Development of a biotechnology platform for enzymatic sulfation of industrial products based on polysaccharide sulfotransferases
Lead Research Organisation:
University of Liverpool
Department Name: Biochemistry
Abstract
A wide range of household products as diverse as foodstuffs, cleaning materials and personal care products, rely on the ability to modify starting materials on an industrial scale to generate products with the desired properties. One key requirement in many cases is the introduction of charged groups, to bestow the desired characteristics such as the ability to gel, to bind other materials or to behave as detergents. This can often be achieved by the addition of charged groups and one key way to do this is to add a sulfate group. The problem is that this is done currently using toxic and environmentally damaging chemicals.
The global market for such household products is huge and growing, for example, for personal care products is $ 7.35 Bn with annual growth of 7%. Our industrial collaborator, Unilever, with whom we have a long and well-established working relationship, is a major global player, with around 50% of the market share.
Consumer sensitivity to environmental concerns, particularly with existing petroleum-based products and the use of harsh chemicals, arising from their resistance to biological degradation, the generation of greenhouse gases and other environmental issues during their production or disposal, has culminated in commercial pressure to develop sustainable alternatives.
The current method of achieving sulfation industrially, involving aggressive chemicals which show poor selectivity and are environmentally damaging, needs to be replaced with a one employing renewable resources without damaging the environment. Together with Unilever, we aim to develop methods by which sulfation can be achieved using enzymes, thereby avoiding these problems. The route we propose - engineering enzymes to carry out this modification - offers both better control of the process and, crucially, enables environmentally responsible production of biodegradable products and waste.
Until now, the application of enzymes to these areas has been hindered by the problems of readily detecting the modifications that have been made and, owing to the cost of some of the materials involved, also of developing a commercially feasible method of adding sulfate groups. Now, however, as a result the combination of preliminary work carried out by ourselves and Unilever, as well as other technological advances, both of these problems can be solved. This project will exploit these improved technologies, together with our established expertise in enzyme production to achieve two principal aims:
(i) to assemble the technology (termed the high throughput enzyme-engineering platform) with which to produce and optimise enzymes that will be suitable for application to a wide range of enzyme-driven processes of industrial relevance and,
(ii) to illustrate the use of this platform to select and optimise suitable enzymes, using a class of enzymes that can add sulfate groups to naturally-occurring and renewable starting materials such as complex sugars (polysaccharides) and lipids (glycolipids) from plants.
The potential for industrial application of these sulfated products will then be assessed by Unilever, a major global company with a developed sustainability agenda that, in the future, will enable delivery of clean, renewable products.
The global market for such household products is huge and growing, for example, for personal care products is $ 7.35 Bn with annual growth of 7%. Our industrial collaborator, Unilever, with whom we have a long and well-established working relationship, is a major global player, with around 50% of the market share.
Consumer sensitivity to environmental concerns, particularly with existing petroleum-based products and the use of harsh chemicals, arising from their resistance to biological degradation, the generation of greenhouse gases and other environmental issues during their production or disposal, has culminated in commercial pressure to develop sustainable alternatives.
The current method of achieving sulfation industrially, involving aggressive chemicals which show poor selectivity and are environmentally damaging, needs to be replaced with a one employing renewable resources without damaging the environment. Together with Unilever, we aim to develop methods by which sulfation can be achieved using enzymes, thereby avoiding these problems. The route we propose - engineering enzymes to carry out this modification - offers both better control of the process and, crucially, enables environmentally responsible production of biodegradable products and waste.
Until now, the application of enzymes to these areas has been hindered by the problems of readily detecting the modifications that have been made and, owing to the cost of some of the materials involved, also of developing a commercially feasible method of adding sulfate groups. Now, however, as a result the combination of preliminary work carried out by ourselves and Unilever, as well as other technological advances, both of these problems can be solved. This project will exploit these improved technologies, together with our established expertise in enzyme production to achieve two principal aims:
(i) to assemble the technology (termed the high throughput enzyme-engineering platform) with which to produce and optimise enzymes that will be suitable for application to a wide range of enzyme-driven processes of industrial relevance and,
(ii) to illustrate the use of this platform to select and optimise suitable enzymes, using a class of enzymes that can add sulfate groups to naturally-occurring and renewable starting materials such as complex sugars (polysaccharides) and lipids (glycolipids) from plants.
