(Re)design of the choroplast genome - towards a synthetic organelle
Lead Research Organisation:
University of Cambridge
Department Name: Plant Sciences
Abstract
Plants and algal cells contain a compartment (or organelle) not found in animal cells - the chloroplast. This is the site of photosynthesis and other important biosynthetic processes, and it contains its own genetic system that is a legacy of the chloroplast's evolution from a free-living photosynthetic bacterium. Over evolutionary time, the circular genome of the chloroplast (the 'plastome') has been massively reduced in size, with the loss of most of its genes. What remains is a tiny genome that contains only a hundred-or-so genes. About half of these encode components of the photosynthetic apparatus, whilst the remainder are genes for housekeeping functions such as gene expression. The plastome therefore represents a naturally reduced genome that could be readily re-designed using synthetic biology approaches to gain insights into minimal requirements for an entire genome. At the same time this would optimize the plastome as a platform (a chassis) for future engineering efforts, such as production of high value products in the chloroplast or re-engineering the photosynthesis process. Genetic engineering of the plastome is well-established for several plant species, and for the single-celled green alga Chlamydomonas reinhardtii, which has served for many years as a model system for studying chloroplast biology. C. reinhardtii is particularly suited for a plastome redesign project as, unlike plant cells, it contains just a single chloroplast and can dispense completely with photosynthesis when grown on acetate as a source of carbon. Furthermore, the generation of chloroplast-engineered strains takes weeks rather than months. Recently, we have developed new tools for engineering the C. reinhardtii plastome and will apply these to address the following questions:
i) by systematically deleting all regions of the plastome known to contain photosynthetic genes and other dispensable DNA, can we define the minimal size and gene content for the plastome?
ii) Can we re-introduce the genes for a particular photosynthetic complex as a single refactored gene cluster, thereby allowing a modular 'plug-and-play' approach to studying how gene changes influence photosynthetic performance.
iii) Can large gene clusters be engineered into the plastome to allow the reprogramming of the chloroplast as a site for synthetic of high-value products such as vitamin B12?
iv) can we design and build an entirely synthetic minimal plastome and introduce this into the chloroplast, replacing the native plastome and thereby 'rebooting' the DNA software of the organelle?
v) can we integrate into our plastome technology the capacity to tune up or down the expression of target genes using different combinations of chemicals in the growth medium, allowing us to control gene clusters or test pairs of gene variants in the same chloroplast by switching expression from one to the other?
The project will provide essential basic understanding of the challenges of synthetic reprogramming of organelle genomes in plants and animal cells, and serve as a platform for future "designer organelle" studies.
i) by systematically deleting all regions of the plastome known to contain photosynthetic genes and other dispensable DNA, can we define the minimal size and gene content for the plastome?
ii) Can we re-introduce the genes for a particular photosynthetic complex as a single refactored gene cluster, thereby allowing a modular 'plug-and-play' approach to studying how gene changes influence photosynthetic performance.
iii) Can large gene clusters be engineered into the plastome to allow the reprogramming of the chloroplast as a site for synthetic of high-value products such as vitamin B12?
iv) can we design and build an entirely synthetic minimal plastome and introduce this into the chloroplast, replacing the native plastome and thereby 'rebooting' the DNA software of the organelle?
v) can we integrate into our plastome technology the capacity to tune up or down the expression of target genes using different combinations of chemicals in the growth medium, allowing us to control gene clusters or test pairs of gene variants in the same chloroplast by switching expression from one to the other?
The project will provide essential basic understanding of the challenges of synthetic reprogramming of organelle genomes in plants and animal cells, and serve as a platform for future "designer organelle" studies.
