Biotransforming Phenylpropanoids derived from Biorefining: a Toolkit Approach

The use of plants as renewable feedstocks to replace petrochemicals requires similar levels of efficiency in terms of refining and recovery of useful products. In biofuel production, which is currently the most advanced refining applied to recover platform chemical from plant biomass, a considerable amount of material is non-fermentable and remains as a low value by-product at the end of the process. Fractionating this residue and adding value to its components would enhance the efficiency economics and ultimately sustainability of biorefining. One relatively abundant and easily recoverable chemical in the non-fermented material is the secondary metabolite ferulic acid, a phenylpropanoid (PP) used extensively in plants for making biologically active polyphenolic compounds. In this project we propose to use ferulic acid and other PPs as precursors to produce dihydrochalcones and their glycosylated derivatives through fermentation in metabolically engineered yeast. These dihydrochalcones are widely used as artificial sweetners and flavour masking agents in the food industry and are currently made by non-chemical transformation methods. In our process, we will reconstruct pathways which function to produce polyphenolics in plants and engineer them into yeast, which on feeding with PPs will then produce these artificial flavouring agents through a sustainable biological process. As artificial flavouring agents, the use of genetic modification to produce these compounds will not affect their market value, as they are not 'natural' products. Furthermore, the modular assembly of our engineered pathway and the feeding of specific PP pre-cursors gives us the potential to produce novel dihydrochalcones which have the potential to be developed into new flavour enhancing products for the food industry. The programme therefore will therefore add value to existing fermentation processes which utilise plant material. In addition the process would also be highly compatible with processes aimed at deconstructing lignin, which is a major non-fermentable product derived from woody biomass composed of PP intermediates. Thus the project is both of immediate utility and establishes useful technologies of longer term value to the biorefining industries which have identified lignin deconstruction and the use of the respective products as a major future commercial opportunity.

Phenylpropanoids (PPs) are metabolites found in all terrestrial plants which are used in nature to make a range of polyphenolic compounds, with dietary functions as flavourings and antioxidants. In this project, we propose to copy the pathways which exist in nature and take PPs left over as low value by products of biofuel and plant fibre production and biotransform them into high value polyphenolic intermediates of value to the food industry. The biosynthetic process proposed would be assembled in yeast , using enzyme pathways which can be assembled together in a modular manner to generate a wide range of end products from the limited range of PPs available from biorefining. To illustrate the technology, we will generate glycosylated dihydrochalcones which are 100s of time sweeter than sucrose and already extensively marketed as artificial flavour enhancers. While our process will use renewable feedstock and biological processing, currently the synthesis of such compounds requires non-sustainable chemical transformations. In terms of novelty, our programme has the capability of generating end products derived from PPs which have not been described in nature , but which based on chemical precedence would have a strong liklihood of being biologically active as flavourings or neutraceuticals. Therefore in addition to providing an alternative route to existing successful products, through using a modular approach for metabolic engineering we have the potential to generate new market leading products in the future. The proposed process involves a series of individual enzyme steps , each of which serves as a biotransformation module (BM), which can then be linked to the next biosynthetic step to produce the compounds of interest. These BMs will be co-ordinately expressed in different combinations using polyprotein expression technology, essentially enabling the directed reconstruction of plant secondary metabolic pathways in yeast.

The groupings who will benefit from the work have been identified as 1. UK industry, notably in companies developing biorefining and more broadly in the industrial biotechnology sector. By deriving high value products from waste streams, the biofuels companies would be able to add value to existing processes with minimal disruption. In addition, the production of high value fine chemicals by a biotechnological route has the potential to generate new businesses in fine chemicals production which could physically cluster with large scale fermentation. Importantly, as these food additives will be entering a market dominated by synthetic compounds, their origins through the genetic modification of yeast will not have the same negative connotations with end users or the regulatory bodies as would be the case if they were supplanting plant derived natural products. As part of a BBSRC industry club, the project would immediately benefit from the close links with its membership of private companies and dissemination agreement. The PI also has close links with the industrial sector outside the club and continues to work with the Bioscience knowledge transfer network . There is therefore excellent scope for the work to be brought to the attention of the private sector. 2. The region's economy, through the commercialisation of the process or the licensing of the patented technology to a third party. With other on-going projects, the programme offers a long term potential to establish a spin out in industrial biotechnology and fine chemicals manufacture to be located in the region. As an alternative strategy the work will be patented and the IP licensed to generate wealth for the University and region. 3. Schools and public engagement. The concept of the biofactory, using living cells to produce chemicals from sustainable sources is an inspirational example of the power of biotechnology and offers a simple entry level into the science for school teachers and children. Similarly, the message is also readily understandable to a 'science hungry' general public who want to find out more of the research work going on in the University. Dissemination to the schools will be via the teachers in clustered in Science Learning Centres and to the school children through Academies and the Science in to Schools programme where staff and students go out into local schools and through the COMPACT scheme where the children get experience of working in the University labs. Engagement with the general public is organized through a public lecture series and most recently through the Institute of Advanced Studies, which holds debating classes around contemporary issues. The PI is particularly interested in developing best practice in ELSIs and as such is a member of the cross research council oversight group for public dialogue on synthetic biology.