The potential for industrial application of these sulfated products will then be assessed by Unilever, a major global company with a developed sustainability agenda that, in the future, will enable delivery of clean, renewable products.
Technical Summary
Sulfation is one way to improve the properties of materials such as polysaccharides and lipids for many industrial applications, ranging from food products (e.g. gelling polysaccharides) to personal care products (e.g. surfactants). The global market for these products is huge and growing, e.g. for personal care is $ 7.35 Bn with 7% growth p.a. Our industrial collaborator, Unilever, with whom we have a well-established working relationship, is a global player with 50% market share.
The current method of sulfating polysaccharides and surfactants industrially involves aggressive chemicals (sulfur trioxide and sulfamic acid) which show poor selectivity and are environmentally damaging. Together with Unilever, we aim to develop enzymatic methods to achieve sulfation, offering better control of the process and the products generated, as well as, crucially, enabling environmentally responsible production of biodegradable materials and waste products.
The application of sulfating enzymes (sulfotransferases; STs) to these sugar-containing materials has been hindered by the problem of detecting sulfation (it has no readily detected absorption characteristics, e.g. in the UV region) and the expense of generating the sulfate donor, PAPS. These challenges have now been met by the combination of our high throughput screening method and the development of recyclable PAPS systems. This project will exploit these advances, together with our established expertise in enzyme manipulation and expression, to:
(i) assemble a generic enzyme-engineering HT platform, suitable for application to a wide range of enzyme-driven processes and,
(ii) to illustrate the use of the platform, using STs to modify polysaccharides and glycolipids, both examples of high-value renewable materials.
The potential for application of these sulfated products will be assessed by Unilever a global company with a developed sustainability agenda, aligning with the BBSRC's strategic aims.
The current method of sulfating polysaccharides and surfactants industrially involves aggressive chemicals (sulfur trioxide and sulfamic acid) which show poor selectivity and are environmentally damaging. Together with Unilever, we aim to develop enzymatic methods to achieve sulfation, offering better control of the process and the products generated, as well as, crucially, enabling environmentally responsible production of biodegradable materials and waste products.
The application of sulfating enzymes (sulfotransferases; STs) to these sugar-containing materials has been hindered by the problem of detecting sulfation (it has no readily detected absorption characteristics, e.g. in the UV region) and the expense of generating the sulfate donor, PAPS. These challenges have now been met by the combination of our high throughput screening method and the development of recyclable PAPS systems. This project will exploit these advances, together with our established expertise in enzyme manipulation and expression, to:
(i) assemble a generic enzyme-engineering HT platform, suitable for application to a wide range of enzyme-driven processes and,
(ii) to illustrate the use of the platform, using STs to modify polysaccharides and glycolipids, both examples of high-value renewable materials.
The potential for application of these sulfated products will be assessed by Unilever a global company with a developed sustainability agenda, aligning with the BBSRC's strategic aims.
Planned Impact
Impact Summary
Investment in basic research, alongside that designed to move discovery to a position where transition to Technology Readiness Level (TLR) 4, or beyond is feasible, underpins improvements in prosperity and societal benefit. The strategic plans of BBSRC highlight the importance of securing diverse impacts from funded research.
Our proposal exploits new tools and technology developed with previous funding from BBSRC and EC to evaluate the addition of a specific chemical group (sulfate), to biological molecules. Although biologically importanct, it had received little strategic funding but developments of high-throughput methods to measure enzymatic sulfation and interest from industry in sustainable routes to sulfation are changing this landscape. Our proposed platform builds on existing investment and will have substantial impact on the production and modification of enzymes to replace existing catalysts, as well as to access, new, sustainable & high value chemicals. This approach and the involvement of a major global player, Unilever, will have game-changing effects on the transition to sustainable and biodegradable chemicals and materials.
