21EBTA Engineering specialised metabolism and new cellular architectures in plants
Lead Research Organisation:
John Innes Centre
Department Name: Biochemistry and Metabolism
Abstract
Plants have a rich endogenous metabolism that can be reprogrammed by genetic transformation. Transient expression techniques have allowed their development as photosynthetic chassis for low-cost high-yield production of vaccines, therapeutic proteins and metabolites. In recent years, several commercial large-scale facilities for transient plant-based bioproduction have been constructed, with products now reaching the market. However, it has proved difficult to engineer high yields of engineered products in true-breeding plant lines, which would pave the way for low-cost bioproduction and new generations of crop varieties.
In this project, we will use fast transient expression systems to design and assemble an artificial synthetic pathway for the production of beta-amyrin, a precursor for many different types of triterpene. The triterpenoids are a large, chemically diverse group of natural products (over 20,000 reported to date), with a wide range of applications in the agricultural, food, cosmetic and pharmaceutical sectors. We will transfer this DNA-encoded pathway into the genome of a new model plant system, the liverwort Marchantia polymorpha. Marchantia is arguably the simplest and easiest land plant to work with at this time. We have constructed many new tools for work with Marchantia, and will use these to trigger beta-amyrin production to specialised oil body containing cells. Oil bodies are natural safe containers for accumulation of high concentrations of compounds in cells. We will then use known developmental regulators to engineer oil cell proliferation in Marchantia tissues to create high-yield harvestable plant organs. We believe the project will provide a prototype for whole organism engineering, and crop improvement.
In this project, we will use fast transient expression systems to design and assemble an artificial synthetic pathway for the production of beta-amyrin, a precursor for many different types of triterpene. The triterpenoids are a large, chemically diverse group of natural products (over 20,000 reported to date), with a wide range of applications in the agricultural, food, cosmetic and pharmaceutical sectors. We will transfer this DNA-encoded pathway into the genome of a new model plant system, the liverwort Marchantia polymorpha. Marchantia is arguably the simplest and easiest land plant to work with at this time. We have constructed many new tools for work with Marchantia, and will use these to trigger beta-amyrin production to specialised oil body containing cells. Oil bodies are natural safe containers for accumulation of high concentrations of compounds in cells. We will then use known developmental regulators to engineer oil cell proliferation in Marchantia tissues to create high-yield harvestable plant organs. We believe the project will provide a prototype for whole organism engineering, and crop improvement.
Technical Summary
This proposal will harness our expertise in large scale assembly of co-regulated gene systems and rapid testing by transient assay in tobacco, and direct these at the challenges of safely transplanting a foreign, high-yield biosynthetic pathway into the chemical and cellular anatomy of a new plant variety.
This project requires (i) the design and assembly of a core synthetic pathway for the production of beta-amyrin, a triterpene scaffold, (ii) optimising the architecture of novel regulatory elements for co-expression of multiple genes, (iii) the modification of endogenous metabolic pathways to maximise availability of substrates, (iv) tissue engineering and targeting of the pathway to specialised cells in order to minimise negative effects on plant growth and maximise yields of beta-amyrin, (v) characterisation of modifying enzymes to decorate and diversify the core triterpene scaffold, and (vi) sharing the emerging toolkits and technologies with research communities in the UK and internationally to underpin future development.
This project requires (i) the design and assembly of a core synthetic pathway for the production of beta-amyrin, a triterpene scaffold, (ii) optimising the architecture of novel regulatory elements for co-expression of multiple genes, (iii) the modification of endogenous metabolic pathways to maximise availability of substrates, (iv) tissue engineering and targeting of the pathway to specialised cells in order to minimise negative effects on plant growth and maximise yields of beta-amyrin, (v) characterisation of modifying enzymes to decorate and diversify the core triterpene scaffold, and (vi) sharing the emerging toolkits and technologies with research communities in the UK and internationally to underpin future development.
Organisations
Publications
Patron NJ
(2023)
Editorial Overview:Engineering plants and plant products for a green bioeconomy.
in Current opinion in plant biology
Méteignier LV
(2023)
Emerging mechanistic insights into the regulation of specialized metabolism in plants.
