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

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description In this work we have developed metabolic pathways for menthol, providing biocatalysis and synthetic biology routes to menthol and related monoterpenoids found in Mentha (mint) essential oils. The era of synthetic biology heralds in a new, more "green" approach to fine chemical and pharmaceutical drug production. Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell.
We have developed a one-pot (bio)synthesis of menthol and neomenthol using recombinant Escherichia coli extracts containing the biosynthetic genes. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds.We also developed a chemoenzymatic approach providing access to all four intermediates in the peppermint biosynthetic pathway between limonene and menthone/isomenthone, including a number of non commercially available intermediates. We have complemented our experimental work with the implementation of machine learning algorithms to model the bacterial ribosome binding site sequence-phenotype relationship from representative subsets of large combinatorial bacterial ribosome binding site libraries allowing the accurate prediction of optimal high-producers. This methodology is potentially compatible with any biochemical pathway and provides a powerful tool toward predictive design of bacterial production chassis. Ultimately, synthetic biology approaches to microbial monoterpenoid production may revolutionize "natural" compound formation.
Exploitation Route Patent filing for menthol production; translational work is being pursued to develop a production platform for this group of chemicals
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description Follow on funding for commercialisation being pursued and the experimental techniques disseminated to the industrial biotechnology community.
First Year Of Impact 2015
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Economic

 
Description GSK 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Provided expertise, research time and equipment.
Collaborator Contribution Provided expertise and funding.
Impact Synthetic biology methods for production of fine chemicals.
Start Year 2011
 
Description MIB Open Day Stands/Tours 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact At Institute Open Day members of research group presented exhibits on topics of enzyme catalysis, synthetic biology, light activated biology and 'proteins' in general. Also demonstrated use of laboratory equipment on lab-tours run for attending students. Event was well received by both students and their teachers and seemed to inspire interest in the subject.

No defined impacts realised to date
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description NSEW Science Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Members of research group presented exhibit on topics of enzymes and proteins. Event was well received by both students and their teachers and seemed to inspire interest in the subject.
Additional stand on SynBio for flavours and fragrances hosted by the iGEM team (2016)

No defined impacts realised to date
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description Science Spectacular 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Members of research group presented exhibit on topics of enzymes and proteins. Event was well received by both students and their teachers and seemed to inspire interest in the subject.

No defined impacts realised to date
Year(s) Of Engagement Activity 2013,2014,2015,2016