Unlocking the potential of engineered C-C bond forming enzymes for biocatalysis
Lead Research Organisation:
University of Bristol
Department Name: Biochemistry
Abstract
The future success of the global pharmaceutical and chemical industries is dependent on the successful development of efficient, selective and sustainable ways of making new organic molecules with useful properties. Historically, such methods have centered on the use of synthetic organic chemistry to fuse and tailor simple chemical building blocks into a vast array of complex molecular architectures, which may in-turn be used as the basis for amongst other things new drugs, crop protection agents, or materials. Despite the undoubted success of synthetic chemistry, problems exist, including the over-reliance on certain types of reactions that have led to an excessive representation of molecules with predominantly flat, 2D architectures, which are of limited value as drugs. In contrast to chemical catalysts, biological catalysts, termed enzymes, are able to perform challenging chemical reactions that can rapidly build complex 3D chemical structures with multiple bonds under precise stereochemical control. In addition, enzymes can perform such reactions under ambient conditions and without any requirement for environmentally damaging reagents. For these reasons there is significant interest in developing biocatalytic routes to current and future pharmaceuticals and similarly important molecules. A carbon-carbon bond forming reaction known as the Diels-Alder reaction is an effective means of building complex 3D molecules in a single step. However, a limitation of this reaction is that to achieve high yields, stereoselectivity and regioselectivity, the electronic properties of the reactants need to be complementary and often harsh reaction conditions are required. This research project builds on our exciting recent discovery of a naturally evolved co-factor independent enzyme that catalyzes the Diels-Alder reaction at room temperature and on substrates which cannot be transformed using conventional synthetic organic chemistry. This discovery opens up the possibility of using this enzyme to generate a whole new series of complex molecules that could form the basis of new drugs or similarly important chemical compounds. During this project we will establish the practical and theoretical limits of the reactions that this and other related Diels-Alderases can catalyze, we will also rationally re-engineer these enzyme to purposefully change their function to allow access to an even greater variety of products. We will partner these engineered biocatalysts with auxiliary enzymes which catalyze further ring forming reactions to develop routes to industrially useful molecules. This project is a strategically important partnership between the University of Bristol and the pharmaceutical company AstraZeneca, and we will work together to develop natural and engineered enzymes and deploy them to generate a vast array of new 3D molecules that can be used as the basis for new drugs to treat a diverse array of human diseases.
Technical Summary
The Diels-Alder reaction, a [4+2] cycloaddition of a conjugated diene to a dienophile, is widely recognised as one of the cornerstone synthetic organic reactions of the 20th century. It is commonly employed in the synthesis of bioactive natural products and in the rapid construction of sp3 rich cyclic and polycyclic compounds. This reaction opened the way to the synthesis of numerous important pharmaceuticals including the anti-viral agents Tamiflu and Peramivir, the anti-fungal Tolciclate, and opiates including Morphine and the smoking cessation aid Varenicline. It has also been widely used in the preparation of vitamins, steroid hormones, agrochemicals (Isopyrazam, Bixafen, Cycocel), and numerous fragrance and flavour compounds. Despite its unquestionable usefulness, the versatility of this reaction is limited by its distinctive steric and electronic requirements, along with the harsh reaction conditions which often must be employed. By contrast, the development of protein catalysts for this reaction remains a major goal, as access to enzymes capable of catalyzing Diels-Alder reactions under ambient conditions and in the absence of co-factors, would enable new, green routes to a wide variety of valuable chemical building blocks, natural products and lead scaffolds. In this academic-industrial LINK project, which builds on substantive collaborative research undertaken by the academic PI and Co-Is in the identification and characterisation of natural Diels-Alderases and associated ring forming enzymes, the researchers will:(i) Answer pressing, unresolved fundamental questions, regarding the enzymology of naturally evolved Diels-Alderases, (ii) undertake structure and simulation-guided reengineering and directed evolution of these enzymes; and (iii) deploy this portfolio of biocatalysts, in combination with auxiliary enzymes, to enable the preparation of chemical building blocks, pharmaceutical lead scaffolds and bioactive natural products.