The early description (Year 1) of individual platform components and their exploitation for enzyme discovery and optimisation will be afforded by the initial cycle of STs through the platform, enabling IP positions to be defined. With Unilever (Year 1-2), we will then maximise visibility by publication in high-profile open access journals in chemical biology, biochemistry/biotechnology and through open access databases (Uniprot, Genebank). GeneMill will engage with the wider community, including Unilever and its extensive supply chain, to provide training in platform use. Many enzymes of biotechnological interest lack high-throughput activity screens, e.g., cyclases, and SB's expertise in Eu-based host-guest sensors will enable the scope of the platform to include many enzyme classes.
Conferences for scientists and the public (Years 1-2) provide the first route to general dissemination, while presenting at subject-specific conferences allows the power of our platform for analysis and development of STs and other enzyme classes to be targeted effectively. A global approach to enzyme modification and optimisation provides greater understanding and rapid transition to industrial relevance. All the research teams are active in conference support and delivery, across diverse audiences, including scientists (biochemistry, biotechnology, chemical biology & chemistry) and non-scientists (non-science undergraduates, schools & museums). These groups will be contacted during the project via distinct activities at UoL and at 'Pint Of Science', and Liverpool Museum open days, during which clear explanations of the role of sulfation, how well-known sulfated high value products are made currently and how our platform enables a transition to sustainable chemicals, will be provided through a portable display suitable for departmental, school, museum or national exhibitions.
All those involved will receive training in impact delivery as appropriate from BBSRC and the PIs. The applicants strongly advocate public-private partnerships to unlock commercial potential e.g. IBCarb awards (IB & EY), DF's long-term support from Allergan Inc. & SB's commercialisation of nucleotide luminescent sensors. By collaborating with groups such as 'Sense About Science', we will explain to the public how basic research on enzymes can transform the production of high value chemicals in well-known products and meet the social ambition of sustainability.
The PDRA will engage in related disciplines in this industry-facing project and develop their skills in high throughput techniques that underpin the discovery and improvement of enzymes as catalysts. Given the strong support and training available from the team, this provides for the PDRA's evolution into a scientific leader in a key area.
Investment in basic research, alongside that designed to move discovery to a position where transition to Technology Readiness Level (TLR) 4, or beyond is feasible, underpins improvements in prosperity and societal benefit. The strategic plans of BBSRC highlight the importance of securing diverse impacts from funded research.
Our proposal exploits new tools and technology developed with previous funding from BBSRC and EC to evaluate the addition of a specific chemical group (sulfate), to biological molecules. Although biologically importanct, it had received little strategic funding but developments of high-throughput methods to measure enzymatic sulfation and interest from industry in sustainable routes to sulfation are changing this landscape. Our proposed platform builds on existing investment and will have substantial impact on the production and modification of enzymes to replace existing catalysts, as well as to access, new, sustainable & high value chemicals. This approach and the involvement of a major global player, Unilever, will have game-changing effects on the transition to sustainable and biodegradable chemicals and materials.
The early description (Year 1) of individual platform components and their exploitation for enzyme discovery and optimisation will be afforded by the initial cycle of STs through the platform, enabling IP positions to be defined. With Unilever (Year 1-2), we will then maximise visibility by publication in high-profile open access journals in chemical biology, biochemistry/biotechnology and through open access databases (Uniprot, Genebank). GeneMill will engage with the wider community, including Unilever and its extensive supply chain, to provide training in platform use. Many enzymes of biotechnological interest lack high-throughput activity screens, e.g., cyclases, and SB's expertise in Eu-based host-guest sensors will enable the scope of the platform to include many enzyme classes.
Conferences for scientists and the public (Years 1-2) provide the first route to general dissemination, while presenting at subject-specific conferences allows the power of our platform for analysis and development of STs and other enzyme classes to be targeted effectively. A global approach to enzyme modification and optimisation provides greater understanding and rapid transition to industrial relevance. All the research teams are active in conference support and delivery, across diverse audiences, including scientists (biochemistry, biotechnology, chemical biology & chemistry) and non-scientists (non-science undergraduates, schools & museums). These groups will be contacted during the project via distinct activities at UoL and at 'Pint Of Science', and Liverpool Museum open days, during which clear explanations of the role of sulfation, how well-known sulfated high value products are made currently and how our platform enables a transition to sustainable chemicals, will be provided through a portable display suitable for departmental, school, museum or national exhibitions.
