Developing platforms for the production of diterpenoids (TSB application reference 43970-304155)
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
Plants produce a wide range of diterpenoids, many of which are used commercially such as a paclitaxel, employed in the treatment of cancers, and steviol glyclosides which are used as zero-calorie natural sweeteners. Many other useful diterpenoids cannot yet be commercially exploited due to their limited availability and/or high production costs. Using synthetic biology it is now possible to engineer organisms such as yeast so that they are able to convert simple sugars to high-value chemicals. This project will develop yeast, algae and higher plant species as "chassis organisms" which can be used for the scalable production of diterpenoids. Our project will focus in particular on compounds which could be used in the treatment of cancers, or used in skin products such as sunscreens to protect skin against harmful UV light. These biological production systems will also be useful in producing many other diterpenoids that are found in nature.
Technical Summary
The project will address issues of supply and high production costs of selected high value chemicals of particular interest to its industrial partners (GSK, Unilever and Croda) and will provide advances in technology for wider applications across UK research and industry.
Currently a number of plant diterpenoids are used in the pharmaceutical, agricultural, food additive and fragrance industries. These are sourced from a limited number of species, restricting their supply and greatly increasing their cost. Using synthetic biology and metabolic engineering, the project will develop new production systems for high value diterpenoids by exploiting recent gene-based discoveries and novel platforms developed by the academic partner laboratories.
The aim of this project is to develop scalable heterologous production systems for diterpenoids. First, "chassis organisms" will be constructed that can supply elevated levels of the common diterpenoid precursor geranylgeranyl pyrophosphate. Genes for secondary metabolism will then be introduced into these chassis to create strains producing specific target compounds. We will then scale up laboratory production processes to pilot scale (eg 100 litre fermenter) and develop processes for the purification of the target compounds. Although we will focus primarily on specific diterpenoids of interest to the industrial partners within our consortium, the GGPP producing chassis will be universal in that they could also be adapted to produce a wider range of diterpenoids of interest to the pharmaceutical, personal care, food, fragrance and agricultural industries.
The overall team has a very good balance of 'push and pull' and is well placed academically, technically and commercially to deliver a high impact project for the IB Catalyst.
Currently a number of plant diterpenoids are used in the pharmaceutical, agricultural, food additive and fragrance industries. These are sourced from a limited number of species, restricting their supply and greatly increasing their cost. Using synthetic biology and metabolic engineering, the project will develop new production systems for high value diterpenoids by exploiting recent gene-based discoveries and novel platforms developed by the academic partner laboratories.
The aim of this project is to develop scalable heterologous production systems for diterpenoids. First, "chassis organisms" will be constructed that can supply elevated levels of the common diterpenoid precursor geranylgeranyl pyrophosphate. Genes for secondary metabolism will then be introduced into these chassis to create strains producing specific target compounds. We will then scale up laboratory production processes to pilot scale (eg 100 litre fermenter) and develop processes for the purification of the target compounds. Although we will focus primarily on specific diterpenoids of interest to the industrial partners within our consortium, the GGPP producing chassis will be universal in that they could also be adapted to produce a wider range of diterpenoids of interest to the pharmaceutical, personal care, food, fragrance and agricultural industries.
The overall team has a very good balance of 'push and pull' and is well placed academically, technically and commercially to deliver a high impact project for the IB Catalyst.
Planned Impact
As described in the proposal submitted to TSB.
Publications
Crozet P
(2018)
Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.
in ACS synthetic biology
Czechowski T
(2022)
Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.
in Proceedings of the National Academy of Sciences of the United States of America
Forestier E
(2021)
Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation
in Plant Biotechnology Journal
Forestier ECF
(2021)
Engineering Production of a Novel Diterpene Synthase Precursor in Nicotiana benthamiana.
in Frontiers in plant science
King AJ
(2016)
A Cytochrome P450-Mediated Intramolecular Carbon-Carbon Ring Closure in the Biosynthesis of Multidrug-Resistance-Reversing Lathyrane Diterpenoids.
in Chembiochem : a European journal of chemical biology
Moses T
(2017)
Synthetic biology approaches for the production of plant metabolites in unicellular organisms.
in Journal of experimental botany
Pérez-Escobar OA
(2020)
Untapped resources for medical research.
in Science (New York, N.Y.)
Zhan C
(2020)
Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance.