Planned Impact
This project will deliver (i) detailed molecular insight into a poorly understood and hitherto unexploited group of naturally evolved biocatalysts, (ii) a suite of engineered variants of these enzymes with modified substrate selectivities and catalytic properties, and (iii) a portfolio of new bioactive molecules and lead compounds for further investigation and exploitation. As such we consider this research to have potential broad ranging intellectual, economic and potential clinical impact. The outlined research programme will contribute substantially to the UK's global leadership in the areas of biocatalysts and drug discovery, and aligns well with current BBSRC strategic priorities and cross-council initiatives. The applicants are committed to ensuring that the outputs from this research impact upon policy-makers, funding bodies, academic institutions, and industry by providing clear evidence of the value of strategically aligned fundamental and applied interdisciplinary research.
This is a collaborative LINK project with the multi-national pharmaceutical company AstraZeneca (AZ), with whom we will work in partnership to realise the impacts of this research. This will be through the generation of IP and licensing agreements centered on the enzymes and small molecules that will be generated. The project will establish a strategically important partnership between UK academia and a multi-national pharmaceutical company in an application area that is of major economic significance to the UK. The outcomes of this project are of direct strategic relevance to AZ's future research strategy and will deliver significant industrial and commercial impact. Should any of the new compounds isolated during the course of this study prove to be useful drug leads we will work with AZ to deliver the potentially significant resulting medical and industrial impact. A key output of this work will also be the establishment of general methods to advance our fundamental understanding of enzyme catalysis and our ability to manipulate naturally evolved enzymes to produce new chemical compounds. These discoveries will impinge on the emerging field of synthetic biology, and have the potential to transform industrial processes and practices specifically in the areas of biotechnology and pharmaceutical development. We will maintain an open dialogue with key industrial stakeholders and policy makers during the lifetime of the project, such that they are informed and in a position of readiness to act appropriately as outputs emerge.
The outlined programme will offer those involved (PDRAs, students, etc.) experience and training at the chemistry-biology interface, providing them with a range of technical and intellectual skills required to succeed in careers in academia, industry or the third sector. The research outputs from this program will be reported to the wider scientific community through publication in leading peer-reviewed journals, presentation at national and international conferences and at meetings with industrial and academic collaborators. Training will be given to those involved in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of widely transferable skills. Further, significant opportunities exist for the presentation of research findings by all those involved to a more general audience through public engagement activities organised by the University of Bristol and AZ (school talks, science festivals, etc.). Such activities will ensure that as broad an audience as is feasible will be informed of our ongoing research.
This is a collaborative LINK project with the multi-national pharmaceutical company AstraZeneca (AZ), with whom we will work in partnership to realise the impacts of this research. This will be through the generation of IP and licensing agreements centered on the enzymes and small molecules that will be generated. The project will establish a strategically important partnership between UK academia and a multi-national pharmaceutical company in an application area that is of major economic significance to the UK. The outcomes of this project are of direct strategic relevance to AZ's future research strategy and will deliver significant industrial and commercial impact. Should any of the new compounds isolated during the course of this study prove to be useful drug leads we will work with AZ to deliver the potentially significant resulting medical and industrial impact. A key output of this work will also be the establishment of general methods to advance our fundamental understanding of enzyme catalysis and our ability to manipulate naturally evolved enzymes to produce new chemical compounds. These discoveries will impinge on the emerging field of synthetic biology, and have the potential to transform industrial processes and practices specifically in the areas of biotechnology and pharmaceutical development. We will maintain an open dialogue with key industrial stakeholders and policy makers during the lifetime of the project, such that they are informed and in a position of readiness to act appropriately as outputs emerge.
