Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensembles

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

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 e.g engineering metabolism or assembly of cDNAs from a metagenomic library to synthesize novel small molecules, building them by an iterative approach is laborious and difficult. Nature has however evolved mechanisms to deal with such complexity. Here we propose 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. The core of our technology will be based on a bacterial integrons, which are natural cloning and expression systems that assemble multiple open reading frames (gene cassettes), using site-specific recombination and conversion to functional genes by expression from an internal promoter. The ability to capture disparate individual genes and physically link them in arrays suitable for co-expression is a trait unique to these genetic elements. The result is an assembly of functionally coordinated genes facilitating the rapid evolution of new phenotypes.We propose to develop a novel technology platform to revolutionise the process of engineering complex multigenic functions by harnessing the power of integrons for continuous directed evolution. Specifically, we will 1) construct and characterise a synthetic integron-based system (syntegron) for continuous directed evolution, 2) develop principles for effectively using syntegron technology via proof-of-principle experiments and computational optimisation, and 3) use synegron technology to assemble and optimise complicated, multi-gene, biosynthetic pathways for natural products (e.g., Taxol). A key step in the creation of syntegrons will be the generation of a toolbox of well-characterized integron integrases with efficient, controllable recombination frequencies and a range of diverse but specific insertion sites. In order to further enhance the potential diversification of the syntegron system we will develop a tunable, inducible lateral gene transfer technology based around conjugative exchange of plasmids and transduction-mediated transfer of phagemid. We will use two complementary approaches to develop strategies for deployment of syntegrons in plants 1) Iidentification and exploitation of functional equivalents of microbial integron platforms in plants 2) test and utilize microbial syntegron elements in plants using plastid-based technology.Bioinformatic approaches will be used to characterize components of our directed evolution in syntegrons (DES) system and the experimental products of this system. A fundamental principle of Systems Engineering is that, as a system become more complex and the number of parameters chosen by the designer increases, intuition breaks-down and computer-aided design (CAD) becomes essential. This principle is just beginning to be appreciated in the field of Synthetic Biology. In this project, we aim to make a step change in this direction, by embedding computational modeling and optimization tools in the heart of the proposed experimental framework.One of the primary uses of the Syntegron technology will be the construction of metabolic pathways to improve known pathway metabolic flux and unknown metabolic pathways. To demonstrate the usefulness of Syntegrons, we will begin with a known challenging metabolic pathway - the mevalonate-based isoprenoid biosynthetic pathway. After establishing that the syntegron system functions in model applications, we will attempt to evolve a novel multigenic function - the biosynthesis of the economically and medicinally valuable molecule Taxol in bacteria demonstrating the utility of the syntegron platform for evolving a wide variety of complex multigenic functions that could not feasibly be constructed or discovered by other means.

