Enhanced Research Capacity

Lead Research Organisation: John Innes Centre
Department Name: Contracts Office

Abstract

Heterologous expression systems are a key resource for our ISP as a whole. In the previous ISP we developed numerous such systems for generation and study of specific metabolites, proteins and whole pathways, and for pathway engineering. Examples include the production in N. benthamiana leaves of gram quantities of the triterpene ß-amyrin as a substrate for further experiments; investigation in N. benthaminana of proteins that regulate chlorophyll degradation in pea; production of the iridoid alkaloid intermediate strictosidine in yeast and the use of Streptomyces superhosts for expressing biosynthetic gene clusters from intractable bacteria. The need for multiple, flexible systems will increase as more, and more diverse, pathways and products are discovered, characterised and engineered. The N. benthamiana transient expression system has proved of particular value for the production of virus-like particles (VPLs) because of its safety, low cost and speed. Project Leader Lomonossoff has shown that these particles, which lack endogenous nucleic acid, can be used in diagnostics and for production of human and farmed animal vaccines. However, issues including scale, product stability and problems of appropriate expression of multiple genes must be solved in order to optimise the N. benthamiana system for this and other purposes.
In this Project we will enhance and optimise our capacity to use heterologous expression systems. The advent of the Leaf Systems® facility (2017) will provide a unique opportunity to produce proteins and metabolites in N. benthamiana on a scale suitable for our research and for investigation of the translational potential of our discoveries. We anticipate that a single batch of plants in this facility will produce up to 10 g of a given protein and 1 g of a natural product. To capitalise on this opportunity we need to improve and refine methods for production of VLPs (Objective 1), and tailor N. benthamiana for specific purposes (Objective 2), for example by removing leaf enzymes that modify metabolites of interest. We also need to increase the capacity and flexibility of other heterologous expression systems, to scale up production of low-abundance proteins and metabolites (Objective 3).

Publications

10 25 50
 
Description 2017-2018
We have determined that plant-based transient expression is a highly effective method for the production of virus-like particles. These are suitable of for near atomic resolution cryo-EM analysis which is enabling hitherto unknown virus structures to be elucidated. We have also had success with the production of secondary metabolites in plants.

2018/2019
Objective 4.1: Virus-like particle production in N. benthamiana

Transient expression has been used produce VLPs derived from enveloped viruses, particularly Dengue virus, serotype 1. Co-expression proteins PrM and E, which contain the antigenic determinants of the virus, resulted in the production of subviral particles. These are currently being assessed immunologically in collaboration with Public Health England (PHE).

Members of the family Luteoviridae cause important viral diseases and are usually phloem-limited, resulting in very low virus titres within infected plants. For this reason, there is little information available regarding the structure of their capsids. Transient expression of the coat proteins of members of the family results in the formation of virus-like particles (VLPs), the structures of which are being determined to near atomic resolution by cryo-EM (in collaboration with the University of Leeds). Such VLPs can potentially be used to develop novel diagnostic reagents and we are investigating this possibility using VLPs from the well-characterised virus, cowpea mosaic virus (CPMV). We have also used a combination of infection and transient expression to elucidate the 3-dimensional structure of a geminivirus by near atomic resolution cryo-EM and develop a model for assembly of these unique twinned particles (Hesketh EL, Saunders K, Fisher C, Potze J, Stanley J, Lomonossoff GP, Ranson NA..The 3.3 Å structure of a plant geminivirus using cryo-EM.Nat Commun. 2018 Jun 18;9(1):2369. doi: 10.1038/s41467-018-04793-6.

Research on CPMV has revealed that VLPs can be used to specifically encapsidate bespoke, non-viral RNA molecules (Kruse I, Peyret H, Saxena P, Lomonossoff GP. Encapsidation of Viral RNA in Picornavirales: Studies on Cowpea Mosaic Virus Demonstrate Dependence on Viral Replication. Virol. 2019 Jan 4;93(2). pii: e01520-18. doi: 10.1128/JVI.01520-18. Print 2019 Jan 15. Such encapsidated RNAs can be used for structural studies on RNA folding and can serve as in-tube diagnostic controls. Regarding the latter application, they have attracted the interest of several commercial entities.

