Industrial chemicals of the monoterpenoid class realised through synthetic biology and pathway engineering

Industrial biotechnology in chemicals manufacture is one of the key enabling technologies in the 21st Century. The European chemical industry is the largest in the world and biotechnology is poised to make a substantial impact on chemicals production (both bulk and fine chemicals) as we harness the biocatalytic potential of enzymes to develop green, innovative and competitive chemical processes. The manufacture of chiral molecules is a particular challenge: microorganisms are tremendously rich sources of compounds and metabolic pathways of use to the chemicals industry or as new pharmaceuticals. In this research programme we will develop new biocatalysts to synthesise an important group of organic compounds (the monoterpenoids) that have high commercial value in the food and fragrances industries. A serious obstacle to exploiting the most promising monoterpenoids is their limited availability from natural resources. Extraction and distillation of these molecules is expensive, low-yielding and requires substantial expenditure of natural resources. Seasonal variation in supply and environmental conditions also affect production. Formation of product racemic mixtures is also a major drawback in the manufacture of monoterpenoids by classical chemical synthesis. This can be mitigated by use of expensive chiral catalysts or by starting with materials from the chiral pool, but for terpenoids these building blocks are not available in sufficient abundance from natural resources. Enzyme-catalysed synthesis is attractive: regio- and stereo-selective biotransformation, use of 'green' reaction conditions and lack of toxic by-products combine to make biosynthesis attractive in sustainable manufacturing. US and European legislation acknowledges that products from biosynthetic manufacturing processes are considered 'natural'. This adds significant market value, especially in foods and flavourings manufacture. Identifying enzymatic routes to terpenoids is important because of (i) the requirement for 'natural products'; (ii) the inability to access them readily by chemical synthesis; (iii) environmental impact of classical synthesis; (iv) limited availability from natural resources.

Our work will unite knowledge-based application of new enzyme catalysts with synthetic biology to design new metabolic pathways to the monoterpenoids. We will generate pathways for monoterpenoid synthesis in whole cell synthetic microorganisms by genetic manipulation of E. coli strains to (i) upregulate enzymes in isoprenoid precursor biosynthesis and (ii) incorporate non-native genes to create novel pathways for monoterpenoid production. We will create several novel biosynthetic pathways, namely for the biosynthesis of (-)-limonene; (5R)-carvone and derivatives; (+)-pulegone; (+)-menthofuran; perillyl alcohol and derivatives; menthol compounds. We will generate novel E. coli strains (each containing upregulated isoprenoid precursor biosynthesis genes) into which additional heterologous/engineered genes encoding component pathway enzymes for monoterpene synthesis will be introduced. Strains will be optimised for enzyme expression, growth and monoterpenoid biosynthesis to enable efficient whole-cell biotransformation through systems-based analysis and modelling. Engineered strains will be used sequentially to generate monoterpenoids without need for enzyme isolation or exogenous addition of cofactor recycling systems. The programme will transform the industrial synthesis of commercially important monoterpenoids. It delivers on the BBSRC Knowledge Based Bio-Economy strategic priority and exploits synthetic/systems biology to underpin next generation biocatalysis for industry.

We will produce new bacterial strains with the capability of manufacturing monoterpenoid compounds, which are valuable industrial products. We will develop a synthetic biology programme in which we will engineer artificial pathways for monoterpenoids based on knowledge of the activities and structures of component enzymes (or their variants) that display desired catalytic properties. Our programme integrates synthetic biology and systems-based approaches, with biocatalysis and analysis of enzyme structures and mechanisms. We have expressed and purified a number of the required component enzymes. A number of these enzymes have been characterised in detail at the level of substrate biotransformation and kinetics, and structure determination, enabling us to define precisely the reactions catalysed. We are therefore in a strong position to take our monoterpenoid biocatalysis programme forward using synthetic biology approaches, underpinned by knowledge of component enzyme structure, mechanism and chemical specificity. The programme builds on our expertise with redox enzyme systems and offers green and simplified routes to monoterpenoids, thereby circumventing many of the limitations associated with current industrial processes that are (i) reliant on scarce natural resources, (ii) give rise to poorly controlled stereochemical outcomes and (iii) utilise toxic reagents. The work programme is strategically important in the industrial biocatalysis area and maps directly onto the KBBE strategy identified by BBSRC.

Beneficiaries: (i) The chemicals manufacturing industry - with improved biocatalytic/synthetic biology manufacturing processes for generating a variety of industrially important and high value monoterpenoids that mitigate risk associated with the limited supply of reagents from natural resources. 'Natural synthesis' avoids use of toxic reagents with consequent environmental benefits and high acceptability in the food and fragrance manufacturing industries. Biosynthesis avoids costly work-up and poor stereospecific control associated with manufacturing based on classical chemical synthesis. (ii) The pharmaceutical/drug development industry - our enzymatically synthesized limonene based products will provide bioactive monoterpenoid scaffolds with high enantiopurity. Expansion of enzyme substrate specificity will enable chemical elaboration of derived monoterpenoids in a controlled and predictable manner, facilitating exploration of new monoterpenoid structures in pharmaceutical development.

Exploitation: We anticipate that our newly designed biocatalytic strains will have commercial impact. Our strategy for translating the technology is to establish IP protection through UMIP (Manchester's IP office) and work in partnership with UMIP for exploitation. We anticipate IP protection at an early stage, with second generation and further IP protection occurring at additional stages later in the grant. Our strategy will be either to license the use of bespoke biocatalysts/strains, or spin out from Manchester a start-up based on this IP position. We will actively pursue follow up funding [ 'Manchester Fund' (a venture capital fund for Manchester based activities], UMIP proof-of-principle funding or BBSRC follow on funding. We have established collaborations with biocatalysis companies (e.g. Chirotech, GSK and partner companies of CoEBio3) with whom we will also explore licensing agreements. We communicate through networking events with external stakeholders (industry, other University groups, venture capital groups, policy groups) through structured workshops, showcase events and industry within MIB and associated Centres. We will use these events to extend the outreach of our work and to demonstrate the utility of our new biocatalysis design platform and the general impact it will have in the design of new industrially useful biocatalysts.

Outreach: We will take advantage of 'Discover days' hosted by Manchester to introduce school children to the science underpinning biocatalysis. We will write web pages on 'Biocatalysis: the new chemicals manufacturing industry' for the 'Children's University at Manchester'. We also host University open days for the general public and we will showcase the impact of biocatalysis and synthetic biology in manufacturing processes at these events. Scrutton lectures to school children at science focus meetings on enzymes. He has written short articles in 'Biological Science Reviews' - a magazine targeted at school children, and we will continue to write articles on the topic of catalysis. Café scientifique events are organised on campus and the team (appointed PDRAs) will engage in these events. We have contributed widely to making our work accessible to non-specialist audiences e.g. radio interviews, general science articles in popular science journals, contributed chapters to popular science books, assisting journalists in reporting in national press (e.g. Economist, Guardian) and hosting workshops and similar in the field. This emphasises our commitment to outreach activities to non-specialists.