The outlined programme will offer those involved (PDRAs, students, etc.) experience and training at the chemistry-biology interface, providing them with a range of technical and intellectual skills required to succeed in careers in academia, industry or the third sector. The research outputs from this program will be reported to the wider scientific community through publication in leading peer-reviewed journals, presentation at national and international conferences and at meetings with industrial and academic collaborators. Training will be given to those involved in the preparation of papers, posters and oral presentations to ensure that, alongside their scientific knowledge and skills, they are developing a portfolio of widely transferable skills. Further, significant opportunities exist for the presentation of research findings by all those involved to a more general audience through public engagement activities organised by the University of Bristol and AZ (school talks, science festivals, etc.). Such activities will ensure that as broad an audience as is feasible will be informed of our ongoing research.
Publications
Nair AV
(2020)
Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide ß-methyl branch incorporation.
in Scientific reports
Barringer R
(2023)
Domain shuffling of a highly mutable ligand-binding fold drives adhesin generation across the bacterial kingdom.
in Proteins
Williams S
(2023)
Discovery and biosynthetic assessment of 'Streptomyces ortus' sp. nov. isolated from a deep-sea sponge
in Microbial Genomics
Back CR
(2021)
A New Micromonospora Strain with Antibiotic Activity Isolated from the Microbiome of a Mid-Atlantic Deep-Sea Sponge.
in Marine drugs
Rooms LD
(2020)
Unlocking the Therapeutic Potential of Antimicrobial Natural Products with Synthetic Biology
in International Biopharmaceutical Industry
Marucci L
(2020)
Computer-Aided Whole-Cell Design: Taking a Holistic Approach by Integrating Synthetic With Systems Biology.
in Frontiers in bioengineering and biotechnology
Zorn K
(2023)
Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases
in ChemBioChem
Maddock RMA
(2020)
Enzyme-catalysed polymer cross-linking: Biocatalytic tools for chemical biology, materials science and beyond.
in Biopolymers
Williams SE
(2020)
The Bristol Sponge Microbiome Collection: A Unique Repository of Deep-Sea Microorganisms and Associated Natural Products.
in Antibiotics (Basel, Switzerland)
Stennett HL
(2022)
Derivation of a Precise and Consistent Timeline for Antibiotic Development.
in Antibiotics (Basel, Switzerland)
Devine AJ
(2023)
The Role of Cytochrome P450 AbyV in the Final Stages of Abyssomicin C Biosynthesis.
in Angewandte Chemie (Weinheim an der Bergstrasse, Germany)
Devine AJ
(2023)
The Role of Cytochrome P450 AbyV in the Final Stages of Abyssomicin C Biosynthesis.
in Angewandte Chemie (International ed. in English)
Description | The future success of the global pharmaceutical and chemical industries is dependent on the successful development of efficient, selective and sustainable ways of making new organic molecules with useful properties. Historically, such methods have centered on the use of synthetic organic chemistry to fuse and tailor simple chemical building blocks into a vast array of complex molecular architectures, which may in-turn be used as the basis for amongst other things new drugs, crop protection agents, or materials. Despite the undoubted success of synthetic chemistry, problems exist, including the over-reliance on certain types of reactions that have led to an excessive representation of molecules with predominantly flat, 2D architectures, which are of limited value as drugs. In contrast to chemical catalysts, biological catalysts, termed enzymes, are able to perform challenging chemical reactions that can rapidly build complex 3D chemical structures with multiple bonds under precise stereochemical control. In addition, enzymes can perform such reactions under ambient conditions and without any requirement for environmentally damaging reagents. For these reasons there is significant interest in developing biocatalytic routes to current and future pharmaceuticals and similarly important molecules. A carbon-carbon bond forming reaction known as the Diels-Alder reaction is an effective means of building complex 3D molecules in a single step. However, a limitation of this reaction is that to achieve high yields, stereoselectivity and regioselectivity, the electronic properties of the reactants need to be complementary and often harsh reaction conditions are required. This research project builds on our exciting recent discovery of a naturally evolved co-factor independent enzyme that catalyzes the Diels-Alder reaction at room temperature and on substrates which cannot be transformed using