Final Report Summary - SUSY (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions)
Executive Summary:
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 (www.glycosusy.eu) for more background information and project outcome.
Project Context and Objectives:
Glycosylation or the process of fusing carbohydrates to specific pharmaceutical or food compounds offers the ability to improve the quality of certain drug and food compounds. Many ways of performing this process do exist, either via the chemical way or by applying enzymes, but they all have an elevated cost price in general. In this European FP7 project, we therefore aim to develop a more cost-effective enzyme-based alternative, which will be up-scaled and transferred to interesting industrial stakeholders. The production costs will drastically be reduced by recycling of the expensive intermediary substrates, which are generated throughout the process through coupling of three-enzyme catalysed reactions. In this story, the role of the glycosyl transferase sucrose synthase (SuSy), the central enzyme in this reaction process, is crucial. Because of its favourable thermodynamics, this enzyme can be exploited to produce the expensive nucleotide-activated sugars that are necessary for glycosylation, starting from sucrose as a cheap and abundant substrate. Moreover, by employing sucrose analogues produced by fructansucrase (FS) instead of sucrose, this strategy can be expanded towards other nucleotide sugars, proving the broad applicability of our platform technology. However, an extensive mutagenesis effort will be needed to overcome the FS’s bottlenecks, e.g. low efficiency in sucrose analogues production due to severe hydrolysis and side product formation.
To enable efficient production of the SuSy-enzyme, various expression systems will be evaluated. This analysis comprises testing different expression hosts (bacterial or yeast strains), different plasmids (constitutive vs. inducible expression) and different expression conditions (medium, pH, temperature, time). In parallel, the stability of SuSy and other glycosyl transferase (GT) enzymes at elevated temperatures and in co-solvents should be optimized to fit the operational conditions, either through enzyme engineering by mutagenesis or by immobilization via multi-point covalent attachment. Tailor-made methods need to be developed with regard to the enzymes’ structure, activity and flexibility.
Another important part of the project deals with the engineering of the selected target enzymes in order to widen or change their substrate specificity. This protein engineering effort will allow more efficient synthesis of sucrose analogues, UDP-sugars and glycosylated compounds by FS, SuSy and other GT enzymes, respectively.
The available wild type and engineered enzymes will be then used to glycosylate a variety of interesting molecules as case study and synthesize a series of model compounds (nothofagin, phlorizin, davidioside, confusoside, resveratrol diglucoside) and isolate them at a small scale (<100 mg). The knowledge and the practical experiences gained during the lab-scale GT-SuSy cascade reaction will be analysed in detail to harvest important information that can be translated to process conditions for larger scale productions. As such, the critical parameters for successful operation of large-scale (100-1000 g of isolated product) glycosylation processes will be defined based on these smaller scale glycosylation experiments.
In the final stage the chosen glycosylation (i.e. UDP-glc production and Nothofagin) reactions will be up-scaled to produce the glycosylated products at 100g-1kg scale, which demonstrates proof-of concept of their industrial feasibility.
In summary, the major achievements of the overall project are:
1. Optimizing the production of sucrose analogues and nucleotide-sugars (WP1)
2. Optimizing the recombinant expression of SuSy and GT enzymes (WP2)
3. Develop co-immobilization protocol to increase its long-term stability of the enzymes (WP2)
4. Improve the characteristics of the biocatalysts by protein engineering (WP3)
a. Improve the stability of the target enzymes (SuSy, FS and GT)
b. Change the substrate specificity of the target enzymes (SuSy, FS and GT)
5. Development of optimal Process conditions (WP4) that will be further elaborated glycosylation potential of the enzymes. This part also includes optimization of Downstream Processing (DSP)
6. Up-scale the production (WP5) and purification to 100g-1kg scale has been performed during the final stage of the project. The purity of the products will be analysed and the process evaluation by Life Cycle Assessment (LCA).
Project Results:
During the earlier stage of the project, 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. 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 have be transfered amongst the consortium partners to enable their implementation in the later stage, namely Process development and Process scale-up, which had already been initiated at the end of reporting period II and were continued and finalized during the final period, allowing us to reach our final goal.
In terms of Process Development, multiple tasks have been fulfilled. The coupling of GT catalyzed glucosylations with UDP-glucose regeneration by GmSusy in a series of model reactions has been evaluated. Several proof-of-concept examples (nothofagin, phlorizin, davidioside, confusoside and a resveratrol diglucoside) were synthesized and isolated at a small scale (<100 mg). Based on these experiences, the critical parameters for successful operation of these and similar processes on a larger scale for production of 100-1000 g of isolated product were defined.