in Nature plants
| Description | The objective of this research project is to build important large-scale biosynthetic pathways and to integrate these into the metabolism, cellular body plan and life-cycle of plants, focusing on triterpenes as the target metabolites. This research exploits the technical groundwork established by the OpenPlant Synthetic Biology Research Centre and Earlham Biofoundry; through which we developed established sophisticated DNA toolsets, building blocks for in vivo chemistry, methods for rapid prototyping in Nicotiana benthamiana and a uniquely simple and facile testbed in the Marchantia polymorpha system. This project involves (i) the design and assembly of a core synthetic pathway for the production of ß-amyrin, a triterpene scaffold, (ii) optimising the architecture of novel regulatory elements for co-expression of multiple genes, (iii) the modification of endogenous metabolic pathways to maximise availability of substrates, (iv) tissue engineering and targeting of the pathway to specialised cells in order to minimise negative effects on plant growth and maximise yields of ß-amyrin, (v) characterisation of modifying enzymes to decorate and diversify the core triterpene scaffold, and (vi) sharing the emerging toolkits and technologies with research communities in the UK and internationally to underpin future development. To date we have evaluated strategies for boosting triterpene biosynthesis in the Nicotiana benthamiana transient expression system, and have demonstrated increased production of the simple triterpene scaffold beta-amyrin (WP1, Objective 1.1). We have delivered constructs for gene editing to silence competing pathways (WP 1, Objective 1.2). We have made substantial progress in quantifying the effects of construct architecture on plant expression constructs (WP 1, Objective 1.3). The outcomes of our initial experiments to optimise the architecture of plant expression constructs were applied to an ongoing project at EI and included in a publication which is currently in revision: Kallam et al., bioRxiv preprint doi: https://doi.org/10.1101/2022.06.15.496242. We have selected and designed genetic elements and synthetic genetic circuits for controlling triterpene biosynthesis in Nicotiana and Marchantia, and testing is now underway (WP2, Objective 2.1). We have established a computational strategy for large-scale genome mining for discovery and classification of plant sugar transferases (UGTs), and have cloned ~150 UGT genes into plant transient expression constructs for use in combinatorial biosynthesis experiments (WP3, Objective 3.1). We have also identified, cloned and heterologously expressed triterpene acyl transferases from marigold enabling the production of triterpene fatty acid esters in Nicotiana benthamaiana (WP3, Objective 3.2). |
| Exploitation Route | In order to share the emerging toolkits and technologies with research communities through WP4, we have focused on i) web portal development to share DNA collections and toolkits; ii) training and protocol development for democratisation of automation using low-cost OpenTrons liquid handlers; iii) community building activities. To date we have hosted an online workshop with a dozen international experts in DNA design tools to explore design best practices for sharing synthetic biology toolkits and are currently working with a data visualisation studio to develop the web portal. We have collaborated on the design and delivery of an in-person automation training workshop in Cambridge (18 attendees) and set up an open access repository (https://github.com/openplant/openplant_automation_protocols) to assemble validated OpenTrons automation protocols for distribution amongst the synthetic biology community. Since December, we have begun to build out a wider international community of researchers, with a focus on openly sharing knowledge and resources around existing OpenPlant-funded DNA collections as blueprints for future communities that would use the toolkit emerging from this project. Thus far, up to 30 international researchers have attended each session of our online series of monthly community meetings, and where we have heard from four researchers who have used and/or developed upon the DNA collections for low-cost diagnostics, reagent and hardware manufacturing. |
| Sectors | Agriculture, Food and Drink,Chemicals,Digital/Communication/Information Technologies (including Software),Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Description | The PI and co-Is on this award have been active participants in workshops, round tables and meetings (with ministers, GOS, BEIS, DSIT) and in regular meetings of the Engineering Biology Leadership Council Science and Technology subgroup to discuss and advise on the future of the UK Engineering Biology programme. |
| First Year Of Impact | 2022 |
| Sector | Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Policy & public services |
| Description | Attended Roundtable meeting to discuss how Government might further help in the UK's engineering biology sector. |
| Geographic Reach | National |
| Policy Influence Type | Contribution to a national consultation/review |
| Description | Visit by Minister of State in the Department for Science, Innovation and Technology |
| Geographic Reach | National |
| Policy Influence Type | Participation in a guidance/advisory committee |
| Description | Building global collaborations to create reagent distribution hubs for open science and innovation - University of Cambridge EPSRC Impact Acceleration Account |
| Amount | £46,200 (GBP) |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 02/2023 |
| End | 03/2023 |
| Title | OpenPlant Automation Protocols Github Repository |
| Description | The Open Bioeconomy Lab and the Earlham Institute are collaborating to validate and make available a library of protocols and automation scripts for the Opentrons OT2 platform via github. We hope this collection of protocols can inspire more researchers to adapt their workflows for automation, enabling a higher throughput and efficiency in general molecular biology work. The current protocols cover minipreps using magnetic beads and custom labware design files. |