All those involved will receive training in impact delivery as appropriate from BBSRC and the PIs. The applicants strongly advocate public-private partnerships to unlock commercial potential e.g. IBCarb awards (IB & EY), DF's long-term support from Allergan Inc. & SB's commercialisation of nucleotide luminescent sensors. By collaborating with groups such as 'Sense About Science', we will explain to the public how basic research on enzymes can transform the production of high value chemicals in well-known products and meet the social ambition of sustainability.
The PDRA will engage in related disciplines in this industry-facing project and develop their skills in high throughput techniques that underpin the discovery and improvement of enzymes as catalysts. Given the strong support and training available from the team, this provides for the PDRA's evolution into a scientific leader in a key area.
Publications
Lima MA
(2022)
Phosphorylation and sulfation share a common biosynthetic pathway, but extend biochemical and evolutionary diversity of biological macromolecules in distinct ways.
in Journal of the Royal Society, Interface
London JA
(2021)
Synthesis and toxicity profile in 293 human embryonic kidney cells of the ß D-glucuronide derivatives of ortho-, meta- and para-cresol.
in Carbohydrate research
Wheeler S
(2022)
Anion binding to a cationic europium( iii ) probe enables the first real-time assay of heparan sulfotransferase activity
in Organic & Biomolecular Chemistry
| Description | A wide range of household products as diverse as foodstuffs, cleaning materials and personal care products, rely on the ability to modify starting materials on an industrial scale to generate products with the desired properties. One key requirement in many cases is the introduction of charged groups, to bestow the desired characteristics such as the ability to gel, to bind other materials or to behave as detergents. One key way to add charged groups is to add a sulfate group. The problem is that this is done currently using toxic and environmentally damaging chemicals, which is often compounded by using fossil fuel-derived starting materials. This represents a serious problem, which we sought to solve by adopting a green approach based on enzymes. In the project we collaborated with Unilever to: (i) create the technology (termed the high throughput enzyme-engineering platform) with which to produce and optimise enzymes that will be suitable for application to sulfation, as well a wide range of other enzyme-driven processes of industrial relevance. (ii) illustrate the use of this platform to select and optimise suitable enzymes, using a class of enzymes that can add sulfates to renewable starting materials, such as complex sugars (polysaccharides) and fats (glycolipids) from plants. The High Throughput Enzyme-Engineering Platform Identification and production of enzymes; the enzyme pipeline. We identified a range of sulfatransferases (STs, enzymes that can add sulfate groups from small molecule PAPS to a different material) with suitable properties and set up robotic high throughput (HTP) system for optimising their production in a common biotechnology bacteria E. coli that allowed us to test 1000s of different conditions. With the robotic platform we identified expression conditions for 10 algal, 8 mammalian and two microorganism STs, and although the expression level and protein stability varied we have seamless transfer to large scale production of these enzymes. The highest expression level included that for 6 STs and their production was scaled up for the activity measurements. Main outcomes: large-scale expression of 6 STs (10-20mg/L). Enzymatic activity. We set up an analytical pipeline that has three complementary activity measurements. Our novel high-throughput Eu-PAPS/PAP luminescent sensor-based assay (recently published, DOI 10.1039/d1ob02071d) provides a direct fluorescent, real-time readout, a PAPS recycling assay that utilises inexpensive PNPS and provides a real-time coupled reaction colourimetric readout, and direct Nuclear Magnetic Resonance (NMR) monitoring of PAPS hydrolysis and substrate sulfation. Together, the three assays allow effective screening of multiple conditions, validation of the reaction and scale-up. The ST activities were tested against a diverse range of commercially-relvant poly-, oligo- and monosaccharides, (heparin, carrageenan, xyloglucan, mannan, galactan, CM-cellulose and rhamnose) and renewable sugar-based bio-detergents. The majority of the produced STs have been active against heparin (a mixture of different sulfation states), but showed selective activity towards the specific sulfation forms. With the Eu-sensor we detected lower activity against some plant polysaccharides. Of these the NST enzyme, which also showed high activity against non-sulfated heparin (its physiological substrate) was one of the most active. The common, but not universal requirement for pre-sulfation at particular sites of many of