Team:Exeter/Human Practices/impact

Biological Vs. Chemical Synthesis

Polysaccharides are undoubtedly complex, with varying side-groups on different sugar units as well as the same sugar units, and even the location of the ether linkage in bonding two saccharides together affecting its physicochemical properties. This diversification of polysaccharides is important in nature, but the synthesis of such polysaccharides are incredible difficult chemically. This difficulty is the result of the protection reactions for all the side-groups except the side-group intended for the specific linkage, and then subsequently the deprotection reactions needed to remove hindering protecting groups. This is a laborious process and results in high costs, a lowering yield as the repeat unit is polymerised and high amounts of waste products. Biological synthesis of polysaccharides using glycosyltransferases is a much easier approach to polysaccharide production. Enzymes are extremely specific, such that even a different stereoisomer of the same sugar monomer will prevent a reaction from taking place due to locations of active site amino acids. This inherent biochemistry and selectivity will lower costs, reduce waste products and result in high yields of the correct stereoisomer.

Our discussions with Dr. Mark Wood led us to a simple example to demonstrate how biological synthesis is a better approach than chemical synthesis of polysaccharides in the construction of the simple disaccharide α-D-glucopyranosyl-α-D-glucopyranose.

Chemical synthesis would involve, at its simplest, purchasing 2,3,4,6-Tetra-O-benzyl-D- glucopyranose with benzyl protecting groups already positioned around the side-groups that need protecting. 2,3,4,6-Tetra-O-benzyl-D-glucopyranose is the only available chemical we could find with protecting groups present and therefore chemical synthesis would be restricted to only α-D-glucopyranosyl-(1->5)-α-D-glucopyranose disaccharide. From Sigma Aldrich, the cost is:

• 1g of 2,3,4,6-Tetra-O-benzyl-D-glucopyranose = £29.80.
• In moles this is: 1g/540.650 = 0.00185mol.
• In pounds/moles (£/mol): £29.80/0.00185 = £16,108.11 per mol.

2,3,4,6-Tetra-O-benzyl-D-glucopyranose has a molecular weight of C₃₄H₃₆O₆, but not the whole structure is used in the bonding of the two glucosyl units together because the four benzyl units (C₂₄H₂₄) will need to be lost as the polysaccharide needs deprotecting, an integral part of the formation of the disaccharide. This loss now becomes a waste product. This waste product is extremely costly, and that is without even taking into consideration environmental risks and proper disposal (since benzene is believed to be carcinogenic). The molecular weight of C₂₄H₂₄ is 312.456g/mol, so loss in terms of money is:

• Take 1g of 2,3,4,6-Tetra-O-benzyl-D-glucopyranose = £29.80 again.
• In moles this is: 1g/312.456 = 0.00320mol.
• In pounds/moles (£/mol): £29.80/0.00320 = £9312.50p per mol lost as a waste product.

These calculations imply a loss of over half the amount in buying the chemical in the first place.

The loss of molecular weight as a waste product is one of the main reasons why many scientists are deterred from making even simple disaccharide. Synthesis of the starting material 2,3,4,6-Tetra-O-benzyl-D-glucopyranose is also incredibly complicated involving many steps, and is produced in low yields. In fact, construction of this simple disaccharide would actually be a racemic mixture of trisaccharides, tetrasaccharides and so on, decreasing in yield as the polymerised disaccharide repeat unit increases. This was also with a simple monosaccharide sugar unit. Imagine if the repeat unit we wanted was a trisaccharide or tetrasaccharide with completely different monosaccharides making up the repeat unit? How would this be polymerised? Both these questions have similarities: they are hugely expensive and hugely inefficient.