in Nature plants
| Description | The project aimed to address the challenge 'production of fine and speciality chemicals and natural products' by developing robust production platforms focused on diterpenoids, which have a diverse range of industrial uses including as a component in fragrances, pharmaceuticals, vitamins, sweeteners and plant growth regulators, and are linked by a common precursor in their biosynthetic pathway (geranylgeranyl pyrophosphate - GGPP). Chassis organisms optimised for the production of GGPP, casbene and a number of casbene derived diterpenoids were delivered, however achieving a step change in production levels and in selecting downstream products of interest for onward development by industry proved challenging, due to the complexity of the biochemical pathways involved in production of the different classes of casbene-derived diterpenoids. Progress was achieved to deliver a suite of interesting diterpenoid compounds that are of ongoing interest to industry, with one of the industrial partners currently evaluating these for relevant bioactivities as part of an ongoing collaboration with the PI's laboratory. |
| Exploitation Route | Progress was achieved to deliver a suite of interesting diterpenoid compounds that are of ongoing interest to industry, with one of the industrial partners currently evaluating these for relevant bioactivities. |
| Sectors | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |
| Description | Commercial validation of a Saccharomyces cerevisiae production platform for bioactive diterpenes |
| Amount | £49,998 (GBP) |
| Funding ID | POC-HVB-2020/01 (GRAHAM) |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 04/2021 |
| End | 03/2022 |
| Description | Production of diterpenes using Yarrowia lipolytica |
| Amount | £34,992 (GBP) |
| Funding ID | Unique ID 718629265 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 03/2021 |
| End | 11/2021 |
| Title | MoClo Toolkit |
| Description | From Crozet et al. (2018): " we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology." |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2018 |
| Provided To Others? | Yes |
| Impact | This kit is available to the community, to allow Chlamydomonas to become the next chassis for sustainable synthetic biology approaches. Based at the University of Cambridge, PM and AGS of this project were involved in the research to develop the kit. |
| URL | https://www.chlamycollection.org/product/moclo-toolkit/ |
| Description | AS talk at Microbiology Society Annual Meeting in Edinburgh |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof Alison Smith (project co-investigator) gave a talk titled 'Plug and play - developing tuneable gene expression in microalgae using synthetic biology approaches'. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Ian Graham was an invited Major Symposium Speaker at the ASPB President's Symposium on Translational Science in 2018. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Ian Graham was an invited Major Symposium Speaker at the ASPB President's Symposium on Translational Science, which was held at the start of the ASPB Plant Biology 2018 meeting in Montreal, Canada. He spoke on the subject of 'Unlocking the secrets of medicinal crops'. |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://community.plantae.org/article/4938184912814475104/aspb-president-s-symposium-translational-s... |
| Description | Invited Special Seminar given by Professor Ian Graham at the University of Melbourne, Australia |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Professor Ian Graham gave an invited Special Seminar at the Molecular Science & Biotechnology Institute at the University of Melbourne, Australia. His talk was titled 'Molecular breeding of medicinal crops and discoveries along the way'. |
| Year(s) Of Engagement Activity | 2015 |
| URL | http://www.bio21.unimelb.edu.au/professor-ian-graham-presents-molecular-breeding-medicinal-crops-and... |
| Description | Invited speaker (IAG) at 16th Euro Fed Lipid Congress, Belfast. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Ian Graham gave an invited talk titled 'Production platforms for high value terpenoids from plants', where he reflected on different production routes for high value chemicals from plants, citing his diterpenoid research as an example. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Invited speaker at Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Shanghai, China |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Professor Ian Graham gave an invited talk at the Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Shanghai, China, in July 2017. Earlier in the same week he was an invited plenary speaker at the ICPM2017 conference in Dalian, China, and he was invited to extend his trip to also give a talk at the Institute in Shanghai. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Invited speaker at International Conference on Natural Product Biotechnology (ICNPB), held in Aberdeen |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Professor Ian Graham gave an invited talk at ICNPB conference held in Aberdeen in September 2017. The audience was 50:50 from research and industry, and very much an interdisciplinary meeting, with participants aiming to get an overview of developments across different parts of the bio-economy. The ICNPB conference was described by the organisers as a research, knowledge transfer and business development conference for the Life Science and Biotechnology community internationally, building on the understanding of Natural Product resources, sustainable technologies, the circular economy, and established research and business links. |
| Year(s) Of Engagement Activity | 2017 |
| URL | http://www.icman-iuphar-np-2017.org/about/icnpb-2017/ |
| Description | Invited speaker at School of Biosciences, University of Kent, Canterbury |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | In December 2017 Ian Graham gave an invited seminar on his research at the School of Biosciences, University of Kent. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.kent.ac.uk/bio/news.html?view=1757 |
