Team:Exeter/Human Practices/impact

Biological vs. Chemical Synthesis

The synthesis of polysaccharides is incredibly difficult chemically. This difficulty is the result of the requirement for protection reactions for all the reactive side-groups except the side-group intended for the specific linkage. Subsequently, de-protection reactions are also needed to remove these protecting groups. This is a laborious process with high costs. Moreover, yield is decreased as the repeat unit is polymerised and high amounts of waste products are generated. Biological synthesis of polysaccharides using glycosyltransferases could be a much easier approach to polysaccharide production. Enzymes are extremely specific. 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 may lower costs, reduce waste products and result in high yields of the correct stereoisomer.

Our discussions with Dr. Mark Wood and querying of a number of research papers (including Crich et al, 2004) led us to a simple example to demonstrate how biological synthesis may be a better approach than chemical synthesis 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, as of the 5^th September 2012, 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 only part of the structure is used in the bonding of the two glucosyl units because the four benzyl units (C₂₄H₂₄) will be lost as the polysaccharide needs deprotecting, an integral part of the formation of the disaccharide. This loss now becomes a waste product and is extremely costly. 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. 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, as of the 5^th September 2012, 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.

What does industry think of our idea?

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.

We also met with the head of technical services at a national food producer to discuss the impact our products could have on the food industry. Their 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.

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.

Dr David Parker, from Shell, also kindly met with us to discuss our business impact. Dr Parker 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 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!

Our discussion with Dr Parker also highlighted the fact that 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. 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.

Impact

The aim of our system is to enable bespoke production of polysaccharides, 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.

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.

Conclusions

In theory, specifically desired physicochemical properties would lead to the production of completely new polysaccharides not found in nature. We feel 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 the feedback we had from our Human Practices panel, we believe this technology will revolutionise glycobiology and manufacturing.

Our project has evolved through our various meetings and the human practice panel to incorporate all human practice considerations. If our E coli or polysaccharide were to be accidentally or deliberately released, it would cause very little harm to the environemnt or public health. The benefits our system would have on every business sector has been recognised and outweighs any potential risk.