The Optimal Reactor Design for the use of (immobilized) enzymes has been determined. By performing the glycosylation reaction with the co-immobilized derivatives in comparison with the behaviour in the glycosylation of the enzymes immobilized in different supports, the difference on the efficacy in the recycled of UDP and the efficiency in the glycosylation have been proven, being the best option the use of co-immobilized derivative. Very low quantity of UDP is necessary for carrying out the reaction with the co-immobilized derivative. On one hand, in the stirred tank reactors there are a high variance in the efficiency of the reaction, as it is done with the co-immobilized derivative, or with the separate derivatives. On the other hand, in the packed bed reactors, there are not too much difference between the use of co-immobilized derivatives or the use of separate derivatives, as long as these derivatives are very well mixed in the column. Accordingly, the best option of reactor is the stirred tank reactor with the co-immobilized derivatives until now, and the worst option would be the packed bed reactor with the separate derivatives without mixing.
In close collaboration the partners intensively studied various strategies for isolation or Downstream Processing (DSP) of the glycosylation products. TUG established efficient preparative reversed phase HPLC protocols for the isolation of resveratrol glucosides, quercetin galactoside and nothofagin, yielding highly pure products (≥98%) with good recovery (70 – 90%). However, high solvent consumption and high costs of the HPLC resin limited the scalability of preparative HPLC, which was therefore best suited for isolation of very small product quantities (≤10 mg). To enable efficient DSP of larger quantities of glycosylation products, ACIB established size exclusion (SEC)-free and chromatography-free DSPs which are readily scalable.
After the development of the processes, determination of the best reaction conditions and Downstream Processing protocols, two glycosylation reactions were up-scaled to produce the glycosylated products at 100g-1kg scale, which demonstrates proof-of concept of their industrial feasibility.
1) UDP-glc production at 100 g scale (purity ≥90%) was realized by efficient whole-cell synthesis and an innovative, chromatography-free DSP. The high expression level of SuSyAc (480 U/gCDW; ~350 mg/Lmedium), the excellent performance metrics of the UDP-glc production (100 gproduct/L, 86% yield, TTN of 103 gUDP-glc/gCDW, STY of 10 g/L/h), and a DSP without chromatography will guarantee easy scale-up also to kilogram quantities. Our UPD-Glc has been included in Carbosynth's (Compton, Berkshire, UK) commercial catalog (www.carbosynth.com product code: MU08960). Supply with several batches of UDP-glc (ACIB; 125 g UDP-glc in total), which were requested by Carbosynth, demonstrates that our UDP-glc meets the quality requirements of suppliers and end-users. Carbosynth is interested in further cooperation.
2) Nothofagin production at 100 g scale by GT-SuSy coupling was realized through intensification of biocatalysis (using a phloretin/2-hydroxypropyl-β-CD inclusion complex) and innovative SEC-free DSP. The nothofagin production had excellent performance metrics of 97% yield and ~50 gproduct/L at a STY of 3 g/L/h. The low solvent consumption of only ~0.6 L per g of purified nothofagin in the advanced AEC step and the absence of SEC guarantee the scalability of the DSP. The high nothofagin concentration obtained at a moderate catalyst loading facilitates the DSP, and furthermore is beneficial for the scalability of the GT-SuSy process.[23] The use of equipment (e.g. AEC column, rotary evaporator, centrifuge and centrifuge bottles) suitable for large-scale production, will guarantee scale-up also to kilogram quantities.
To ensure that our nothofagin meets the quality requirements of potential customers, quality control was performed. A market price evaluation of the SuSy produced UDP-Glc and Nothofagin have been performed.
To evaluate the environmental impact of the newly developed biocatalytic processes (UDP-glucose and nothofagin), a Life Cycle Assessment (LCA) was performed, using an impact assessment based on the Sustainable Process Index®. All steps of the process were taken into account (“cradle-to-grave”), including the production of raw materials, the parameters of the enzymatic conversion, and the disposal of waste. An important notice is that the LCA clearly shows that the energy used is the most important factor for the ecological footprint of the entire process. An eco-friendly energy mix significantly reduces the ecological footprint.
