Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensembles
Lead Research Organisation:
John Innes Centre
Department Name: UNLISTED
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
Operons (groups of physically linked and co-regulated genes) are common features of bacterial genomes. Until recently gene order in eukaryotes has been assumed to be random. However there are now many examples of āoperon-likeā gene clusters in eukaryotes. These include gene clusters for use of different nutrient sources in yeasts, for synthesis of antibiotics and toxins by filamentous fungi, for innate and adaptive immunity in animals and for production of defense compounds in plants. Phylogenetic evidence indicates that these gene clusters are unlikely to have arisen by horizontal gene transfer and that in most cases they have been assembled within lineages in recent evolutionary history by gene duplication, acquisition of new function and genome reorganization. Selection for the formation of such gene clusters is likely to be intense, driven by the need to rapidly adapt to changing environmental conditions, and implies remarkable genome plasticity. Elucidation of the mechanisms by which these adaptive clusters form will enable us to understand how eukaryotes rapidly adapt to changes in their environment through modification of their metabolism.
This project will involve investigation of mechanisms of formation and/or the regulation of operon-like metabolic gene clusters in cereals. The project is part of a multi-disciplinary EPSRC-funded collaborative research project on the development of methodology for synthetic evolution of complex genetic ensembles in bacteria and plants that includes groups in the UK (Dr Susan Rosser, University of Glasgow; Professor Paul Freemont, Imperial College, London; Professor Declan Bates, University of Leicester) and the US (Professor Josh Leonard, Northwestern University; Professor Jay Keasling, UC Berkeley).
This project will involve investigation of mechanisms of formation and/or the regulation of operon-like metabolic gene clusters in cereals. The project is part of a multi-disciplinary EPSRC-funded collaborative research project on the development of methodology for synthetic evolution of complex genetic ensembles in bacteria and plants that includes groups in the UK (Dr Susan Rosser, University of Glasgow; Professor Paul Freemont, Imperial College, London; Professor Declan Bates, University of Leicester) and the US (Professor Josh Leonard, Northwestern University; Professor Jay Keasling, UC Berkeley).
Planned Impact
unavailable
Organisations
People |
ORCID iD |
| Anne Osbourn (Principal Investigator) |
Publications
Colloms SD
(2014)
Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.
in Nucleic acids research
Delis C
(2011)
Role of lupeol synthase in Lotus japonicus nodule formation.
in The New phytologist
Field B
(2011)
Formation of plant metabolic gene clusters within dynamic chromosomal regions
in Proceedings of the National Academy of Sciences
Field B
(2012)
Order in the playground: Formation of plant gene clusters in dynamic chromosomal regions.
in Mobile genetic elements
Kemen AC
(2014)
Investigation of triterpene synthesis and regulation in oats reveals a role for Ć-amyrin in determining root epidermal cell patterning.
in Proceedings of the National Academy of Sciences of the United States of America
Kliebenstein DJ
(2012)
Making new molecules - evolution of pathways for novel metabolites in plants.
in Current opinion in plant biology
Mugford ST
(2013)
Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat.
in The Plant cell
NĆ¼tzmann HW
(2014)
Gene clustering in plant specialized metabolism.
in Current opinion in biotechnology
Osbourn A
(2012)
Finding and analyzing plant metabolic gene clusters.
in Methods in enzymology
Osbourn AE
(2012)
Synthetic biology. 4th New Phytologist Workshop, Bristol, UK, June 2012.
in The New phytologist
| Description | A grand challenge in synthetic biology is the need for technologies that enable the construction of novel and complex functions in biological systems. When these functions involve the expression and coordination of multiple genes, building them becomes increasingly difficult. Assembling multigenic functions in an organism by an iterative approach is both laborious and difficult, since the engineered genes and their products often interact strongly with both one another and the pre-existing native functions in the organism. Such challenges have posed major hurdles to efforts to engineer metabolism in microbes and plants. The goal of this project was to develop a synthetic system that harnesses the power of multiple natural mechanisms to enable synthetic biologists to generate, diversify, and refine complex multigenic functions. As part of this project we have developed a recombinase-based platform for the assembly and engineering of metabolic pathways in microbes. We have also exploited the discovery that the genes for the biosynthesis of diverse natural product pathways in plants are organised in clusters like beads on a string (in a manner reminiscent of bacterial operons) to mine genomes for new natural product pathways. |
| Exploitation Route | Our findings open up unprecedented opportunities for genome mining for discovery of entirely new metabolic pathways and chemistries of agronomic and medicinal importance. The genes of new candidate clustered pathways can be synthesised, assembled and fed into microbial or plant expression platforms for functional analysis so providing a high throughput platform for natural product discovery. The discovery of new clusters will provide parts, biocatalytic assemblies, biosensors and orthogonal regulatory circuits for metabolic engineering and other synthetic biology applications. |
| Sectors | Agriculture Food and Drink Chemicals Creative Economy Education Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |
| URL | https://www.jic.ac.uk/directory/anne-osbourn/ |
| Description | Our findings have opened up unprecedented opportunities for genome mining for discovery of entirely new metabolic pathways and chemistries of agronomic and medicinal importance. The genes of new candidate clustered pathways can be synthesised, assembled and fed into microbial or plant expression platforms for functional analysis so providing a high throughput platform for natural product discovery. The discovery of new clusters will provide parts, biocatalytic assemblies, biosensors and orthogonal regulatory circuits for metabolic engineering and other synthetic biology applications. This work has been instrumental in enabling the lead PI to develop a prominent international profile in the international synthetic biology community and in securing prestigious funding for plant synthetic biology and metabolic engineering projects, e.g. Professor Osbourn is the co-Director of the BBSRC/EPSRC-funded OpenPlant synthetic biology research centre led by the University of Cambridge and the John Innes Centre; she was also recently awarded funding as part of an NIH 'Genome to natural products (GNPN)' network consortium to establish a pipeline for mining plant genomes for new metabolic clusters. |
| First Year Of Impact | 2012 |
| Sector | Agriculture, Food and Drink,Chemicals,Creative Economy,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Cultural Societal Economic |
| Title | Transient plant expression technology for triterpene production at preparative scale |
| Description | We have develop a method for transient heterologous expression of biosynthetic enzymes in N. benthamiana for production of high-value triterpenes. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. We have developed a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2017 |
| Provided To Others? | Yes |
| Impact | Generation of gram-scale quantities of 98% pure triterpenes and demonstration that we can rapidly carry out combinatorial expression of enzymes from our triterpene toolkit to generate known and new-to-nature compounds. This had attracted considerable interest from industry and led to four new projects directly funded by different companies in the pharma, ag, food and drink and home and personal care sectors. |
| URL | https://www.jove.com/video/58169/transient-expression-nicotiana-benthamiana-leaves-for-triterpene |
| Description | SAW Seminar - Sowing the seeds for science outreach |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | Presented creative outreach projects and ways to capture impact at the department seminar to encourage scientists to be bold with their outreach plans and to demonstrate the value of documenting the process and the outcomes for reporting, reflection and to improve the method. |
| Year(s) Of Engagement Activity | 2020 |