Innovative Routes to Monoterpene Hydrocarbons and Their High Value Derivatives

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

Propelling chemicals/natural products production towards 'green' and more sustainable manufacturing processes will require the harnessing of the power of Synthetic Biology (SynBio) and integration with more traditional biocatalytic and chemo-catalytic processes. SynBio is a potentially disruptive technology, but major de-risking is required before the SynBio of chemicals manufacture can replace traditional manufacturing processes. In part, this is reflected in the major costs associated with the development of new manufacturing plant in the industrial sector, which is required to transition from conventional petrochemical/natural resource-based manufacturing to SynBio-based chemicals manufacturing.

In this programme we will design, build and integrate new SynBio technology that accelerates delivery of bespoke SynBio solutions for chemicals/natural products synthesis. Our focus is on a major group of industrially valuable compounds, the monoterpenoids, which offer new opportunities for bio-based production using SynBio. By adopting modular 'plug-and-play' platform approaches and a production pipeline that embraces the 'design-build-test-deploy' life-cycle we will turn knowledge assets into innovative chemicals production solutions for monoterpenoid compounds. We will recruit and design bespoke biological parts (e.g. enzyme catalysts; transporters; membrane components) and assemble them in novel ways to create a bio-based production pipeline contained within a synthetic, engineered microbial biofactory. Iterative design-build-test-deploy life-cycles will be used to optimise production of monoterpenoid chemicals in defined proof-of-principle projects. Using these Demonstrator Projects, we will deliver innovative SynBio approaches for chemicals/natural products biosynthesis and promote leading capability development that will stimulate interactions with industry and other stakeholders. These projects will require the unique, multidisciplinary environment (including bioscience, engineering, chemistry and computational science) and collaborative working culture represented by the team of applicants.

Enzyme-catalysed SynBio-based 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, as is the case in the production and use of monoterpenoids. The SynBio approaches we develop will also generate new chemical entities not readily accessible using traditional synthetic approaches. This will provide new opportunities for industrial exploitation, including the synthesis of new chemical libraries that will support industrial and academic drug discovery programmes. More broadly, the programme will provide general tools, technology platforms and SynBio 'know-how' that will impact widely in sustainable manufacture of chemicals and natural products and de-risk SynBio approaches for more rapid take-up and development by the industrial sector.

Technical Summary

Terpene cyclases/synthases (TC/S) catalyse the most complex reactions in biology and are the entry points to the most structurally and stereochemically diverse group of natural products, the terpenoids. Despite its rich chemical diversity, the terpenome is derived from common metabolites through a series of complex reactions initiated by the TC/S enzyme family. This application is focussed on cyclic/acyclic monoterpene hydrocarbons and their high value derivatives (monoterpenoids). These compounds are found in plant extracts and essential oils, and can be derived from petrochemical feedstocks. They are synthesised through the activity of the large TC/S family, which catalyse 'high energy' cyclisation reactions involving unstable carbocation intermediates.

Through plug-and-play engineering we will link knowledge of TC/S chemistry with metabolic engineering to create novel biosynthetic pathways for important monoterpenoids. We will generate tools and knowledge to develop robust microbial strains for monoterpenoid biosynthesis. We will integrate expertise across several knowledge areas including: enzyme/pathway building block discovery; design and engineering for creation of a library of parts; refactoring of novel metabolic pathways; genome-scale metabolic modelling and experimental pathway validation; chassis engineering and end-product yield optimisation. A feature of the programme is the need to integrate knowledge of enzyme mechanism with bio/chemo-catalysis, systems biology (SysBio) and SynBio, including pathway/chassis engineering.

Our vision is to propel the production of monoterpenoids towards 'green' and sustainable manufacturing by harnessing the power of SynBio and linking this to detailed understanding of TC/S chemistry. We will provide essential tools, resources and basic science 'know-how' that de-risks the application of SynBio to monterpenoid production and open up SynBio routes to synthesis of the chemically diverse monoterpenome.

