13TSB_SynBio A synthetic biology-based approach to engineering triterpenoid saponins and optimisation for industrial applications

Saponins have already found industrial application as foaming agents in the beverage, food and cosmetics industries. However, the breadth of triterpene saponin structures has not been fully explored or exploited, and their application limited by accessibility and availability from plant extracts, depite broad-ranging physical and bioactive properties documented in the literature. This project will demonstrate the optimisation of triterpene saponin structures for efficacy and functionality for applications in the chemical industries. Saponin derivatives will be engineered using a synthetic biology tool kit and metabolic pathway expression in a Nicotiana benthamiana host. Extracted saponins will be evaluated for physical properties and in application screens. Structure-function rules will be built to iteratively optimise the saponin structures for functionality. The options for a biotechnology platform, scale-up and commercial production of the lead saponins will be assessed.

This project will systematically explore the structural diversity of triterpenoid saponins for greater efficacy and new applications in the chemical industries. The project will generate (a) known and novel triterpenoid saponins, (b) data driven models of the structural basis of saponin functionality of industrial relevance and (c) a business case and an assessment of potential routes to commercial production for lead saponins from the programme
(a)The project will use a synthetic biology tool kit for triterpene engineering that is proprietary to the John Innes Centre. The JIC tool kit includes gene sequences encoding (i) a collection of triterpene synthases to generate various triterpene scaffolds and (ii) downstream tailoring enzymes which modify the basic scaffold, including glycosyl transferases, acyl transferases and oxidases, as well as rapid and effective methods for triterpene production in planta using synthetic biology approaches for triterpene pathway construction and transient expression systems. The project will also develop new tools for scaffold tailoring, specifically by the isolation and characterisation of additional glycosyl transferases
(b) Saponins will be extracted and purified from transformed plant leaves and chemically analysed by TLC, GC-/LC-MS, and, as appropriate, by NMR. Sufficient quantities will be generated for physical characterisations (surface and interfacial tensions, foam quality, quantitity and longevity, zeta potential), formulation studies (including phase behaviour) and the application of specific, proprietary evaluations including performance screens and bioactivity assays. The data will be used to generate structure-function models, which will be used to iteratively optimise the engineered saponin structures for key functional attributes. Further structure-function insights will be generated through chemical derivatisatisation.
c) The feasibility of commercial production of lead saponins will be investigated.

Synthetic biology offers huge potential benefits when applied in plants, from producing high-value chemicals and superior polymers for industry and ensuring food security through crop improvement (e.g. the recent funded synthetic biology projects at JIC on engineering the first steps for nitrogen fixation into wheat led by Professor Giles Oldroyd and on metabolic engineering of wheat for resistance to take-all disease, led by the JIC PI on this proposal, Professor Anne Osbourn), to production of vaccines for medicine. As an example of the latter, the CPMV-HT synthetic biology technology developed at the JIC has already been used to produce a vaccine for the H5N1 avian flu virus.
The output of the research will potentially replace non-sustainable oil based material with biologically derived chemicals that can be environmentally sustained with an enhanced carbon footprint. There is a wide range of potential uses for these bio-synthesised chemical.
The project, through the integration of academic and industrial research and disciplines, will provide excellent training for the PDRA appointed to this position.

It will be important for scientists to take the opportunity to 'frame' the debate around synthetic biology at an early stage as was arguably failed to achieve by scientists working on GM in earlier decades. Professor Osbourn, has made significant contributions in this area. In June 2012 she organised a New Phytologist international synthetic biology workshop aimed at enhancing awareness of synthetic biology in the plant community (see workshop report - Osbourn et al. 2012, New Phytologist 196: 671). Videos of presentations from this workshop can be viewed online at http://www.newphytologist.org/synthetic. Synthetic biology encourages scientists to work together with others to identify grand challenges faced by society and to collectively find solutions. To do this effectively it is essential that there is meaningful and productive engagement between scientists who engage in synthetic biology and the wider public. Professor Osbourn has established an educational initiative - the Science, Art and Writing (SAW) Initiative - that makes science accessible to school children and adults alike (see www.sawtrust.org). SAW projects have already taken place on the theme of synthetic biology in local primary schools by the University of East Anglia iGEM team (see http://www.youtube.com/watch?v=5e7Ro1R0J1g). JIC will continue to develop SAW as a mechanism for making synthetic biology more accessible to the interested public.