conventional synthetic organic chemistry. This discovery opens up the possibility of using this enzyme to generate a whole new series of complex molecules that could form the basis of new drugs or similarly important chemical compounds. During this project the University of Bristol team have been working in partnership with the pharmaceutical company AstraZeneca to study natural and engineered Diels-Alderases, deploying these biocatalysts to generate a vast array of new 3D molecules that can be used as the basis for new drugs to treat a diverse array of human diseases. Although this project is still ongoing, the team have already made some very exciting discoveries about natural Diels-Alderases, including the ability of these enzymes to accept and act upon non-natural substrates. We are actively exploring industrial applications of these enzymes with our partner AZ. |
Exploitation Route | This is a multi-disciplinary academic-industrial LINK project being undertaken collaboratively between the University of Bristol and the globally-leading pharmaceutical company AstraZeneca (AZ). The outcomes of this project are of direct strategic relevance to AZ's future research strategy and will deliver significant industrial and commercial impact. The project represents a strategically important partnership between UK academia and a multi-national pharmaceutical company in an application area that is of major economic significance to the UK. The importance of the outlined research programme to AZ is reflected in their commitment to fund >50% (>£1.0M) of the overall project cost. AZ's commitment reflects a widely accepted future industrial vision that the development of biosynthetic and biocatalytic routes to high value products will allow industry to transition away from its dependence on petrochemical feedstocks, protecting against price-volatility and reducing carbon footprint and environmental impact. Further, the outputs from this project will enable AZ to access a suite of biocatalysts for reactions that are impractical to perform using synthetic chemistry, opening up new avenues in pharmaceutical lead scaffold generation. A formal collaboration agreement is in place between the project partners, which ensures that the project outcomes, e.g. new enzymes, molecules, drugs leads, etc. can be expediently assessed and exploited by AZ, delivering maximum impact from the research. |
Sectors | Chemicals Environment Healthcare Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
Description | Advisor to the UK Prime Minister's Council for Science and Technology, National Engineering Biology Project. |
Geographic Reach | National |
Policy Influence Type | Participation in a guidance/advisory committee |
Description | Assessment of novel natural product drug combinations for the treatment of multidrug-resistant and extensively drug-resistant TB |
Amount | £25,860 (GBP) |
Organisation | Medical Research Council (MRC) |
Department | Medical Research Foundation |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 06/2021 |
End | 06/2022 |
Description | Automated crystal imaging to support discovery science and structure-based drug discovery. |
Amount | £797,525 (GBP) |
Funding ID | MC_PC_MR/Y000781/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2023 |
End | 03/2024 |
Description | BBSRC Innovation Fellowship: Building C(sp3)-rich complexity by combining biocatalytic cycloaddition and C-C cross-coupling reactions |
Amount | £5,837 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2021 |
End | 06/2021 |
Description | BrisEngBio: From Synthetic to Engineering Biology at Bristol |
Amount | £1,517,913 (GBP) |
Funding ID | BB/W013959/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2022 |
End | 01/2024 |
Description | Harnessing natural product diversity to combat multidrug-resistant pathogens |
Amount | £1,431,248 (GBP) |
Funding ID | MC_PC_MR/T029579/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2020 |
End | 03/2023 |
Description | Harnessing the Power of Diels-Alderases in Sustainable Chemoenzymatic Synthesis |
Amount | £863,005 (GBP) |
Funding ID | BB/Y000846/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 12/2023 |
End | 11/2026 |
Title | Library of natural Diels-Alderases. |
Description | We have established a library of naturally evolved Diels-Alderases, which we are currently screening for biocatalytic activity. Members of our enzyme library have been shown to catalyse novel cycloaddition reactions that are of major interest to academic and industrial researchers. |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | No |
Impact | Our collection has yielded new enzymes with novel functions. We are currently exploring the commercial potential of members of this enzyme library. |
Description | Research collaboration with AstraZeneca |