In general, the major achievements in the SuSy-project so far as described above reflect the high quality research that has been performed in our consortium and the changing success of the experiments reflects the challenging nature of our research. The high quality of our research can be deducted from the 16 peer-reviewed papers that have been accepted for publication in top journals. Several other manuscripts have been submitted for peer-review or are currently in preparation, we estimate another 5 publications will result from the research project. Due to the collaborative nature of the project, multiple papers are joint publications with co-authors from different participants, also highlighting the close collaboration that was necessary to reach the ambitious goals.
Glycosylation or the process of fusing carbohydrates to specific pharmaceutical or food compounds offers the ability to improve the quality of certain drug and food compounds. Many ways of performing this process do exist, either via the chemical way or by applying enzymes, but they all have an elevated cost price in general. In this European FP7 project, we therefore have developed a more cost-effective enzyme-based alternative, which has been up-scaled and transferred to interesting industrial stakeholders. The production costs could be drastically reduced by recycling of the expensive intermediary substrates, which are generated throughout the process through coupling of three-enzyme catalysed reactions.
In summary, the major achievements of this overall project are:
1. Optimizing the production of sucrose analogues and nucleotide-sugars
2. Optimizing the recombinant expression of SuSy and GT enzymes
3. Development of (co-)immobilization protocols to increase stability of the enzymes
4. Improve the characteristics of the biocatalysts by protein engineering
a. Improve the stability of the target enzymes (SuSy, FS and GT)
b. Change the substrate specificity of the target enzymes (SuSy, FS and GT)
5. Development of optimal Process conditions allowed further elaboration of the glycosylation potential of the enzymes. This part also included optimization of Downstream Processing (DSP)
6. In the final stage, the production and purification of two chosen compounds (UDP-glc production and Nothofagin) have been up-scaled to 100g scale. The purity of the products has be analysed and the process has been evaluated by Life Cycle Assessment (LCA).
7. Our UPD-Glc has been included in Carbosynth's (Compton, Berkshire, UK) commercial catalog (product code: MU08960), demonstrates that our UDP-glc meets the quality requirements of suppliers and end-users.
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.
List of Websites:
http://www.glycosusy.eu/
Prof. Tom Desmet
Unit for Biocatalysis and Enzyme Engineering
Faculty of Bioscience Engineering, Ghent University
Coupure Links 653, 9000 Gent, Belgium
Tel. +32(0)9/264.99.20
http://www.biocatalysis.ugent.be
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 (www.glycosusy.eu) for more background information and project outcome.
Project Context and Objectives:
Glycosylation or the process of fusing carbohydrates to specific pharmaceutical or food compounds offers the ability to improve the quality of certain drug and food compounds. Many ways of performing this process do exist, either via the chemical way or by applying enzymes, but they all have an elevated cost price in general. In this European FP7 project, we therefore aim to develop a more cost-effective enzyme-based alternative, which will be up-scaled and transferred to interesting industrial stakeholders. The production costs will drastically be reduced by recycling of the expensive intermediary substrates, which are generated throughout the process through coupling of three-enzyme catalysed reactions. In this story, the role of the glycosyl transferase sucrose synthase (SuSy), the central enzyme in this reaction process, is crucial. Because of its favourable thermodynamics, this enzyme can be exploited to produce the expensive nucleotide-activated sugars that are necessary for glycosylation, starting from sucrose as a cheap and abundant substrate. Moreover, by employing sucrose analogues produced by fructansucrase (FS) instead of sucrose, this strategy can be expanded towards other nucleotide sugars, proving the broad applicability of our platform technology. However, an extensive mutagenesis effort will be needed to overcome the FS’s bottlenecks, e.g. low efficiency in sucrose analogues production due to severe hydrolysis and side product formation.
To enable efficient production of the SuSy-enzyme, various expression systems will be evaluated. This analysis comprises testing different expression hosts (bacterial or yeast strains), different plasmids (constitutive vs. inducible expression) and different expression conditions (medium, pH, temperature, time). In parallel, the stability of SuSy and other glycosyl transferase (GT) enzymes at elevated temperatures and in co-solvents should be optimized to fit the operational conditions, either through enzyme engineering by mutagenesis or by immobilization via multi-point covalent attachment. Tailor-made methods need to be developed with regard to the enzymes’ structure, activity and flexibility.
Another important part of the project deals with the engineering of the selected target enzymes in order to widen or change their substrate specificity. This protein engineering effort will allow more efficient synthesis of sucrose analogues, UDP-sugars and glycosylated compounds by FS, SuSy and other GT enzymes, respectively.