Technical Summary
Synthetic biology offers an unprecedented opportunity both to consider (re)designing biological systems for useful outputs, but also the ability to dissect existing biological systems, to establish the minimum requirements of a process, a genome, or even an entire organism. However, to fulfill this promise the system under investigation needs to be tractable in terms of manipulation and analysis, and ideally to be amenable to incremental changes. In this proposal we aim to establish what is needed for a minimal chloroplast genome of the alga Chlamydomonas reinhardtii, and then to design, build and test a synthetic version. In the process we will also redesign it to develop a system for exploring fundamentals of photosynthetic complex assembly and function, and for expression of heterologous genes. C. reinhardtii can live heterotrophically and thus photosynthetic genes are completely dispensable. To establish which other parts of the 204 kb C. reinhardtii plastome (CP) can be removed, we will carry out systematic deletions, and in the process identify any cryptic essential sequences, as well as gaining information on the minimum size needed for CP stability and maintenance. We will test the efficiency of refactoring the five pet genes for the cytochrome b6f complex, and use operons for increasing numbers of enzymes of the biosynthetic pathway for vitamin B12 as proxies for heterologous gene clusters. We will take advantage of a system we have developed using nucleus-encoded trans-acting factors required for stability of chloroplast transcripts to tune expression of the introduced genes. These experiments will inform the design of a completely synthetic minimal CP, lacking all non-essential genes. This will be introduced into a recipient host strain that had been previously pretreated to reduce CP copy number, and use several strategies to facilitate complete substitution of the endogenous plastome with our synCP-v1.0.
Planned Impact
The full exploitation of synthetic biology (SynBio) within the rapidly expanding bioeconomy requires an understanding of how SynBio can be applied to organisms beyond the model systems of E. coli and yeast. Importantly, the manipulation of phototrophic organisms (plants, algae and cyanobacteria) is fundamental to globally important areas such as food and feed production, biofuel generation, sustainable production of phytochemicals and novel bioactives, and biological carbon capture. This project aims to define the parameters for minimizing and redesigning the chloroplast genome (plastome), with the overall goal of reprogramming an algal chassis with a completely synthetic plastome. An ability to carry out such reprogramming would open up possibilities for making designer chloroplasts with a plethora of novel properties, and would therefore benefit academic researchers and industries across a wide spectrum. Examples include: i) photosynthesis researchers and those aiming to improve photosynthetic performance in food crops, and in organisms grown for biofuels; ii) industrial biotechnologists developing plants and algae as light-driven platforms for low costs synthesis of recombinant proteins and valuable metabolites; iii) synthetic biologists interested in introducing novel organelle compartments into eukaryotic cells; iv) evolutionary biologists interested in how organelle genomes have been shaped over evolutionary time, and genome researchers investigating the miniaturization of genomes.
The project will also contribute to researcher training and capacity building in algal biotechnology and synthetic biology - two priority areas for BBSRC as attested by its funding of the PHYCONET NIBB and the SynBio Centres. This will help to ensure that there are skilled researchers for UK's growing Industrial Biotech sector. Ultimately, the growth of this sector will create jobs and provide economic benefit to the country.
There is a great interest amongst the general public, students and 'lay scientists' regarding synthetic biology and green technologies, and a growing recognition of the need to develop sustainable solutions to the global challenges of providing food, feed, fuels and pharmaceuticals to an ever-increasing population. Through the various engagement activities embedded within the project (detailed in the Pathway to Impact) we will grow this interest and awareness. Furthermore, the dialogue with these groups will help provide a holistic overview of our research and it relevance to society. Finally, we have established links with Government offices, trade organisations and business support bodies (e.g. InnovateUK), Societies (e.g. Microbiology Society, British Phycological Society) and algal associations (e.g. European Algal Biomass Association), and so are able to contribute positively to the framing of legislation regarding the control and exploitation of algal synthetic biology. Lobbying and involvement in the drafting of roadmaps and policy documents are already a key activity for both PI's and this will continue under this project.
The project will also contribute to researcher training and capacity building in algal biotechnology and synthetic biology - two priority areas for BBSRC as attested by its funding of the PHYCONET NIBB and the SynBio Centres. This will help to ensure that there are skilled researchers for UK's growing Industrial Biotech sector. Ultimately, the growth of this sector will create jobs and provide economic benefit to the country.
There is a great interest amongst the general public, students and 'lay scientists' regarding synthetic biology and green technologies, and a growing recognition of the need to develop sustainable solutions to the global challenges of providing food, feed, fuels and pharmaceuticals to an ever-increasing population. Through the various engagement activities embedded within the project (detailed in the Pathway to Impact) we will grow this interest and awareness. Furthermore, the dialogue with these groups will help provide a holistic overview of our research and it relevance to society. Finally, we have established links with Government offices, trade organisations and business support bodies (e.g. InnovateUK), Societies (e.g. Microbiology Society, British Phycological Society) and algal associations (e.g. European Algal Biomass Association), and so are able to contribute positively to the framing of legislation regarding the control and exploitation of algal synthetic biology. Lobbying and involvement in the drafting of roadmaps and policy documents are already a key activity for both PI's and this will continue under this project.