Biological synthesis, using inducibly activated enzymes on a single plasmid and the Wzy- dependent pathway, is much more flexible in producing the disaccharide glucopyranosyl-α- D-glucopyranose. This is because we have enzymes in GlycoBase that selectively add UDP- glucose to a glucose acceptor to form a 1->2, 1->3, 1->4 and 1->6 linkage. Taking the enzyme WaaR which forms glucopyranosyl-(1->2)-α-D-glucopyranose, the only thing that needs adding to the media is the inducer (in this example, we can say that WaaR is behind a pBAD/AraC promoter which is induced by L-arabinose). UDP-glucose is a very common diphosphonucleotide sugar which won’t need to be added as a substrate. Again, from Sigma Aldrich and working with the same calculations as before, the cost is:

• 25g of L-arabinose = £28.70
• In moles this is: 25g/180.16 = 0.13877mol
• In pounds/moles (£/mol): £28.70/0.13877 = £206.82p per mol.

The biological synthesis of glucopyranosyl-α-D-glucopyranose is over 75-fold cheaper than the chemical synthesis of the disaccharide.

Whilst the generation of the recombinant plasmid containing these inducible glycosyltransferase genes, in this project, was initially costly (>£10,000) and fermenter costs may raise this cost furthermore, the long-term benefits of this technology is hugely significant over the chemical synthesis of bespoke polysaccharides in terms of: cost, time, labour, yield of specific stereoisomer and length, generation of waste products and short-term flexibility of producing a wide range of polysaccharides.

Letters of Commendation

We met with Dr Timothy Atkins of DSTL, who believed our technology would “Take a large step forwards in translating scientific research into exploitable output”. His interest was particularly focussed on production of conjugate polysaccharide vaccines. A member of our team is meeting with the head of technical services at a national food producer to discuss the impact our products could have on the food industry. Thei interest was primarily in cyclodextrin. Cyclodextran has cholesterol reducing properties, but we could also potentially make anti-freeze xylomannan and food preservatives and flavourings for their products if we were able to overcome some of the food restrictions and food safety barriers.

Dr. Cliff Rush runs a company which specialises in bespoke peptide synthesis called ISCA Biochemicals Ltd. The potential of linking polysaccharide units to his peptides for customers would revolutionise his own company and is one of his current endeavours.

Other businesses from whom we have received letters of commendation include a veterinary surgery, aware of the impact that better surgical glue, vaccine programmes and drug delivery systems could have on their business, as well as the environmental impact that a cheap and easy to produce vaccine could have against tuberculosis which is a large problem in our local area. We continue to take meetings and correspondance with other companies in our local area and beyond.

Impact

This massive advantage of biological synthesis of polysaccharides over chemical synthesis and the technology that we are developing is more than likely going to impact on all levels, from chemical companies currently synthesising polysaccharides through to businesses where there is a need for bespoke polysaccharides. This could even be extended to the general public from incorporation of manufactured polysaccharides into food-stuffs to increase shelf-life, to changing blood groups to improve efficacy of blood transfusions (see Applications). Clearly our new system for the synthesis of bespoke polysaccharides will affect jobs, business opportunities and economy.

Funding for our project in the future will be pivotal and we look to remain at the University of Exeter when progressing with our technology beyond iGEM. This is for two reasons:

Firstly, the University of Exeter is renowned for funded impact-led research. UK research- intensive universities, including the University of Exeter, are scored periodically based upon quality of research. The ‘Research Excellence Framework’ (REF) which will succeed the ‘Research Assessment Exercise’ (RAE) in 2014, is undertaken by four UK funding bodies. Each assessment takes into account the quality of research over previous years and the results inform funding bodies to allocate funding to specific higher education institutions that scored highly. After our discussions with Dr. Matthew Baker and Dr. Teresa Penfield, we discovered that 20% of the REF score is through impact of research on society. The University of Exeter is notorious for impact-led research and we feel that by presenting case studies of the quality of research from the university, funding opportunities will be better secured. Matt and Teresa also pointed us to further funding opportunities provided by the ‘Developmental and Alumni Relations Office’ (DARO) which generates donations which is used to fund specific projects at the University of Exeter.