Objective 4.2: Tailoring N. benthamiana
We have used N. benthamiana as a platform for the production and engineering of triterpenoids on a gram-scale (Stephenson MJ, Reed J, Brouwer B, Osbourn A. (2018).Transient expression in Nicotiana benthamiana leaves for triterpene production at a preparative scale. Journal of Visualized Experiments issue 138: Article Number: e58169 Published: AUG 2018 10.3791/58169. ; Reed J, Osbourn A. (2018). Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant Cell Reports 37 (10): 1431-1441. N. benthamiana lines with edits to knock-out peroxidase and competing alkaloid production have been generated and are being characterized. Additional glycosyl transferase candidates for silencing are currently being evaluated by a combination of expression analysis and in vitro activity assay. Additional edited lines in which 2-5 of these glycosyl transferases are knocked out will be generated starting in March-April 2019.
Exploitation Route The method is already of huge interest to structural virologists and several collaborations have already been established. Likewise the methods for the production of secondary metabolites will be of interest to the field and to numerous collaborators..
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Objective 4.1 A collaboration with Plant Bioscience Ltd (PBL) and Leaf Expression Systems (LES), both situated on the Norwich Research Park (NRP) has been developed to commercially exploit plant-based expression.This collaboration has focused on the production of virus-like particles of enveloped viruses, such Dengue virus, with the aim of producing novel vaccines against emerging viral threats. The availability of such vaccines clearly has both economic and societal benefits. They have also collaborated with external commercial entities on the deployment on in-tube controls for PCR-based diagnostics. Again, this will have economic benefits, particularly in the field of agriculture. A collaboration has been developed with the University of Leeds on the determination of the structure of virus-like particles (VLPs) from viruses which are either difficult, dangerous or impossible to isolate by conventional means. The availability of such novel structures provides improved understanding of virus architecture which lead to novel ways of controlling infections. Moreover, the VLPs can be used for the production of synthetic diagnostic reagents, such as ones based on adhirons (synthetic equivalents of antibodies), for the diagnosis of disease. Objective 4.2 Nicotiana benthamiana has been developed as a platform for engineering triterpenoids, compounds with numerous agronomic, medicinal and industrial applications. . Lines of N. benthamiana with edited genomes which could be used as an alternative to wild-type plants for protein production are under development. Such lines will likely have benefits in terms of the quality and quantity of the proteins with the concomitant economic impact.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic,Policy & public services

 
Description Attendance at Department for International Trade meeting
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
Impact Development of export opportunities
 
Description EC H2020
Amount € 8,286,008 (EUR)
Funding ID 774078 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 11/2017 
End 10/2021
 
Description EC H2020
Amount € 7,199,560 (EUR)
Funding ID 760331 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2018 
End 06/2022
 
Description Collaboration with Leaf expression Systems, NRP 
Organisation Leaf Expression Systems
PI Contribution We are collaborating with Leaf expression Systems (LES) to scale-up the production of several VLPs which have previously made on a small scale at JIC. these include eVLPs from CPMV and poliovirus. The constructs originally produced at JIC have been transferred to LES.
Collaborator Contribution The use of facilities and technology for the scaled-up production of VLPs with particular emphasis on process development and quality assurance.
Impact The collaboration has been active for less than 1 year so there are no outcomes to report yet.
Start Year 2017
 
Description Cryo-EM of VLP structures 
Organisation University of Leeds
Department Astbury Centre for Structural Molecular Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Production of VLPs in plants
Collaborator Contribution Cryo-EM analysis of the structure of plant-produced VLPs
Impact This is a multi-disciplinary collaboration using plant-based expression (JIC) to produce VLPs. The structure of these can be determined by near -atomic resolution cryo-EM (Leeds).
Start Year 2017
 
Description PBVAB Conference Presentation 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation "Combining transient expression and high resolution cryo-electron microscopy to produce novel virus-like particles" by Walden M., Meshcheriakova Y., Marsian J., Hesketh E., Ranson N., Lomonossoff G. P. PBVAB 2017, Albufeira, Portugal, ,5-7th June 2017
Year(s) Of Engagement Activity 2012,2014,2017
 