Planned Impact

Commercial Beneficiaries. The Syntegron Platform is a generic technology for Synthetic Biology applications and will therefore be of significant interest to a wide range of global companies who are involved in the sale of enabling research technology e.g. Invitrogen and Promega. In addition companies (e.g. Amyris Biotechnologies, Dow, Genencor, Genzyme, Novozymes) who use metabolic and pathway engineering for strain improvement, enhanced or novel natural product production would also benefit from syntegron technology due to the potential for significantly speeding up R&D, expanding product range and enhancing cost effectiveness. Ultimately syntegron technology will be of great interest to a large number of companies in application areas like Bioenergy, Biopharmaceuticals, Pharmaceuticals, Agriculture and Bioremediation via research collaborations and access to IP and know-how via licensing deals. As a consequence the UK and US economies will benefit from the formation of spin out companies and/or industrial collaborations to commercialise the proposed Syntegron technology. Communication and engagement with industrial beneficiaries. The PI team have between them a great deal of experience and an excellent track record in collaborating with industry and transitioning academic research into the commercial sector. JK is a co-inventor of 4 patents and 17 pending patent applications, and is the founding scientist of Amyris Biotechnologies. PF is co-founder of the spin-out company Equinox Pharma specialising in in silico drug discovery and has 2 patents under review. AO holds a patent on genes for natural product synthesis in plants with 3 further patent applications pending. DB has collaborations with leading figures across the European aerospace control research community, including Airbus, Volvo, Alstom, DEIMOS and the European Space Agency. SR holds 1 patent and has 2 under review and led a large multidisciplinary BBSRC industrial LINK project with Arcadis GMI an international environmental consultancy and a major car manufacturer. The project PIs are committed to continued engagement with industry and will ensure that any IP generated will be fully protected and exploited. Capacity building beneficiaries. The project will contribute to undergraduate and postgraduate training in Synthetic Biology, providing exposure to the interdisciplinary developments of the project. For example in the UK the EPSRC Centre for Synthetic Biology and Innovation at Imperial College organises a final year UG module in Synthetic Biology (PF) and a one years masters programme in Synthetic Biology. The annual International Genetically Engineered Machine (iGEM) competition provides further training for undergraduate students. Four of the PIs on this proposal (PF, JK, JL, SR) have experience in co-ordinating and mentoring iGEM teams. Outreach beneficiaries. The project will contribute through engagement with schools and more broadly with society. The Science, Art and Writing (SAW) initiative, established by AO, is an established cross-curricular science education programme for (www.sawtrust.org). SAW uses themes and images from science to stimulate scientific experimentation, art and creative writing. The initiative provides opportunities for scientists to go into schools to work with teachers, children and other experts in innovative ways. AO and JL, with their research groups, will take research science into primary schools in the UK and the US with Synthetic Biology as the theme. Projects will be evaluated via monitoring in the schools and feedback forms. The outputs will be disseminated through the children's work, events, publications and the SAW Trust website. The participating schools will also be encouraged to share their experiences and interpretations of synthetic biology through their own websites and through links between the schools. Benefits realisation will be monitored by the Project Management Group

Publications

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Boutanaev AM (2015) Investigation of terpene diversification across multiple sequenced plant genomes. in Proceedings of the National Academy of Sciences of the United States of America

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Bowyer J (2016) Mechanistic Modeling of a Rewritable Recombinase Addressable Data Module. in IEEE transactions on biomedical circuits and systems

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Bowyer J (2015) Development and experimental validation of a mechanistic model of in vitro DNA recombination. in Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference

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Delis C (2011) Role of lupeol synthase in Lotus japonicus nodule formation. in The New phytologist

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Fogg PC (2014) New applications for phage integrases. in Journal of molecular biology

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Kemen AC (2014) Investigation of triterpene synthesis and regulation in oats reveals a role for ß-amyrin in determining root epidermal cell patterning. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have demonstrated that genetic constructs for metabolic pathways can be assembled rapidly, efficiently and cheaply. This technology is now being used as part of an industrial collaboration funded by the TSB with the SME Ingenza.
We have identified novel mechanisms in the way that the recombinase PhiC31 recombines DNA.
We have made significant discoveries about the prevalence, structure and regulation of plant gene clusters encoding secondary metabolic pathways.
We have developed computation tools to exploit these recombinases and genetic clusters.
Exploitation Route These technologies have general use in DNA assembly and also in the production of commercially important plant secondary metabolites.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description We have demonstrated that genetic constructs for metabolic pathways can be assembled rapidly, efficiently and cheaply. This technology is now being used as part of an industrial collaboration funded by the TSB with the SME Ingenza.
First Year Of Impact 2012
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Industrial Biotechnology Innovation Centre Accelerate Award
Amount £219,170 (GBP)
Organisation Industrial Biotechnology Innovation Centre 
Sector Academic/University
Country Unknown
Start 10/2015 
End 11/2016
 
Description Responsive Mode
Amount £1,757,302 (GBP)
Funding ID EP/K034359/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 04/2013 
End 03/2018
 
Title Novel methods for gene assembly 
Description We have developed (and are continuing to develop) new methods for rapid efficient assembly of DNA fragments using serine integrases. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact First version of the method is published (S.D. Colloms et al., Nucleic Acids Res. 42, e23). 
 
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