The available wild type and engineered enzymes will be then used to glycosylate a variety of interesting molecules as case study and synthesize a series of model compounds (nothofagin, phlorizin, davidioside, confusoside, resveratrol diglucoside) and isolate them at a small scale (<100 mg). The knowledge and the practical experiences gained during the lab-scale GT-SuSy cascade reaction will be analysed in detail to harvest important information that can be translated to process conditions for larger scale productions. As such, the critical parameters for successful operation of large-scale (100-1000 g of isolated product) glycosylation processes will be defined based on these smaller scale glycosylation experiments.
In the final stage the chosen glycosylation (i.e. UDP-glc production and Nothofagin) reactions will be up-scaled to produce the glycosylated products at 100g-1kg scale, which demonstrates proof-of concept of their industrial feasibility.
In summary, the major achievements of the overall project are:
1. Optimizing the production of sucrose analogues and nucleotide-sugars (WP1)
2. Optimizing the recombinant expression of SuSy and GT enzymes (WP2)
3. Develop co-immobilization protocol to increase its long-term stability of the enzymes (WP2)
4. Improve the characteristics of the biocatalysts by protein engineering (WP3)
a. Improve the stability of the target enzymes (SuSy, FS and GT)
b. Change the substrate specificity of the target enzymes (SuSy, FS and GT)
5. Development of optimal Process conditions (WP4) that will be further elaborated glycosylation potential of the enzymes. This part also includes optimization of Downstream Processing (DSP)
6. Up-scale the production (WP5) and purification to 100g-1kg scale has been performed during the final stage of the project. The purity of the products will be analysed and the process evaluation by Life Cycle Assessment (LCA).
Project Results:
During the earlier stage of the project, 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. 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 have be transfered amongst the consortium partners to enable their implementation in the later stage, namely Process development and Process scale-up, which had already been initiated at the end of reporting period II and were continued and finalized during the final period, allowing us to reach our final goal.
In terms of Process Development, multiple tasks have been fulfilled. The coupling of GT catalyzed glucosylations with UDP-glucose regeneration by GmSusy in a series of model reactions has been evaluated. Several proof-of-concept examples (nothofagin, phlorizin, davidioside, confusoside and a resveratrol diglucoside) were synthesized and isolated at a small scale (<100 mg). Based on these experiences, the critical parameters for successful operation of these and similar processes on a larger scale for production of 100-1000 g of isolated product were defined.
The Optimal Reactor Design for the use of (immobilized) enzymes has been determined. By performing the glycosylation reaction with the co-immobilized derivatives in comparison with the behaviour in the glycosylation of the enzymes immobilized in different supports, the difference on the efficacy in the recycled of UDP and the efficiency in the glycosylation have been proven, being the best option the use of co-immobilized derivative. Very low quantity of UDP is necessary for carrying out the reaction with the co-immobilized derivative. On one hand, in the stirred tank reactors there are a high variance in the efficiency of the reaction, as it is done with the co-immobilized derivative, or with the separate derivatives. On the other hand, in the packed bed reactors, there are not too much difference between the use of co-immobilized derivatives or the use of separate derivatives, as long as these derivatives are very well mixed in the column. Accordingly, the best option of reactor is the stirred tank reactor with the co-immobilized derivatives until now, and the worst option would be the packed bed reactor with the separate derivatives without mixing.
In close collaboration the partners intensively studied various strategies for isolation or Downstream Processing (DSP) of the glycosylation products. TUG established efficient preparative reversed phase HPLC protocols for the isolation of resveratrol glucosides, quercetin galactoside and nothofagin, yielding highly pure products (≥98%) with good recovery (70 – 90%). However, high solvent consumption and high costs of the HPLC resin limited the scalability of preparative HPLC, which was therefore best suited for isolation of very small product quantities (≤10 mg). To enable efficient DSP of larger quantities of glycosylation products, ACIB established size exclusion (SEC)-free and chromatography-free DSPs which are readily scalable.
After the development of the processes, determination of the best reaction conditions and Downstream Processing protocols, two glycosylation reactions were up-scaled to produce the glycosylated products at 100g-1kg scale, which demonstrates proof-of concept of their industrial feasibility.