Description Audio Interview BBC Radio Norfolk 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Interview by G.P. Lomonossoff on Matthew Gudgin show on BBC Radio Norfolk, 27th Oct 2017. This was an interview based on the "Desert Island Discs" format and last 1 hour from 18:00-19:00.
Year(s) Of Engagement Activity 2017
 
Description Emerging viruses of zoonotic and veterinary importance Meeting 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk entitled "Can plant-based transient expression be used to combat emerging viral diseases?" G.P. Lomonossoff, Churchill College Cambridge, Emerging viruses of zoonotic and veterinary importance, 24th-26th July 2017.
Year(s) Of Engagement Activity 2017
 
Description Freethink media film 2018 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Web-based film about plant-based expression produced by Freethink media.
Year(s) Of Engagement Activity 2017
URL https://www.facebook.com/freethinkmedia/videos/1698762333516461/
 
Description Geminivirus talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk entitled "The Structural Features of a Begomovirus Particle Revealed by Cryo-Electron Microscopy" Saunders K., Hesketh E., Fisher C., Ranson N., Lomonossoff GP. The 2017 meeting of the American Society for Virology meeting at the University of Wisconsin-Madison, USA. 24th - 28h June 2017.
Year(s) Of Engagement Activity 2017
 
Description Glycosylation, Manchester 2017 
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 Presentation entitled "The mechanism of glycosylation and the consequences of glycoform on therapeutic protein functionality" by G.P.Lomonossoff. Manchester, UK,.Establishing International Partnerships in Industrial Biotechnology and Bioenergy - Improved glycoform-based biopharmaceutical production in plants. 18-20th Sept. 2017
Year(s) Of Engagement Activity 2017
 
Description HVCfP, Manchester 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk "Translating academic research - The Leaf Systems Experience" by G.P. Lomonossoff, 2017 High Value Chemicals from Plants annual meeting, Manchester, UK, 19 Oct 2017
Year(s) Of Engagement Activity 2017
 
Description JIC 50 Open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk entitled "Using plants as factories - from basic virology to applications" and participation in JIC 50 Alumnus Open day,17 Sep 2017
Year(s) Of Engagement Activity 2017
 
Description JIC ASM 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk entitled "Well, we Built it - now will they come?" by GP Lomonossoff, JIC Annual Science Meeting 2017, 12 Oct 2017
Year(s) Of Engagement Activity 2017
 
Description Lives in Science Workshop 2018 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Interaction with Year 11 Art students visiting JIC, 15 Jan 2018
Year(s) Of Engagement Activity 2017
 
Description Meeting on Norwich Research Park 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Presentation "Transient expressions of synthetic biology in plants" at Earlham Institute, Norwich Research Park, Meeting "Decoding and Recoding Biological Systems", 19/05/17
Year(s) Of Engagement Activity 2017
 
Description Norwich Bio Makers 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Involvement by GP Lomonossoff in Norwich Bio Makers "Building Nanostructures in Plants", St. Andrews Brew House, Norwich, UK 13 Dec 2017
Year(s) Of Engagement Activity 2017
 