Secondly, the University of Exeter is able to fund intellectual property (IP rights). There is currently no technology like the one we are developing in the market and we are not aware of any research seeking to design a system for the synthesis of bespoke polysaccharides. We believe we have the freedom to operate because there are no existing patents to prevent our technology from expanding. The nature of iGEM and open source code means that all ideas of the project have to be disclosed. However, as discussed with Mr. Mark Kelly, the process can be disclosed but individual polysaccharides can then be patented. As a result of the conversations we have had with Mark, Matt and Teresa throughout the project, we plan to patent specific high-quality polysaccharides which will be extremely valuable in industry under the University of Exeter’s IP rights.

David Parker, from the oil company Shell, kindly met with us to discuss our business impact.

David drew our attention to another way of producing our polysaccharides rather than having to produce and then isolate them from inside our E coli, he suggested that we could actually use the bacterium to produce the glycosyltranferase enzymes and then isolate these from the E coli. If stored and handled properly the enzymes would last a couple weeks and could be used to produce the polysaccharides in a test tube extracellularly and so avoid risking damaging the polysaccharides in extraction from the cells. This could also overcome some of the stigma surrounding GM products since the polysaccharides are not being made directly by the E coli but by their enzymes, which many products we already use e.g. detergents and beer!

For new products on the market, consumer habits need to be changed. Companies will not necessarily use what is the best product for them but what they are most familiar with and it’s the consumer’s attitude that we need to change. For our technology there is no competitor, nothing to compare our technology to, so we are filling a niche, but we still need to demonstrate that our products work and are worth investing in. This also involves calculating the market value of our product and pitching our product at the right size and price. This made us think carefully about where our product could be utilised and consider more carefully any GM restrictions in these sectors, particularly in the food industry and how these could be overcome. We also considered how our technology could be scaled up for mass-production and thought through the pros and cons of each.

Bespoke Polysaccharide Manufacture

Our system for the production of polysaccharides is completely bespoke, giving the user a choice in creating any polysaccharide they would like to make. The manufacture of common and known polysaccharides using our technology will clearly be an easier and an alternative route for companies. For example, the cyclical cyclodextrin polysaccharides are well known for depleting cholesterol levels and are additives in a number of food products. Cyclodextrins exist in three forms: α-, β- and ɣ-forms with 6, 7 and 8 membered rings respectively. Whilst in this project we are producing α-cyclodextrin, we are easily able with the Wzy-dependent system to produce both β and ɣ-cyclodextrins if required. We know that companies are interested in our technology for the production of all three of these cyclodextrins because we have had interest from large-scale food businesses such as Ginsters, the UK’s biggest selling pasty maker that also specialises in other savoury products.

However, we believe that the real attraction that businesses will find with our technology is the production of novel polysaccharides to elicit certain physicochemical properties, but also other polysaccharides which cannot yet be made due to barriers in understanding its synthesis. For example, xylomannan is a recently discovered polysaccharide that protects Upis ceramboides (Alaskan beetle) from freezing at temperatures as low as -60^oC. Unfortunately, the genome for U. ceramboides has not been sequenced and prevents any potential for enzymatic synthesis of this valuable polysaccharide. However, using our technology, the possibility of producing xylomannan becomes a reality. The user will be able to choose glycosyltransferases from a variety of organisms to remove this inhibitory barrier and to produce xylomannan. In fact, similar but different polysaccharides could be produced very easily based on the locations of hydrophilic and hydrophobic side-groups giving xylomannan’s unique physicochemical anti-freeze properties.

In theory, specifically desired physicochemical properties would lead to the production of completely new polysaccharides not found in nature. We believe that bespoke polysaccharide manufacture is an innovative technology and after the interest we have had from companies through letters of commendation, the interviews we have had with academics and external persons, and holding a Human Practices panel, this will revolutionise glycobiology and manufacturing.