1) UDP-glc production at 100 g scale (purity ≥90%) was realized by efficient whole-cell synthesis and an innovative, chromatography-free DSP. The high expression level of SuSyAc (480 U/gCDW; ~350 mg/Lmedium), the excellent performance metrics of the UDP-glc production (100 gproduct/L, 86% yield, TTN of 103 gUDP-glc/gCDW, STY of 10 g/L/h), and a DSP without chromatography will guarantee easy scale-up also to kilogram quantities. Our UPD-Glc has been included in Carbosynth's (Compton, Berkshire, UK) commercial catalog (www.carbosynth.com product code: MU08960). Supply with several batches of UDP-glc (ACIB; 125 g UDP-glc in total), which were requested by Carbosynth, demonstrates that our UDP-glc meets the quality requirements of suppliers and end-users. Carbosynth is interested in further cooperation.
2) Nothofagin production at 100 g scale by GT-SuSy coupling was realized through intensification of biocatalysis (using a phloretin/2-hydroxypropyl-β-CD inclusion complex) and innovative SEC-free DSP. The nothofagin production had excellent performance metrics of 97% yield and ~50 gproduct/L at a STY of 3 g/L/h. The low solvent consumption of only ~0.6 L per g of purified nothofagin in the advanced AEC step and the absence of SEC guarantee the scalability of the DSP. The high nothofagin concentration obtained at a moderate catalyst loading facilitates the DSP, and furthermore is beneficial for the scalability of the GT-SuSy process.[23] The use of equipment (e.g. AEC column, rotary evaporator, centrifuge and centrifuge bottles) suitable for large-scale production, will guarantee scale-up also to kilogram quantities.
To ensure that our nothofagin meets the quality requirements of potential customers, quality control was performed. A market price evaluation of the SuSy produced UDP-Glc and Nothofagin have been performed.
To evaluate the environmental impact of the newly developed biocatalytic processes (UDP-glucose and nothofagin), a Life Cycle Assessment (LCA) was performed, using an impact assessment based on the Sustainable Process Index®. All steps of the process were taken into account (“cradle-to-grave”), including the production of raw materials, the parameters of the enzymatic conversion, and the disposal of waste. An important notice is that the LCA clearly shows that the energy used is the most important factor for the ecological footprint of the entire process. An eco-friendly energy mix significantly reduces the ecological footprint.
In general, the major achievements in the SuSy-project so far as described above reflect the high quality research that has been performed in our consortium and the changing success of the experiments reflects the challenging nature of our research. The high quality of our research can be deducted from the 16 peer-reviewed papers that have been accepted for publication in top journals. Several other manuscripts have been submitted for peer-review or are currently in preparation, we estimate another 5 publications will result from the research project. Due to the collaborative nature of the project, multiple papers are joint publications with co-authors from different participants, also highlighting the close collaboration that was necessary to reach the ambitious goals.
Glycosylation or the process of fusing carbohydrates to specific pharmaceutical or food compounds offers the ability to improve the quality of certain drug and food compounds. Many ways of performing this process do exist, either via the chemical way or by applying enzymes, but they all have an elevated cost price in general. In this European FP7 project, we therefore have developed a more cost-effective enzyme-based alternative, which has been up-scaled and transferred to interesting industrial stakeholders. The production costs could be drastically reduced by recycling of the expensive intermediary substrates, which are generated throughout the process through coupling of three-enzyme catalysed reactions.
In summary, the major achievements of this overall project are:
1. Optimizing the production of sucrose analogues and nucleotide-sugars
2. Optimizing the recombinant expression of SuSy and GT enzymes
3. Development of (co-)immobilization protocols to increase stability of the enzymes
4. Improve the characteristics of the biocatalysts by protein engineering
a. Improve the stability of the target enzymes (SuSy, FS and GT)
b. Change the substrate specificity of the target enzymes (SuSy, FS and GT)
5. Development of optimal Process conditions allowed further elaboration of the glycosylation potential of the enzymes. This part also included optimization of Downstream Processing (DSP)
6. In the final stage, the production and purification of two chosen compounds (UDP-glc production and Nothofagin) have been up-scaled to 100g scale. The purity of the products has be analysed and the process has been evaluated by Life Cycle Assessment (LCA).
7. Our UPD-Glc has been included in Carbosynth's (Compton, Berkshire, UK) commercial catalog (product code: MU08960), demonstrates that our UDP-glc meets the quality requirements of suppliers and end-users.
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.
List of Websites:
http://www.glycosusy.eu/
Prof. Tom Desmet
Unit for Biocatalysis and Enzyme Engineering
Faculty of Bioscience Engineering, Ghent University
Coupure Links 653, 9000 Gent, Belgium
Tel. +32(0)9/264.99.20
http://www.biocatalysis.ugent.be