Organisations
People |
ORCID iD |
Alison Smith (Principal Investigator) |
Publications
Geisler K
(2021)
Exploring the Impact of Terminators on Transgene Expression in Chlamydomonas reinhardtii with a Synthetic Biology Approach.
in Life (Basel, Switzerland)
Jackson HO
(2021)
The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism.
in Frontiers in plant science
Description | We have started to build a comprehensive map of the various aspects of the genome in the chloroplast of the green alga Chlamydomonas reinhardtii. This has enabled us to initiate the work to establish which parts might be deleted and still enable the cells to survive. We need to do this before we can design a minimal chloroplast genome, which is the main aim of the project. We have shown that it is possible to refactor (that is reorganise) a set of genes within the chloroplast genome, and also to recode (that is change the genetic code) for two essential genes. Together these three approaches will enable us to build an artificial genome and introduce it back into the chloroplast. We have published two papers and three others are currently in preparation. We have also secured additional funding to allow us to capitalise on our findings and generate a synthetic chloroplast plastome |
Exploitation Route | Those interested in knowing what is required for a minimal genome, as well as those interested in Chlamydomonas itself |
Sectors | Agriculture Food and Drink Manufacturing including Industrial Biotechology |
Description | Improving regulation of chloroplast gene expression in Chlamydomonas reinhardtii |
Amount | £2,100 (GBP) |
Funding ID | SSB-AU032214 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2022 |
End | 09/2022 |
Description | Rewriting The Genetic Code: The Algal Plastome As A Testbed For Basic And Applied Studies |
Amount | £3,146,402 (GBP) |
Funding ID | BB/W003538/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2022 |
End | 03/2027 |
Title | MoClo Toolkit for Chlamydomonas |
Description | A kit of 119 standard DNA parts that can be used for Golden Gate (Modular Cloning, MoClo) cloning to prepare constructs for genetic modification of Chlamydomonas reinhardtii. |
Type Of Material | Biological samples |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | The kit is freely available to the community (for a small distribution fee) and should enable much more rapid preparation of clones and then easy comparison between results from different groups |
Description | AS gave a talk at the 2nd Biodesign Research Conference |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | AS gave a talk at the 2nd Biodesign Research Conference entitled Redesigning the Chlamydomonas chloroplast genome' to an audience from across biological and engineering disciplines, both academics and industrialists. |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.biodesign-conference.com/2021/ |
Description | Agritech East - outreach evening to algal innovation centre by those interested in and working in farming sector |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Industry/Business |
Results and Impact | This is the text from the Agritech East Report after the event: Sainsbury Lab and Algal Innovation Centre - Cambridge University At the end of January we hosted a Young Innovators' Forum (YIF) visit to the Sainsbury Lab and Algal Innovation Centre, Cambridge University. This was visit as part of YIF programme which aims to bring together early career entrants from across farming, science and technology at innovative agri-business and research facilities. On the night we were joined by farmers, scientists, researchers and engineers who were all interested to find out about the work happening in the lab. The Sainsbury Lab is a world class research facility which opened in 2011. The group found out that the research which is carried out is exploring the regulatory systems which control growth and development in plants. We were able to get an insight into the labs as we walked through the building due to the open plan nature of the space. Unlike some research facilities, the Sainsbury Lab is unusual in having individuals from different research groups sat on the same benches, which encourages more conversation and collaboration across different research strands. We heard about the findings of some of the research and particularly enjoyed finding out about the most unusual discovery which was made totally by accident when a plant was found to produce minerals that were previously only believed to be found in asteroids! We then moved onto the Algal Innovation Centre which sits within the Botanic Gardens. A purpose built facility, the AIC provides a secure environment to carry out research into the uses and behaviour of algae. Matt Davey, Senior Research Associate, talked us through some of the different work which has been carried out within the lab. This includes working with a local water company on growing algae on waste nitrate which can then be used to produce energy by putting it into an anaerobic digester. We learnt about other research linked to the health food sector due to the fact that Omega oils, which in the past have been extracted from fish, an actually be captured from the algae which produces it. Some of the work that Matt and his team have been carrying out is how to optimise growing conditions and nutrient supply, especially understanding how different algal strains