the STs is expected, as the biosynthesis of the sulphated polysaccharides usually starts with sulfation at a single position by one enzyme, followed by the addition of sulphate groups to other positions by several sulfation-dependent STs. However, the unexpected detection of activity against non-sulfated substrates by a number of our STs indicates that they can also be optimised for non-sulfated substrates. The NMR experiments demonstrated substrate-dependent PAPS hydrolysis with the rate proportional to the enzyme concentration. The NMR-estimated Km for PAPS was in the 10-100 uM range. In the NMR experiments we validated the activity of the STs against specifically sultated forms of heparin, and several sulphated carrageenans. We also detected sulfation of several sugar-based bio-detergents. Our PAPS regeneration system provides for concomitant cheap, large scale sulfation and, since the conversion of PAP produced by the ST to PAPS is coupled to the generation of paranitrophenol (PNP) from PNPS hydrolysis by SULT1A1, a real-time by measure (absorbance at 405 nm) of the progress of polysaccharide sulfation. Thus, we can both monitor the sulfation reaction and estimate the amount of sulfated sugar product. NMR measurement of the rate of PAP to PAPS conversion provided the means to determine the optimal enzyme and PAP concentration to ensure that PAPS regeneration is not the rate-limited step in the coupled sulfation reaction. The PNPS assay was in full agreement with direct NMR measurement. The Eu-sensor assay allows a real-time direct measure of sulfation and is particularly suited to high throughput screening of sulfation of multiple acceptor substrates with multiple enzymes. With PAPS-regeneration system we successfully scaled-up sulfation of native and, and non-native substrate disaccharide substrates. The NMR analysis demonstrated selective quantitative sulfation at a single position of one sugar residue, creating a well-defined product. The effective sulfation of non-native substrate showed that the enzymes can be further optimised for different types of saccharides, and saccharide-containing materials. In support of this conclusion, we detected sulfation of sugar-based bio-detergents, although it was less effective than for disaccharide substrate due to the formation of detergent micelles. These experiments provided us a lead ST for sulfation of bio-detergents. Main outcomes: Pipeline for enzymatic activity assay. High activity against specifically sulphated heparin and carrageenan by 6 STs. Lower activity against a wide range of substrates non-physiological sugar substrates. A lead ST for bio-detergent sulfation. Location of modifications and key residues in substrate recognition. The understanding of the sulfation pattern and key residues recognised in the acceptor substrate are closely connected. Screening with the panel of heparin derivatives demonstrated that substrate selectivity of many of the tested enzymes is based on the positions of existing sulfate groups and the target hydroxyl acceptor. Using the PAPS regeneration system, we produced mg amounts of the sulfated product and measured NMR spectra to identify sulfation positions. This showed, for example, the sulfation at the 6-OH position by one of the algal STs with the highest activity. This particular enzyme can only be produced as an MBP-fusion, as it is otherwise unstable, which prevents the use of NMR to identify of the key residues binding substrate. However, the recent AlphaFold release allowed us to use modelling. In agreement with our detected 6-OH sulfation, the AlpaaFold structure was remarkably close to the X-ray structure of zebra fish 6-O ST solved in complex with several substrates (PDB 5T03). Using these structures, we modelled the substrate complex for the algal ST and identified 3 pairs of conserved positively charged residues involved in substrate recognition. Similarly, we modelled all other active STs and are investigating their substrate recognition mechanism. Main outcomes: scale-up of the product to mg quantities, identified sulfation sites, defined key substrate recognition residues. Enzyme optimisation. Our aim is to change or broaden substrate specificity and, where appropriate remove the dependence on pre-existing sulfation. The algal 6-O ST has a shallow, open substrate recognition site, which facilitates modification, is produced in high quantities and remains active at room temperature over several days. Therefore, we selected it as our primary modification target. To reduce the number of mutants in screening we adopted a two-stage approach. In the first stage the three pairs of positive changed residue (see above) are separately mutated to 4 others - Ala, Glu, Gln and Tyr, probing the effect of the substitution to different amino acid classes at the main substrate recognition sites. The mutations are introduced into 3 separate DNA fragments that are combined into the full gene using Golden Gate assembly. This produces 125 defined variants. We conducted the parallel cloning of some of the variants and assayed them with the Eu-sensor using our small-scale expression pipeline. From the results of the tests, we select sites for the saturation mutagenesis and testing in the follow-up experiment. Main outcomes: mutagenesis strategy and pipeline, design of mutants, mutant cloning. Summary. The funding allowed us to establish an enzyme-discovery pipeline that includes cloning, production, optimisation, screening and scale-up stages. We applied the pipeline to a range of STs and identified several lead enzymes that are suitable for targeted optimisation. We are taking forward one enzyme for sulfation of plant polysaccharides and one for the sulfation of bio-detergents. |