Planned Impact

Global trends are driving an increasing demand for bio-based chemical production. With the historic volatility of global oil prices, limited and seasonal variation in natural resources, global pollution, consumers becoming eco-aware and desiring to live more sustainably, governments and companies are acting to implement policies/processes to reduce global dependence on existing natural/oil-based products and transition to more sustainable ways of living. This programme is at the heart of this agenda developing bio-based SynBio technology for chemicals manufacture of commercially important monoterpenoids.
Benefits from the research:
(i) People. We will train a new generation of scientists at all levels who can work across discipline boundaries, harnessing expertise in predictive design and the SynBio of chemicals biosynthesis. This will capture innovative approaches being developed in chemo/bio-catalysis process design, SynBio, experimental and computational SysBio, and knowledge-based biocatalyst/component design fields. An ability to integrate skills from these different research areas to develop novel bio-catalytic solutions for industry will be a major part of training. We will enable training and delivery of project milestones in laboratory environments built to facilitate working across biological and chemical disciplines, supported by strengths in computation, biophysics and mechanistic chemistry, structural biology, SynBio and SysBio. Training in platform technologies and Responsible Innovation will produce staff who will become SynBio ambassadors.
(ii) Knowledge. Bio-based chemical synthesis through IB and SynBio has the potential to offer powerful solutions to major societal challenges such as health, energy supply and sustainable manufacture. This will be driven through integration of biological and chemical processes and major changes to chemical production for agriculture, pharmaceuticals, and bulk chemicals manufacturing across a range of sectors. These are burgeoning economies, restricted in scope through our ability to design/re-profile enzyme catalysts, requiring innovative chemo-/bio-catalytic solutions to processing and the coupling of chemo- and bio-catalysis through SynBio approaches to facilitate novel chemical transformations. These new developments will drive wealth creation in chemicals manufacturing and its allied economies. There is strong potential for inward investment from industrial stakeholders, and the possibility of technology licensing/spin out/spin in opportunities for technologies developed in the research programme.
(iii) Economy. The programme is positioned predominantly in the TRLs 1-3 space, and our mission will be to accelerate commercialisation with our partners in a 'market-focused approach' by driving the technology we develop towards the higher TRLs. We have identified goals and strategies to achieve these aims and a number of mechanisms for attaining targets/deliverables. Early IP targets will be focused on new tools for SynBio, including development of novel building blocks/components for redesign, and the engineering of novel biocatalysts, regulatory elements and chassis. Industry will benefit from these tools. Novel processes for chemicals/natural products synthesis based on monoterpene scaffolds will emerge, which will foster economic competitiveness in the UK in the area of chemicals manufacture. The work will impact quality of life by adopting sustainable, green processes for manufacture and provide tools/resources more broadly for the chemicals manufacturing industry over the 5 years of the award.
The Responsible Innovation elements of our programme will include real-time assessment and anticipation of research and innovation trajectories, deliberation and reflection, and collaborative development. This will impact by informing publics, policy-makers and wider stakeholders about the impact and benefits/limitations of SynBio in chemicals manufacture.

Publications

10 25 50

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Arcus VL (2020) Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates. in Annual review of biophysics

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Ascue Avalos GA (2019) From Bugs to Bioplastics: Total (+)-Dihydrocarvide Biosynthesis by Engineered Escherichia coli. in Chembiochem : a European journal of chemical biology

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Bunzel HA (2019) Emergence of a Negative Activation Heat Capacity during Evolution of a Designed Enzyme. in Journal of the American Chemical Society

 
Description A synthetic biology platform for the production of diverse monoterpenoids in Escherichia coli was established through introduction of a heterologous isoprenoid production pathway, subsequently we employed a library of different plant monoterpene synthases in a single 'plug and play' platform system for the production of over 30 different linear, monocyclic, and bicyclic industrially important monoterpenoid scaffolds in E. coli via fermentation on simple sugars. Due to their highly branched reaction mechanism, many monoterpene synthases show a high level of product promiscuity resulting in product mixtures rather than commercially viable pure products. In this work, several plasticity regions were identified in the plant monoterpene synthase family that allow for tuning of product profiles towards alternative products. Variants with alternative product profiles have been created for different monoterpene synthase enzymes with increasingly complex cyclisation cascades. Bacteria are also known to produce terpenoids; and previously two bacterial monoterpene synthases were identified in the soil bacterium Streptomyces clavuligerus. Insertion of these two enzymes in our monoterpenoid production platform yielded both higher product titres as well as fewer by-products. We have solved the crystal structures of these two enzymes, and via a combination of experimental and computational work we were able to understand the nature of the reaction mechanism of these bacterial enzymes that results in lower levels of product promiscuity. These results provide a general basis to guide rational engineering of monoterpene synthases and is an important step towards the predictable and tuneable engineering of terpene synthases for efficient production of desired terpenoids. In addition, using genome mining tools, we have discovered several novel bacterial terpene synthases, including a number of enzymes showing dual mono- and sesquiterpene synthase activity, suggesting that bacteria are a potentially much larger source of monoterpene synthase activity than previously assumed. Further work included the development of a cytochrome P450 toolbox for the oxy-functionalisation of the monoterpenoid scaffolds, increasing the chemical diversity and value of the products; modelling of limonene production in our modified E. coli strain, allowing the identification of bottlenecks in the metabolic process; and the development of an automated pipeline for the screening of diverse monoterpenoids allowing the rapid identification of desired products in large variant libraries for natural product production in synthetic biology programs.
Exploitation Route The synthetic biology platforms developed will be used in further research and eventually converted to industrial applications.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The prototype engineered E. coli strains resulting from this project underpin two patent applications for production of a specific terpene. The strains have been further developed in early translational programs with US and other partners in a consortium formed by C3 biotech (maritime & aerospace ltd). In addition, transition funding for large scale production has been obtained from UK and US defence sector. Expansion of the research programme is planned through joint US-UK funding in 2022.
First Year Of Impact 2020
Sector Aerospace, Defence and Marine,Chemicals,Energy,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Northern Powerhouse
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a national consultation
 
