(Re)design of the chloroplast genome - towards a synthetic organelle.

Lead Research Organisation: University College London
Department Name: Structural Molecular Biology

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

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.

Publications

10 25 50

publication icon
Larrea-Álvarez M (2021) The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants. in International journal of molecular sciences

 
Description A major aim of this project is to determine how to reduce the size of the DNA genome ('plastome') within the chloroplast compartment of algal and plant cells to the minimum size for viability. This minimal chromosome could then be used as a 'naive chassis' onto which we add a suite of genes for making any designer product. The deletion process involves systematically removing all non-essential DNA regions whilst not affecting the replication and functioning of the plastome. We have therefore had to develop new synthetic biology methods for precision removal of target regions (i.e. "DNA surgery") that do not leave behind any unwanted DNA (e.g. a selectable marker gene), as well as developing a simple reporter system based on the nano-luciferase bioluminescent protein for quickly assaying chloroplast gene expression. We have now employed this to create viable transgenic lines that carry large deletions in the plastome. A second part of the project focuses on testing in vivo whether recoding of endogenous plastome genes using a genetic code of only 51 codons is feasible. We have now demonstrated this by recoding seven genes with now apparent phenotypic effect, indicating that a synthetic plastome could be designed based on this 51 codon table. A final part of the project involves developing fast, standardised methods for building novel genes to be inserted into the plastome. We have therefore developed a 'one-step' DNA assembly method termed STEP ("Synbio Toolkit for Engineering the Plastome") that has greatly accelerated and simplified our ability to engineer the chloroplast. Each of these molecular tools represent significant advances towards the ultimate goal of the project to build completely synthetic, designer plastomes for a range of biotechnology applications.
Exploitation Route We intend to publish all our findings in open access journals, and make the tools available to all those interested in using chloroplast engineering for develop algae as light-driven cell factories for synthesising novel products.
Sectors Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
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 01/2027
 
Description On-line presentation at webinar "Algal Biotechnology for Sustainable Aquaculture in 2021 and Beyond" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The webinar was organised by the National Center for Genetic Engineering and Biotechnology (BIOTEC) in Thailand to bring together researchers and industries interested in using algal biotechnologies in the aquaculture sectors. Over 200 participants joined from all round the world and there was significant engagement in the Q&A sessions.
Year(s) Of Engagement Activity 2021
 
Description Online workshop "Algal Biotechnology for Sustainable Aquaculture" 
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 An on-line workshop organised by colleagues in Bangkok with the aim of engaging with researchers and industry. Approximately 200 engaged with the event which led to various follow-up discussions with researchers and industries in SE Asia.
Year(s) Of Engagement Activity 2021
 
Description Presentation at part of the Clean Tech Challenge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Presentation and discussion exploring the use of algae for clean technologies.
Year(s) Of Engagement Activity 2019
URL https://londonevolution.org/homepage/past-events/trips/2019-2/saul-purton-clean-tech-using-microalga...
 
Description Presentation of Algae-UK to delegates attending EIT-Food Course, Cambridge December 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The aim of this professional development course was to provide introductory hands-on training and theory in algal biology, culturing, growth and subsequent molecular and metabolite analysis under laboratory and small to large scale growth facilities, as well as giving business information and networking opportunities specifically tailored towards the algal bioeconomy.
Year(s) Of Engagement Activity 2019
URL https://www.eitfood.eu/programmes/algal-biotechnology-techniques-and-opportunities-for-the-sustainab...
 
Description School visit (Dunottar School, Reigate) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentations and discussion activity given by Prof Saul Purton and Dr Henry Taunt to years 10 & 11 part of their Science Week.
Year(s) Of Engagement Activity 2019
URL https://www.dunottarschool.com/news-events/latest-news/scientists-visit-dunottar-school-to-speak-at-...