| Exploitation Route | Our study established a platform for enzyme development and a methodology for the analysis and scale-up of enzymatic sulfation. This will benefit academic researchers investigating biological sulfation across the kingdoms of life. Many components of the platform can be used for other classes of enzymes and are now available through GeneMill facility of Liverpool University. Successful high-level sulfation of a range of bio-materials demonstrated a way of creating new types of materials from renewable sources and waste materials. We are currently discussing follow-up applications with academic and commercial partners, such as using sulfated polysaccharides for extraction of metals from waste water. The study strengthened our partnership with Unilever. As a follow up we are investigating suitability of some sulfated materials for specific applications. Moreover, our success has led to us joining the DECARBON Prosperty Partnership application, led by UCL, with Manchester, Keele, Northumbria, Unilever and Johnson Matthey, which has been selected after the submission of the preliminary application and the following interview for full application. |
| Sectors | Chemicals,Education,Environment,Manufacturing, including Industrial Biotechology |
| Description | There are two main outcomes. The first is the establishment of the High Throughput Enzyme-Engineering Platform, called iM3. This is now used, in part or entirely in other projects, such as the development of a new protein expression tag. The second, is based on both iM3 and the expertise developed in enzymatic sulfation in this project, which has led to us joining the DECARBON consortium, which seeks to replace the current fossil fuel surfactants on the market with biotechnology derived ones. We are also exploring the potential for IP in our work, and where within the agreement with Unilever we have freedom to operate exploring opportunities with companies involved in the production of platform chemicals. |
| First Year Of Impact | 2021 |
| Sector | Chemicals,Education,Environment,Manufacturing, including Industrial Biotechology |
| Impact Types | Economic |
| Description | MSc Biotechnology module |
| Geographic Reach | Local/Municipal/Regional |
| Policy Influence Type | Influenced training of practitioners or researchers |
| Description | DTP CASE |
| Amount | £81,000 (GBP) |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 10/2020 |
| End | 10/2024 |
| Title | NMR analysis of complex mixtures |
| Description | We developed and implemented 2D NMR methodology for the analysis of complex biomolecules and mixtures. The method is based on a combination of NMR experiments and software for the automated analysis of NMR spectra. |
| Type Of Material | Improvements to research infrastructure |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | Introduced the method into the analysis of surfactant-based formulations and skin fat. |
| Title | Quantitative 2D NMR analysis |
| Description | zNMR software for the quantitative analysis of 2D HSQC NMR spectra |
| Type Of Material | Data analysis technique |
| Year Produced | 2022 |
| Provided To Others? | No |
| Impact | We applied the method to analyse complex biomaterials and biological mixtures. |
| Description | 2D NMR analysis of complex mixtures |
| Organisation | Unilever |
| Department | Unilever UK R&D Centre Port Sunlight |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | We developed a quantitative method based on 2D NMR spectroscopy for the analysis of complex biological mixtures. |
| Collaborator Contribution | Financial contribution funded the analysis software development and method validation. |
| Impact | Prototype software for the integration of the high-resolutions 2D HSQC NMR spectra. Validation of the approach on a set of test mixtures of biodetergents. Pilot analysis of spectra. |
| Start Year | 2022 |
| Description | Polysaccharide sulfation |
| Organisation | Unilever |
| Department | Unilever UK R&D Centre Port Sunlight |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | We conducted the NMR analysis of biomaterials. |
| Collaborator Contribution | In-kind contribution to cover part of the research project. |
| Impact | Publication |
| Start Year | 2021 |