| Description | Invited speaker at first CEPLAS 2017 summer school on Frontiers in Plant Science, at Hennef, near Cologne, Germany |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Professor Ian Graham was an invited speaker at the first summer school of the Cluster of Excellence on Plant Science, (CEPLAS), which is a research initiative of the Max Planck Institute for Plant Breeding Research, the University of Cologne, the Heinrich Heine University Düsseldorf and the Jülich Science Centre. They focus on the analysis and implementation of complex traits in plants in four different research areas covering annual and perennial life histories, C4 photosynthesis, plant-microbe interactions and metabolic interactions. Their main goal is to understand these complex traits to the point, where we can create artificial modules and use them to obtain more reliable food sources and higher yields in the future. The summer school was part of the CEPLAS training activities and was directed towards Ph.D. students and young postdocs. Their aim was to accommodate 80 early career researchers, with half of them having an international background. Thematically the summer school was focused on the understanding and presentation of emerging fields within plant sciences and their possible future directions. Ian was given the honour of presenting the first talk of the event, which was on his high impact work in molecular breeding in two of the worlds major major medicinal crops - Artemisia and poppy - with various discoveries that relate to the metabolism of high value chemicals and implementing these into a breeding programme. |
| Year(s) Of Engagement Activity | 2017 |
| URL | https://www.ceplas.eu/en/training-careers/ceplas-courses-and-excursions/ceplas-summer-school-2017/ |
| Description | Invited talk given by IAG at RBG Kew State of the World's Plants and Fungi Symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Invited presentation given by Ian Graham at the Royal Botanic Gardens Kew 'State of the World's Plants and Fungi Symposium', October 2020. Ian's presentation was entitled 'From medicinal crops to synthetic biology - maximising the use of plant (and fungal) diversity to meet global health challenges'. |
| Year(s) Of Engagement Activity | 2020 |
| URL | https://www.kew.org/science/state-of-the-worlds-plants-and-fungi |
| Description | Poster at the Chemistry and Biology of Natural Products Symposium X, University of Warwick, 30 June - 1 July 2016. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | The CBNP X was a two-day symposium for postgraduate students, PDRAs and young academics but also open to any academic/industrialist working in any areas of natural product chemistry and biology. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Poster presentation by EF at Cytochrome P450 Biodiversity and Biotechnology Conference in York |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | The title of the poster presented was 'Engineering Nicotiana benthamiana for the production of high value diterpenes'. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Poster presentation by EF at TERPNET 2019 conference. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | EF presented a poster titled 'Developing Nicotiana benthamiana stable overexpression lines for the production of high value diterpenoids' at the TERPNET 2019 international conference held in Germany. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Poster presentation by PM at the TERPNET 2019 international conference held in Germany. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | PM presented a poster titled 'Using synthetic biology to produce high value diterpenoids in microalgae' at the international TERPNET 2019 conference held in Germany. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Poster presentation by SS at TERPNET 2019 conference. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | SS presented a poster titled 'A yeast biosynthetic platform for the production of casbene-derived diterpenoids' at the TERPNET 2019 conference held in Germany. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Presentation by AS at 2021 Festival of Genomics and Biodata |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presentation on bioactive diterpenoids from Euphorbiaceae, given by Prof Alison Smith at the 2021 Festival of Genomics and Biodata. The Festival brings healthcare, research, pharma, technology, government, investors, patient groups and others together in a unique, collaborative digital environment. |
| Year(s) Of Engagement Activity | 2021 |
| URL | https://www.festivalofgenomics.com |
| Description | Presentation given by Prof Ian Graham at Cardiff University |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof Ian Graham gave an invited presentation at the School of Chemistry, Cardiff University, titled 'Engineering bioactive diterpenoid biosynthesis - sourcing gene tools from roadside verges to paddy fields'. |
| Year(s) Of Engagement Activity | 2023 |
| Description | Prof Alison Smith gave an invited talk at the Crossing Kingdoms Symposium held in Cambridge. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof Alison Smith (project co-investigator) gave an invited talk where she mentioned the diterpenoid project at the Crossing Kingdoms international synthetic biology symposium, held a the the University of Cambridge. Her talk was titled 'Plug and play - developing tuneable gene expression in microalgae using synthetic biology approaches'. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Prof Alison Smith gave an invited talk at the GRC Plant Metabolic Engineering Conference, USA. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof Alison Smith (project co-investigator) gave an invited talk at the Gordon Research Conference on Plant Metabolic Engineering where she mentioned the diterpenoid project research, and her talk was titled 'Exploiting Metabolism: How Vitamins are Good for Algal Biotechnology'. See: https://www.grc.org/plant-metabolic-engineering-conference/2017/ |
| Year(s) Of Engagement Activity | 2017 |
| Description | Prof Alison Smith was an invited speaker in the MSU Plant Sciences series. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof Alison Smith (project co-investigator) mentioned the diterpenoid research in an invited talk in the Michigan State University (USA) Plant Sciences seminar series, titled 'What can vitamin metabolism do for algal biotechnology?'. |
| Year(s) Of Engagement Activity | 2018 |
| Description | THYME Entrepreneurship Workshop - Mentoring the aspiring biobased business leaders of tomorrow |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Professional Practitioners |
| Results and Impact | Ian Graham mentored the aspiring biobased business leaders of tomorrow using experience gained in conducting world class research with impact. This presentation was part of the BioVale THYME project: https://www.biovale.org/about-us/biovale-projects/thyme/ |
| Year(s) Of Engagement Activity | 2019 |
| Description | Talk given by AS at GRC Chloroplast Biotechnology, Ventura, USA |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Prof Alison Smith (project co-investigator) gave a talk titled 'Developing Synthetic Biology Approaches in Microalgae for Metabolic Engineering', mentioning the diterpenoid project research. |
| Year(s) Of Engagement Activity | 2017 |
| Description | University visit (Xi'an Jiaotong University PR China) by IAG. |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | IG gave a talk titled 'Unlocking the Secrets of Medicinal Crops: From Artemisinin to Morphine, Why are Plants Still Better than Bugs at Making these Drugs?' at Xi'an Jiaotong University PR China. |
| Year(s) Of Engagement Activity | 2019 |