Organisation | AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | Expertise in form of prior knowledge and understanding of natural Diels-Alderases and their study. Intellectual input by conducting research directly funded by the joint BBSRC/AZ award. Training of staff by securing funding for an AZ staff member to undertake a 3 month research placement in Bristol. Access to data, equipment or facilities critical to the delivery of the project objectives, e.g. protein expression plasmids, recombinant proteins, NMR facilities, protocols for in vitro enzyme assays, etc. |
Collaborator Contribution | Expertise in form of prior knowledge and understanding of high throughput screening methods, protein biochemistry and directed evolution. Intellectual input by conducting research directly funded by the joint BBSRC/AZ award. Training of staff by offering laboratory placements to PDRAs and PhD students based in Bristol, within AZ's biocatalysis group.. Access to data, equipment or facilities critical to the delivery of the project objectives, e.g. protein expression plasmids, recombinant proteins, Mass spec facilities, automated chemistry and funding for a PhD studentship in Bristol. |
Impact | DOI's of publications arising from this project to date: 10.3389/fbioe.2020.00942 10.3390/antibiotics9080509 10.1002/1873-3468.13954 10.1038/s41598-020-71850-w 10.1002/bip.23390 This is a multidisciplinary collaboration involving the following disciplines: Chemistry (analytical, synthetic and medicinal) and Biological Sciences (structural biology, enzymology, biophysics), |
Start Year | 2019 |
Description | 8th CSIR Conference, Pretoria, South Africa. 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited talk at international conference. New international collaborations were established as a result of the presentation. |
Year(s) Of Engagement Activity | 2022 |
URL | https://conference.csir.co.za/ |
Description | Co-Chair of Biocatalysis Conference |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Co-chair of RSC Biocatalysis Conference. Conference was organised under the auspices of the RSC Biotechnology Group. |
Year(s) Of Engagement Activity | 2023 |
URL | https://www.rsc.org/events/detail/75566/biotransformations-ii-science-to-industrial-application |
Description | Exhibit at the Somerscience Festival 2023 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Biochemical Society sponsored exhibit at the Somerscience Festival in 2023. Exhibit was titled "Antibiotic Discovery in the Abyss". The exhibit featured interactive displays, models, a microscope and posters, all describing research focused on the discovery of antibiotics from deep-sea microorganisms. The PI, 2x PDRAs and 2x PhD students manned the exhibit throughout the day. |
Year(s) Of Engagement Activity | 2023 |
URL | https://somerscience.co.uk |
Description | International conference presentation - On the Hunt for the Next Generation of Antimicrobial Agents, 2021 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited conference talk, On the Hunt for the Next Generation of Antimicrobial Agents, Lille, France. 9th - 10th Dec |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.rsc.org/events/detail/48140/on-the-hunt-for-next-generation-antimicrobial-agents |
Description | Invited conference presentation - 7th International Conference on Deep Sea Microbiology, 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited lecture at an international conference. The lecture resulted in the establishment of a number of new international research collaborations. |
Year(s) Of Engagement Activity | 2022 |
Description | Invited conference presentation - Applied Biocatalysis Summit 2021 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited international conference presentation, 3rd -4th November 2021. |
Year(s) Of Engagement Activity | 2021 |
URL | https://applied-biocatalysis-summit.com/ |
Description | Research talk - Applied Biocatalysis 2020 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Research talk at international conference. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.rsc.org/events/detail/45060/applied-biocatalysis-summit-2020-virtual-event |
Description | Research talk at Conference; Natural Products - Emerging Opportunities |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | 30 minute research presentation at the Natural Products - Emerging Opportunities Conference. |
Year(s) Of Engagement Activity | 2023 |
Description | Research talk at the Somerscience Festival 2023 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | 30 minute research presentation at the Somerscience Festival focused on the discovery and development of antibiotics from natural sources. |
Year(s) Of Engagement Activity | 2023 |
URL | https://somerscience.co.uk |