13TSB_SynBio - production of chitosan in microorganisms

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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'.

Publications

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Description We have developed a process using microorganisms to produce chitosan, a valuable polymer with many applications in food, personal products, medicine, agriculture, and other areas. Chitosan is normally manufactured from the shells of shrimps and crabs. We hope that our process will allow it to be produced from plant-based materials. Modifications to our engineered pathway may also allow the production of variants with different and useful properties.
Exploitation Route Following further analysis of samples, Unilever has decided not to pursue this process. We are therefore preparing two manuscripts for publication describing our results, and will then seek other industrial partners with an interest in functional polysaccharides. Chitosan and related molecules have many potential applications in the food and pharmaceutical sectors, and our recombinant platform may offer the potential for production of modified products with improved properties.
Addendum, 1 March 2020: we have become aware of a new industrially linked project involving University of Edinburgh researchers to produce chitosan. We will supply them with our chitosan deacetylase clones and assay protocols to assist in their project. This may open new opportunities for collaboration and further development of this project.
Sectors Agriculture

Food and Drink

Chemicals

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Unilever, our industrial partner, has decided not to pursue this approach, but their activity in this area has been informed by our results. We are therefore preparing a publication describing the results, which we hope to submit later this year, and will seek other opportunities for further development. Addendum, 1 March 2020: we have become aware of a new project involving University of Edinburgh researchers and an industrial partner for chitosan production via a diferent route. We will supply them with our chitin deacetylase constructs and assay protocols. This may advance their project and offer new opportunities for collaboration and further development of our project. We have also recently been contacted by researchers at Northumbria University associated with the Hub for Biotechnology in the Built Environment (http://bbe.ac.uk/) funded by Research England, who are also keen to pursue chitosan production as part of their materials program, and we hope this may lead to a new collaboration. I haven't listed either of these under 'Collaborations' as there are no formal collaborations in place yet, but I am hopeful that we will soon be able to move the project forward.
First Year Of Impact 2016
Sector Manufacturing, including Industrial Biotechology
 
Description Further development of chitosan production process 
Organisation BioTangents
Country United Kingdom 
Sector Private 
PI Contribution We are providing advice to Biotangents, an SME which is continuing research with Unilever to determine whether a recombinant chitosan production process is feasible. Note added March 2020: Biotangents is moving in different directions (I am still collaborating with them) and is now focused on veterinary diagnostics. It now seems unlikely that they will want to pursue chitosan production in the foreseeable future.
Collaborator Contribution Unilver is conducting structural and functional assays on material generated by BioTangents using the original GMO produced in this research.
Impact Internal confidential reports.
Start Year 2015
 
Description Further development of chitosan production process 
Organisation Unilever
Department Unilever Research and Development
Country United Kingdom 
Sector Private 
PI Contribution We are providing advice to Biotangents, an SME which is continuing research with Unilever to determine whether a recombinant chitosan production process is feasible. Note added March 2020: Biotangents is moving in different directions (I am still collaborating with them) and is now focused on veterinary diagnostics. It now seems unlikely that they will want to pursue chitosan production in the foreseeable future.
Collaborator Contribution Unilver is conducting structural and functional assays on material generated by BioTangents using the original GMO produced in this research.
Impact Internal confidential reports.
Start Year 2015