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.

Planned Impact

As described in the proposal submitted to TSB.
 
Description Good progress has been made towards the key objectives of the project. Since we began in April 2015 work has focussed on making chassis organisms which can produce the precursor compound (geranylgeranyl pyrophosphate) of the diterpenoid target molecules. This project is a collaborative work, and the chassis organisms being developed include algae (Alison Smith at University of Cambridge), yeast and plants (Ian Graham, project PI).

A set of molecular tools have been collected and used for the optimisation of geranylgeranyl pyrophosphate (GGPP). The project is now developing new production systems for plant, yeast and algal-based production of GGPP. The plant and yeast work is being carried out in Ian Graham's Group, and Alison Smith's Group is developing the new algal strains.

In parallel, the diterpenoid biosynethesis gene clusters have been investigated. This has led to the discovery of a gene cluster from Jatropha curcas. This cluster contains the genes that encode the enzymes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids including the lathyranes jolkinol C and epi-jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon-carbon ring closure (this work was done in collaboration with Geoff Brown, University of Reading). This conversion of casbene to lathyranes will allow the scalable heterologous production of these potential anti-cancer drugs, which can often only be sourced in limited quantities from their native plant.

Diterpene production has been achieved in three heterologous production platforms, namely yeast, micro algae and Nicotiana benthamiana and production is being scaled up in partnership with industry.
Exploitation Route Not applicable this year - project still active.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
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 engi 
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 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 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) 
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