survive in different climates. It was a surprise to learn that algal pigments can be found in everyday products such as Smarties and wine gums! Finally, we were shown around the Plant Growth Facility. In this building there are lots of separate chambers in which all factors needed for plant growth can be controlled, such as humidity and light. This enables researchers to be able to drill down into the specific variables that might influence or affect a particular result. As we walked around the facility, we saw a range of plant species being tested on including wheat, potatoes and rice. These secure, air tight chambers also allow tests into plant pathogens to happen as they can be contained and assessed within a rigorous framework. We were really lucky to be able to explore the Sainsbury Lab and Algal Innovation Centre. It was so interesting to hear about all the research that is going on which could impact the agricultural industry in the future. As with all of our YIF events, attendees then had the opportunity to discuss the tours as well as talk about their experience and knowledge in the area. We chatted to some of the attendees to see what they got from attending the event, here is what they had to say: Matt White, Engineer, Cambridge Consultants The YIF visit was an excellent opportunity for industry members like ourselves to learn about the cutting-edge research being carried out in agricultural sectors. Having the opportunity to speak with leading researchers in algae growth and cultivation was fascinating and highly valuable for expanding our knowledge in this area! These events are great at increasing links between academia and industry to help us develop agricultural technology together more effectively. Pamela Ribone, Researcher, Sainsbury Lab The reason for attending the YIF visit was that I really wanted to try and apply what I have done so far with my research to a more applied science, and this was an incredible opportunity to know more about what is in between really basic science and the final application to a product. The Algal Innovation Centre looks like the kind of things I would like to go for the next step. From the visit I understood that they do laboratory research, but focusing on solving problems. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.agri-tech-e.co.uk/wp-content/plugins/civicrm/civicrm/extern/url.php?u=23903&qid=1491603 |
Description | Conference presentation by KC & PMM |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Posters presented at Synthetic Biology UK meeting describing our work in developing synthetic biology approaches in algae |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.biochemistry.org/events/synthetic-biology-uk-2022/ |
Description | Festival of Algae meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Industry/Business |
Results and Impact | Poster presentation by PMM. Audience interested in approaches developed and potential for exploitation |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.algae-uk.org.uk/events/a-festival-of-algae-2/ |
Description | KG Talk at AlgaeEurope 2020 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Due to the covid-19 pandemic, the event was held online in 2020. Around 250 researchers, industry and business partner with interests in algae biotechnology joined the four-day event. We gave a talk presenting the research in the lab and attended a Q&A session afterwards. Discussions with ongoing partners (e.g. Algenuity) and new partners continued throughout the event. |
Year(s) Of Engagement Activity | 2020 |
URL | https://algaeurope.org/participants-information/?utm_campaign=2911&utm_source=enormail&utm_medium=em... |
Description | Katrin Geisler and Payam Mehrshahi - Lead lecturers for a workshop titled "Expressing transgenes in Chlamydomonas and Phaeodactylum - a guide to commonly used methods for transformation of their nuclear genomes" |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Workshop titled "Expressing transgenes in Chlamydomonas and Phaeodactylum - a guide to commonly used methods for transformation of their nuclear genomes" was held on 10th - 11th December. Funding was secured from EIT-Food and AlgaeUK that enabled travel grants to be awarded to delegates from the UK, Italy, Germany, Turkey, Philippines, Brazil, Ghana, and cover their administration and catering costs. The aim of the workshop was to provide hands-on training in use and application of the latest technologies and methodologies for transformation and engineering of the microalgae Chlamydomonas and Phaeodactylum. The format included a daily programme of presentations at the Department of Plant Sciences teaching laboratory introducing delegates to different algal transformation methods, theory of techniques, standard operating procedures and best practices that result in successful transformation of these algae. The delegates were also introduced to the basics of advanced DNA assembly and cloning strategies. During the hands-on sessions delegates had the opportunity to handle, study under the microscope and prepare microalgae before transforming them with transgenes. The programme provided a great opportunity for postgraduate, postdoctorate and researchers to gain training in algal transformation techniques. We expect to run the course again in 2020. |
Year(s) Of Engagement Activity | 2019 |
URL | https://data.plantsci.cam.ac.uk/PlantMet/index.php/workshop-on-expressing-transgenes-in-chlamydomona... |
