversion is far from ecological. Our SuSy-technology platform aims to reach these goals through “greener” biocatalytic glycosylation reactions and thus forms a very interesting alternative. The extension of our technology towards galacto-, manno- and fucosylation providing immunogenic or prebiotic properties to “functional” oligosaccharides will further broaden the range of synthesized products, and by extension the potential customer base. Potential applications of our technology can be found in the carbohydrate-processing, food, chemical, pharmaceutical and personal care industries, represented by Novozymes, Nestle, Danone, Roche and Novartis.

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