Description UK Fuels
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description Future Biomanufacturing Research Hub
Amount £10,284,509 (GBP)
Funding ID EP/S01778X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 03/2026
 
Description IAA Award - AIBLHiCoS: a novel pKa predictor
Amount £60,175 (GBP)
Funding ID BB/S506692/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 07/2019
 
Description ONRG- Optogenetic fuel production
Amount £193,615 (GBP)
Funding ID N62909-18-1-2137 
Organisation ONRG Office of Naval Research Global 
Sector Public
Country United States
Start 06/2018 
End 09/2021
 
Title Production strain for terpene fuel 
Description We developed a production strain for production of terpene fuel. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Translated to industrial applications through C3Biotech and IP registration 
 
Description Office of Naval Research Global 
Organisation ONRG Office of Naval Research Global
Country United States 
Sector Public 
PI Contribution Contribution of knowledge and materials for synthetic biology of fuels production
Collaborator Contribution Synthetic chemistry for advanced fuels
Impact Creation of ongoing research consortium in area of advanced fuels
Start Year 2017
 
Title Monoterpenoid biosynthesis 
Description This follows on from the early patenting of the biosynthetic routes to menthol and the second patent protects the identity of a key evolved enzyme derived from keto steroid isomerase. 
IP Reference GB1719530.6 
Protection Patent granted
Year Protection Granted 2017
Licensed No
Impact None as yet.
 
Title PROCESS OF PRODUCING MONOTERPENES 
Description The present invention relates to a process of producing a monoterpene and/or derivatives thereof. The process comprises the steps of: a) providing a host microorganism genetically engineered to express a bacterial monoterpene synthase (mTS); and b) contacting geranyl pyrophosphate (GPP) with said bacterial mTS to produce said monoterpene and/or derivatives thereof. The present invention also relates to a microorganism for use in producing a monoterpene and/ or derivatives thereof and a recombinant microorganism adapted to conduct the step of converting geranyl pyrophosphate (GPP) into a monoterpene and/or derivatives thereof by expression of a bacterial mTS. It was shown to produce 1,8 cineole using 1,8 cineole synthase and to produce linalool using linalool synthase, both from Streptomyces clavuligerus. 
IP Reference WO2018142109 
Protection Patent granted
Year Protection Granted 2018
Licensed No
Impact n/a
 
Company Name C3 Biotechnologies Ltd 
Description A licensing company for technology that enables production of bio-propane 
Year Established 2015 
Impact None yet - too early
 
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 2012,2013,2014,2015,2016,2017,2018
 
Description New Scientist Live 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A stand on a stand on DNA and Synthetic Biology of Fragrances at the New Scientist Live which is an award-winning, mind-blowing festival of ideas and discoveries for everyone curious about science and why it matters. For four days in September, it transforms London, the world's most exciting capital city, into the most exciting place in the universe. More than 140 speakers and 100 exhibitors come together in one venue to create an unrivalled atmosphere and energy, packed with thought-provoking talks, ground-breaking discoveries, interactive experiences, workshops and performances.
Year(s) Of Engagement Activity 2018
URL https://live.newscientist.com/
 
Description RI Talk - fuels of the future 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Manchester University Alumni Event: Hear from our panel of leading academics from the Manchester Institute of Biotechnology as they explore the challenges of developing the fuels of the future, the solutions we are delivering and why Manchester is leading the world in industrial biotechnology.

One of the main challenges our society faces is the dwindling level of oil reserves which we not only depend upon for transport fuels, but also plastics, lubricants and a wide range of petrochemicals. As the 21st century progresses, and we move towards more bio-based economies, we need solutions for the manufacture of chemicals that are smarter, more predictable and more sustainable.

From underpinning strategic research to the transfer of technology into the marketplace, The University of Manchester has a range of worldclass activities supporting the need for solutions that can play their part in meeting the global energy challenge.

Just as Manchester was at the heart of the Industrial Revolution, The University of Manchester is now leading the way, both nationally and across Europe, towards a bio-industrial revolution.

Panel: Professor Nigel Scrutton (Chair), Professor of Enzymology and Biophysical Chemistry, Professor David Leys, Professor of Structural Biology, Professor Eriko Takano, Professor of Synthetic Biology
Venue: The Royal Institution of Great Britain, London
Year(s) Of Engagement Activity 2018