Description | Keynote presentation to AlgaEurope 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | AGS gave a keynote address to AlgaEurope 2022, the major conference for the algal biotech sector in Europe |
Year(s) Of Engagement Activity | 2022 |
URL | https://algaeurope.org/ |
Description | PDRA Gonzalo I Mendoza-Ochoa presented poster at: Aptamers in Bordeaux |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Mendoza-Ochoa GI, Mehrshahi P and Smith AG. "Thiamine riboswitches for controlling transgene expression in the alga Chlamydomonas reinhardtii". Poster presented at: Aptamers in Bordeaux; 2018 June 28-29; Bordeaux, France. |
Year(s) Of Engagement Activity | 2019 |
URL | http://www.aptamers-in-bordeaux.com/final-programme-2019/ |
Description | PM presented a talk at the EIT-Food event "Through the keyhole" |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | PM, LA, EH gave joint talk about acitivities in the Algal Innovation Centre. The presentation was titled "Through the Keyhole Algal Innovation Centre" |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.opencambridge.cam.ac.uk/events/through-laboratory-keyhole |
Description | Presentation at Plastid Preview 2019, University of York |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Harry Jackson (UCL), Pawel Mordaka (University of Cambridge) "(Re)design of the choroplast genome - towards a synthetic organelle', Presentation at Plastid Preview 2019, University of York, 2nd-3rd Sept 2019 |
Year(s) Of Engagement Activity | 2019 |
URL | https://www.mackinderlab.com/plastid-preview-2019.html |
Description | Presentations at AlgaEurope 2022 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Several posters were presented highlighting our research and R&D activities to the major conference for the algal biotech sector in Europe |
Year(s) Of Engagement Activity | 2022 |
URL | https://algaeurope.org/ |
Description | SRA Payam Mehrshahi and Dr Lorraine Archer presented via a video walk-through the Algal Innovation Centre for the EIT food workshop ALGAL BIOTECHNOLOGY 2020 - TECHNIQUES AND OPPORTUNITIES FOR THE SUSTAINABLE BIOECONOMY |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Due to Covid-19 restrictions the EIT Food Professional Development course was redesigned so that 3x 2day courses were combined and presented online once over a two-day course. As the lead organisation, the University of Cambridge team (Payam Mehrshahi, Matthew Davey, Lorraine Archer) produced daily programme of lecture/seminars that brought together leading international experts to introduce the theory of techniques, SOPs best practice and live demonstration of equipment and facilities. The course offered insights and examples from an industrial and entrepreneurial perspective, that helped the participants to start or improve their own algal-based business. The video walk-through of the Algal Innovation Centre at the University of Cambridge was designed by Payam and Lorraine to reflect the wide range of research involving algae that is carried out in the university and in this facility. The 30 minute video was followed by a 45 minute Q&A session during which participants asked about technical aspects of equipment, consumables, algae strains, culturing conditions and trouble shooting advice. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.eitfood.eu/media/documents/ALGAL_BIOTECH_DRAFT_TRAINING_COURSE_2020_FINAL_v10.pdf |
Description | SRA Payam Mehrshahi presented a poster at the International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | The conference was organised by the Society of Biological Engineering and brought together scientists and engineers from universities, industry and government working in all aspects of plant synthetic biology, plant bioengineering and plant biotechnology. Payam presented research from the lab that concern the use of synthetic biology to develop tools for regulation of gene expression in microalgae. This poster was well received by those in attendance with multiple questions raised to discuss the technical aspects of the molecular tools generated by our research group. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.aiche.org/sbe/conferences/international-conference-on-plant-synthetic-biology-and-bioeng... |
Description | SRA Payam Mehrshahi presented at the EIT food workshop ALGAL BIOTECHNOLOGY 2020 - TECHNIQUES AND OPPORTUNITIES FOR THE SUSTAINABLE BIOECONOMY |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | In 2020 due to the COVID restrictions, the EIT Food professional development course ALGAL BIOTECHNOLOGY - TECHNIQUES AND OPPORTUNITIES FOR THE SUSTAINABLE BIOECONOMY was held as a hybrid course. This course was developed and led by colleagues at The University of Cambridge (UK), Matis (Iceland) and Fraunhofer (Germany). The course attendees were graduate, postgraduate and industry professionals who are interested in gaining exposure to the latest insight into technical, commercial and policy concepts that impact algal biotechnology. Payam's talk titled "Genetic Engineering Approaches For Algal Biotechnology" was followed by a Q&A session. The range of questions was testament to the diversity of interests and stage in career of those in attendance. |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.eitfood.eu/projects/algal-biotechnology-techniques-and-opportunities-for-the-sustainable... |