Description Oral presentation at AAB Conference "Advances in Plant virology", 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Peyret H., Saxena P., Kruse I., Lomonossoff G. P.(2018) Studies on cowpea mosaic virus show that RNA encapsidation is dependent on replication: implications for the assembly of members of the order Picornavirales. AAB Conference "Advances in Plant virology", Jury's Inn, Birmingham UK 12-13th April 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at AAB Conference "Advances in Plant virology", 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P.(2018) Synthetic virus-like particles and how to make them in plants AAB meeting "Advances in Plant Virology", Jury's Inn, Birmingham 12-13th April 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at ISPMF Helsinki, 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P., Marsian J., Paley R., Hurdiss D. L., Ranson N. A.(2018) Global food security - Plant-made Virus-Like Particles as a candidate vaccine against Nervous Necrosis Virus. Oral presentation at the 3rd meeting of the International Society for Plant Molecular farming (ISPMF), 11-13th June 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at ISPMF meeting, Helsinki 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Peyret H., Lomonossoff G. P.(2018) Using a synthetic biology approach to develop a novel, easy to use, and open access transient expression system
Helsinki, Finland, 3rd ISPMF conference, 11-13th June 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at Ninth International Virus Assembly Symposium, Madeira 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Castells Graells R., Johnson J. E., Lomonossoff G. P.(2018) Studying virus maturation stages with an insect virus Madeira - Portugal, Ninth International Virus Assembly Symposium, 6th-10th May 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at SEB Florence 2018 symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P.(2018) Transient expression of virus-like particles for use in biomedicine and bionanotechology SEB Florence 2018 symposium "Plant Biotechnology for health and nutrition,
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at VLPNPV, Bern 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P., Walden M., Steele J., Meshcheriakova Y., Marsian J., Saunders K., Thuenemann E., Peyret H., Hesketh E., Thompson R.(2018) Plants as a highly effective platform for VLP production VLPNPV Meeting Bern, Switzerland, 25-27th Sept. 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at VLPNPV, Bern 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Saunders K., Hesketh E. L., Fisher C., Ranson N. A., Lomonossoff G. P.(2018) The generation of aberrant geminivirus-like particles in infiltrated plant leaves Inselspital, Universitatsspital, Bern, Switzerland. The Fifth International Conference on: Virus-Like Particle & Nano-Particle Vaccines - VLPNPV 2018 25-27th September 2018
Year(s) Of Engagement Activity 2018
 
Description Presentation by D.Ponndorf, Korea 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Ponndorf D., Peyret H., Meshcheriakova Y., Dobon Alonso A., Lomonossoff G.(2018) Virus-like particles from plants: Versatile tools for research in vaccine development
Korean Society for Plant Biotechnology: from Farming to Pharming .
Year(s) Of Engagement Activity 2018
 
Description Presentation to DBT consortium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plant-expressed Proteins, The technology and its use for BTV Meeting for BBSRC/DBT India Grant Consortium, School of Veterinary Sciences, Sutton Bonnington, University of Nottingham, UK
Year(s) Of Engagement Activity 2018
 
Description Science Innovation Showcase 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Presentation "Growing vaccines in plants" and networking by G.P. Lomonossoff at Science Innovation Showcase (SIS) held at JIC 7- 8th Feb. 2018
Year(s) Of Engagement Activity 2017
 
Description Seminar at NUIG 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Synthetic virus-like particles and how to make them in plants Seminar at National University of Ireland - Galway (NUIG), Galway, Republic of Ireland.
Year(s) Of Engagement Activity 2018
 
Description Seminar, Glasgow 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P.(2018) Plant-based transient expression of virus-like particles for use in biomedicine and bionanotechology Seminar at University of Glasgow, Glasgow, UK
Year(s) Of Engagement Activity 2018
 
Description Talk at APHA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Deliver presentation entitled "A Leaf Expression platform for the production of vaccine candidate materials, mAbs and for in vitro diagnosis" at the Animal and Plant Health Agency (APHA), Weybridge, UK.
Year(s) Of Engagement Activity 2017
 
Description VLPNPV Singapore 2017 - 2 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk "Tackling Emerging Viruses - Plant Made Virus Like Particles As Vaccines Against Enveloped Viruses", Ponndorf D., Peyret H., Dobon Alonso A., Meshcheriakova Y., Lomonossoff G.P., Biopolis, Singapore Virus-like particle and nano-particle vaccines - VLPNV 2017, 29th November - 1st December 2017
Year(s) Of Engagement Activity 2017
 
Description VLPNV Singapore 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk "Designer length TMV-based nano-rods for nanotechnological applications" K. Saunders G.P. Lomonossoff, Biopolis, Singapore Virus-like particle and nano-particle vaccines - VLPNV 2018 29th November - 1st December 2017.
Year(s) Of Engagement Activity 2017
 
Description Vienna Meeting 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation "Transient Expressions of Synthetic Biology in Plants" by G.P. Lomonossoff, 4th International Conference Plant Transformation & Biotechnology, Vienna, Austria 30/06/17
Year(s) Of Engagement Activity 2017
 
Description participation in Royal Society annual "Labs to Riches" event, 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P.(2018) Participation in Royal Society annual "Labs to Riches" event, Royal society, London, UK 20th March 2018
Year(s) Of Engagement Activity 2018