| Type Of Technology | Webtool/Application |
| Year Produced | 2023 |
| Open Source License? | Yes |
| Impact | Only released in 2023 and will be updated throughout the year, no notable impacts expected yet. |
| URL | https://github.com/openplant/openplant_automation_protocols |
| Description | AO attended Synthetic Biology Industrial Translation Showcase @Synbio_Showcase:Synbite Showcase Programme at the Royal Academy of Engineering 17.11.22 |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Industry/Business |
| Results and Impact | The aim of the 1-Day SynbiTECH Showcase was to reflect important issues and changes in the field of synthetic biology/engineering biology. As with the previous SynbiTECH meetings, the focus was primarily be on the development of the U.K.'s industrial and commercial synthetic biology/engineering biology sector. Consequently, the programme did not only reflect industrial and company development, but, also, important associated matters including strategy and policy, finance, policy and biosecurity. |
| Year(s) Of Engagement Activity | 2022 |
| URL | http://www.synbicite.com/news-events/2022/nov/17/synbicite-showcase-2022/ |
| Description | Activity stand at mixed arts festival |
| 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 | We put together an activity for the science marquee at the Latitude Festival, working with colleagues from the University of East Anglia, Food and Farming Discovery Trust, CEFAS, Precious Plastics East and some local artists with the overall topic of microplastics in the environment. Some of the activity was focused on the problems microplastics cause as pollutants in the ocean and in food chains but we also had a section on plastics in our daily lives that enabled exploration of alternative plant-based materials and the potential for greener alternatives from both biological engineering for new materials and better recycling and remaking processes. We feel that by looking at a problem (microplastics in our environment) but then exploring better alternatives we conveyed a sense of agency to the families to seek out and support alternatives in their daily lives. We were particularly pleased to hear many people pledge to give up using clingfilm in favour of beeswax and paper alternatives to wrap their lunch! |
| Year(s) Of Engagement Activity | 2022 |
| Description | Anne Osbourn meeting with George Freeman MP during visit to Norwich Research Park |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | Anne Osbourn met George Freeman MP (Minister of State in the Department for Science, Innovation and Technology) as one of the NRP Entrepreneurial Researchers. Professor Anne Osbourn, Founder of Hothouse Therapeutics, spoke about Unlocking Nature's Inaccessible Chemistry. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Biomaker Training Workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Postgraduate students |
| Results and Impact | Series of Training Workshops for no-code programming and assembly of scientific instrumentation as documented at https://www.biomaker.org. Follow-on participation in open source projects. |
| Year(s) Of Engagement Activity | 2022,2023 |
| URL | https://www.biomaker.org |
| Description | Engagement Showcase |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | This was an 'in-house' event where scientists were invited to put up displays of their engagement resources so that other scientists could come and get ideas, see what is available to share and to encourage more scientists to try public engagement. We took a selection of tried and tested outreach resources, including activities that help to explain genome mining, biosynthetic pathway building and biological engineering. We also took our DNA robot, 'DNA Dave' and his new companion 'RNA Ruth' that definitely seemed to encourage people to be bold and creative with their ideas for the tools they could make to help their research come to life for a general audience. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Independent review of UKRI Roundtable discussion co-hosted by the Campaign for Science and Engineering & Sir David Grant |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Policymakers/politicians |
| Results and Impact | Anne Osbourn was a member of an independent review of UKRI. Roundtable discussion. Co-hosted by the Campaign for Science and Engineering and Sir David Grant. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://www.ukri.org/news/ukri-welcomes-independent-review-report/ |
| Description | Norwich Science Festival satellite event at Diss Corn Hall |
| 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 | We took an activity stand to a science discovery day at Diss Corn Hall. This event was set up as a satellite venue for the very popular Norwich Science Festival to try and reach a broader audience. There were 3 workshop sessions throughout the day for 50 children per workshop and their families, all of which were fully booked! We took a stand that focused on the instructions held within DNA to 'make stuff' which was explained by inviting people to engage with our robot DNA Dave, pushing buttons and turning cogs to complete transcription and translation to make new products. We used examples from plants that people would be familiar with such as menthol, limonoids, vanillin and anthocyanins and then invited children to extract anthocyanins from red cabbage to use to make colour-changing paint. Many of the parents were amazed how easy the process was and were keen to build on the experiment at home with their children to make a colour palette of paints using pigments from plants and acids and bases. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Open-Source Lab Automation Workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | The workshop was a hands-on session with the aim to introduce automation to wet-lab researchers interested in implementing it in their workflows.The session was a collaborative effort led by researchers from both the Earlham Institute and University of Cambridge, and led participants from basic to more advanced applications. The first half of the session was dedicated to introducing what automation is, and how to design protocols using the Opentrons Protocol Designer, demonstrate other protocols from the Protocol Library and how to adapt and implement them into workflows. The latter half was a hands-on session where participants worked on making their own protocols, and the workshop leaders/facilitators helped to troubleshoot, before inviting participants to run their protocols on the OT-2 instrument. One outcome of the workshop was that participants were able to take away their own protocols to use within their own labs. The workshop also allowed participants to network with one another (particularly between academic researchers from different institutes, and those working in the biotech industry), which we hope to follow-up on in the near future. One final key impact was increased interest in the use of automation: participants expressed interest in learning more advanced methods of designing protocols, particularly in programming in Python. Given this interest, one of the proposed next steps is to work with Earlham Institute and Cambridge researchers to design a series of automation/OT education workshops tailored to participants' needs and interests. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://www.engbio.cam.ac.uk/events/open-source-lab-automation-workshop |
| Description | Participation in the Steering Committee for Standards and Metrology in Engineering Biology |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Participation in the steering committee for the development of standards and metrology in engineering biology with the National Measurement Laboratory. This activity is ongoing and is expected to lead to the development of standards to improve the accuracy of measurements, essential for confidence in engineering biology products that are brought to market/the clinic. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Presentation at Ginkgo Grove |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Dr Jenny Molloy gave a talk "Common Threads in Open Science and Decentralised Science" at the Ginkgo Bioworks "Grove" Seminar series, which was attended by around 25 attendees from mostly a business and entrepreneurship background. The talk was followed by a lot of discussion and several requests for additional information about the Open DNA Collections. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Presentation at Global Community Biosummit |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Other audiences |
| Results and Impact | Dr Jenny Molloy gave an invited keynote "Building global collaborations for access to reagents" at the Global Community Biosummit organised by MIT. Around 90 people attended on Zoom, who were an international group of people running community biology labs across all areas of the world, students, artists, educators, entrepreneurs and social scientists. There were multiple questions and discussion afterwards and at least five follow-ups via email or Slack, of which one person has started volunteering regularly for the Reclone community. |
| Year(s) Of Engagement Activity | 2022 |
| URL | https://www.biosummit.org/ |
| Description | Reclone Community Meetings (monthly) |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Since December, we have begun to build out a wider international community of researchers, with a focus on openly sharing knowledge and resources around existing OpenPlant-funded DNA collections as blueprints for future communities that would use the toolkit emerging from this project. Thus far, up to 30 international researchers have attended each session of our online series of monthly community meetings (https://reclone.org/meetings/), and where we have heard from four researchers who have used and/or developed upon the DNA collections for low-cost diagnostics, reagent and hardware manufacturing. The most recent meeting in March saw community members discuss new DNA parts to add to these DNA collections (https://stanford.freegenes.org/collections/open-genes) - these parts will be synthesised and included in a future updated open DNA toolkit collection, which we aim to distribute to up to three institutes in Latin America, Africa, and Asia to act as regional reagent distribution hubs for other local researchers to more easily make use of these collections. |
| Year(s) Of Engagement Activity | 2022,2023 |
| URL | https://www.youtube.com/@reclone_org |
| Description | School Science Art and Writing (SAW) project |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | We delivered a SAW project on the theme of plant defence at Avenues Junior School in Norwich. The children were encouraged to collect samples from the school garden of plants showing symptoms of disease and observe them using microscopes. This enabled them to look closely at plants, learn about a range of pathogens and develop confidence in using microscopes. They moved on to an experiment where they had to identify wheat and oat seedlings by grinding up roots using pestle and mortars and viewing the exudates under UV light to look for the presence of the fluorescent molecule avenacin that is present only in oat and protects it from attack by the 'take all' fungus Gaeumannomyces tritici. We then discussed how scientists are learning how plants make important molecules and reconstructing biosynthetic pathways in model systems and that wheat plants could be engineered to also produce avenacin to protect them from take all disease. The children were then asked to design a defence for a plant to protect it from 1 of 6 potential threats by turning over a 'chance card'. They were very creative and enjoyed dreaming up ideas that could be used for defence. They took the new concepts and vocabulary on to write poetry and create pieces of art that represented the modular building of pathways. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Talk at 'Enterprise Wednesday' event |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Industry/Business |
| Results and Impact | A short talk describing the potential of plants for the discovery of new products for medicine and industry. This was followed by a discussion in which there were numerous follow-up questions from attendees. |
| Year(s) Of Engagement Activity | 2023 |