13TSB_SynBio - production of chitosan in microorganisms

Chitosan is a natural polysaccharide consisting of many molecules of the sugar glucosamine, linked together in a chain. Chitosan has many important agricultural, industrial and biomedical applications, including use to promote blood clotting in wound dressings. Chitosan is normally made by chemical treatment of the shells of crabs and other crustaceans, which contain a similar polysaccharide, chitin, a chain of molecules of the sugar N-acetylglucosamine. Chemical treatment of chitin results in conversion of the N-acetylglucosamine to glucosamine, giving chitosan.

The aim of this project is to free chitosan production from its dependence on marine sources by making a microorganism which can produce chitosan from renewable plant-based materials. Most microorganisms naturally produce N-acetylglucosamine and incorporate it into their cell walls. In the case of fungi, the cell wall is largely made of chitin, but this is not currently an economical source of material for chitosan production, since cell walls are only a small part of the total mass of fungi. Many bacteria also produce polysaccharides made of glucosamine or N-acetylglucosamine, which they use to attach to surfaces.

In this project, we aim to use the techniques of synthetic biology to generate an artificial metabolic pathway to produce large amounts of chitosan. We will assemble sets of genes from fungi and bacteria which encode enzymes which, starting with sugars derived from plant biomass, can synthesise large amounts of the activated form of N-acetylglucosamine, assemble N-acetylglucosamine into long chains, secrete the chains from the cell, and convert the N-acetylglucosamine to glucosamine, giving a product similar to the chitosan derived from crustacean shells. We will assemble many such pathways and select the best for further development. We will then test different growth conditions to determine the best conditions to maximize chitosan production, and test these conditions on a larger scale.

By this means, we hope to develop a novel process for chitosan production which does not rely on marine resources. This project will also lay a solid foundation for the development of similar processes to produce other important polysaccharides with industrial and biomedical applications.

Chitosan is a polymer of beta-1,4-linked glucosamine residues with many useful applications. It is currently prepared by deacetylation of chitin extracted from crustacean shells. The aim of this project is to develop a recombinant microorganism which can produce chitosan from sugars obtained from biomass. Fungal cell walls contain chitin, synthesized by chitin synthase, and deacetylases operating on chitin are known. Furthermore, many bacteria produce cellulose, a glucose polymer produced in the same way as chitin by a related enzyme, as well as extracellular poly-N-acetylglucosamine, a component of biofilms.

In this project, we aim to use the techniques of synthetic biology to generate an artificial metabolic pathway to produce large amounts of chitosan. We will assemble sets of genes from fungi and bacteria which encode enzymes which, starting with sugars derived from plant biomass, can synthesise large amounts of the UDP-N-acetylglucosamine, assemble N-acetylglucosamine into long chains, secrete the chains from the cell, and convert the N-acetylglucosamine to glucosamine, giving a product similar to the chitosan derived from crustacean shells. Using the combinatorial DNA assembly technology of Genabler, we will assemble many such pathways and select the best for further development. We will then test different growth conditions to determine the best conditions to maximize chitosan production, and test these conditions on a larger scale.

By this means, we hope to develop a novel process for chitosan production which does not rely on marine resources. This project will also lay a solid foundation for the development of similar processes to produce other important polysaccharides with industrial and biomedical applications.

Major impacts of this project will include the following:

1-Unilever will benefit from a new process to manufacture chitosan and related polymers from renewable plant-based resources, rather than relying on marine resources (the shells of crabs and other crustaceans). This supports Theme 2 of the UK Roadmap for Synthetic Biology, 'Continuing responsible research and innovation', as well as Theme 3, 'Developing technology for commercial use'.

2-Genabler, and the synthetic biology community, will benefit from the opportunity for a thorough assessment of the GSA technology in the context of an industrial project. This will highlight the benefits of the GSA technology, and also indicate any areas where technology improvements are required. Since rapid combinatorial assembly of modular DNA parts is a key enabling technology for synthetic biology, this supports Theme 1 of the UK Roadmap for Synthetic Biology, 'Foundational science and engineering'.

3-the synthetic biology and biomedical communities will benefit from a clear demonstration of the application of synthetic biology techniques to the production of polysaccharides tailored to particular applications. The techniques developed in this project can easily be applied to the improved production of both known and novel polysaccharides, with a wide variety of applications. Again this supports Theme 3 of the UK Roadmap for Synthetic Biology, 'Developing technology for commercial use', as well as Theme 4, 'Applications and markets'.