An improved bioproduction system for proteins and small molecules

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Global demand for quantities of vaccines and therapeutic molecules is growing. Plants, in particular, are the source of a great diversity of biologically active small molecules and a great many natural products found in plants are used as human therapies. However, these chemicals are often found in low abundance or are produced in species that are difficult to mass-cultivate requiring either chemical synthesis or the transfer of the genetic pathway to an alternative biological host in order to produce compounds at sufficient quantities.

While microorganisms have proven to be exceptionally powerful for manufacturing therapies, some are not easily produced in high yields. There is particular interest in platforms that are able to respond rapidly to new disease threats, for example, the production of vaccines. Plants have been shown to be capable of efficient expression of therapeutic proteins and secondary metabolites. In a process commonly known as 'molecular pharming' plants have been demonstrated to be capable of producing a very large number of vaccine doses in just a few weeks.

Gene expression can be complicated by the endogenous metabolism of the host diverting intermediates or performing unwanted modifications of expressed molecules. Much work has been done to tailor specific strains of bacteria and yeasts to increase production of compounds. However, to date, little effort has been spent on improving the plant production chassis, partly due to a lack of available tools. New technologies now allow us to take targeted approaches to modifying plant genes. We have identified genes expressed by the plant that are likely to be deleterious to heterologous bioproduction of small molecules. We will now make new lines of Nicotiana benthamiana, a relative of tobacco from Northern Australia, that are improved in their ability to produce small molecules of interest. We will then measure the impact of the changes that we have made by assessing the ability of our new lines to produce greater quantities of desirable new proteins and metabolites. This work will add to our knowledge of the metabolism of plants, helping us to understand how it responds to perturbation. It will also lead towards the production of plants that are genetically tailored for the production of different classes of therapeutic molecules

Technical Summary

Nicotiana benthamiana has enormous potential as a host for expression of small molecules and protein but little has been done to optimise this host. A major problem is that the endogenous metabolism performs unwanted modifications of heterologously expressed molecules. Transient expression in N. benthamiana is achieved by agroinfiltration in which cultures of Agrobacterium tumefaciens carrying genes of interest on binary plasmid vectors are infiltrated into leaves. Using transcriptome/RNA-seq data of plants following agroinfiltration of cultures carrying constructs encoding metabolic pathways, we have identified N. benthamiana genes that are upregulated. We have shortlisted several candidate genes in which we will induce mutations and deletions using RNA-guided Cas9 from the CRISPR system. We hypothesise that by knocking out these genes, heterologously expressed small molecules and proteins will not be derivatised. We will also identify additional targets for targeted mutagenesis by screening an expanded set of candidate genes identified in our RNA-seq data using transient approaches to downregulation (Virus Induced Gene Silencing (VIGS)).

Although lab-strains are disarmed for their ability to produce tumours, A. tumefaciens is a plant pathogen and several studies have reported that modulating various aspects of plant immunity can:
i) improve transient expression in Arabidopsis
ii) increase the rate of Agrobacterium-mediated transformation in Arabidopsis and N. benthamiana
iii) allow growth of A. tumefaciens within leaves
We will assess the impact of reducing the pathogen-triggered immune response on yield of heterologous molecules expressed via agroinfiltration.

Our overall aim is to produce lines of N. benthamiana that are able to produce proteins and metabolites at increased yields and purity. We will assess this by testing the abundance and purity of proteins and metabolites heterologous expressed from constructs already in use in our laboratories.

Planned Impact

Genome engineering technologies are widely anticipated to transform fundamental research in the near term. The academic plant community will benefit from the availability of lines of N. benthamiana with specific genes knocked out. Programmable nucleases have been demonstrated in several plant species in the past two years but very few stable knock-out lines of Nicotiana benthamiana are available to the research community at present. Programmable nucleases also promise wider benefits to agricultural productivity as new traits are demonstrated in crops and feed through to breeding programmes. This proposal applies the use of RNA-guided Cas9 for engineering N. benthamiana, a model allotetraploid plant. Our project will create a large number of lines that will help to establish the technology as a routine tool for basic plant science. Moreover, our analyses will add to the understanding of how N. benthamiana responds to the presence of toxic and foreign molecules and perturbation of its secondary metabolism.

There is particular interest in platforms that are able to respond rapidly to new disease threats, for example, for the production of vaccines. Plants have been demonstrated as being capable of producing molecules at commercial scales but, despite this, little effort has been put into improving the plants for bioproduction, particularly of high-value metabolites from other plant species. As one of the first studies aiming to improve a vascular plant chassis for improved production of small molecules our results will give a clear direction of how to achieve this. Ultimately, this project will contribute to the BBSRC's aspirations to develop new approaches and technologies to enable a bioeconomy. We will seek to facilitate this by communicating our results widely, particularly with scientists and commercial enterprises involved in developing plant chassis of bioproduction.

Publications

10 25 50
 
Title Nicotiana, biofoundry. 
Description An artistic interpretation inspired by plant systems for bioproduction of natural products developed with artist and designer, Karen Ingram. 
Type Of Art Artwork 
Year Produced 2018 
Impact Published as part of 'Convergent Visions' Faesthetic #15 produced in partnership with SXSW for the SXSW Art Program 2018. Presented at SXSW Interactive 2018. 
URL http://think.faesthetic.com/archives/9687
 
Description Our primary objective is to improve the plant, Nicotiana benthamiana, as a chassis for the bioproduction of small molecules. N. benthamiana is currently used for heterologous protein expression because young plants (up to 4 weeks) are particularly amenable to 'agroinfiltration', which is transient expression following infiltration into the leaves of a culture of Agrobacterium tumefaciens carrying genes of interest on a binary plasmid vector. The speed and ease of this method meant that it has been adopted for the mass production of therapeutics including vaccines. However, the expression of some proteins results in cell death and the production of high-value small molecules is hampered by unwanted modifications (oxidation, glycosylation, glutathionylation, proteolysis) by endogenous genes.

Our first aim was to identify N. benthamiana genes products likely to interfere with the heterologous expression of proteins and small molecules. We have done this by comparing genes identified as being up-regulated following agroinfiltration against the types of chemical modifications observed on our heterologous-expressed molecules. In many cases, we observed correlations in the types of genes upregulated and in the chemical modifications observed. This enabled us to select candidate genes by (a) their predicted function and (b) expression profile following agroinfiltration.

For the high-confidence targets (or combinations of targets), we designed constructs for targeted mutagenesis using CRISPR/Cas9. We tested these constructs in transient assays to verify their ability to induce mutations at the target. These constructs are being used to produce stable lines. To date, we have built and tested the majority of these constructs and they have been used to produce primary transgenic lines with mutations in target genes. Many of these lines have been genotyped and the seed has been collected. Others are still in the transformation process or are maturing in the greenhouse. In the next generation, we are verifying the genotype and presence of the transgene. In the coming months, we will assess heterologous production in these plants, testing for any differences in chemical modifications of target products.

Many of the enzyme families of interest are very large, meaning that there are hundreds of potential enzymes encoded in the N. benthamiana genome that could perform the identified modifications/derivatisations. Although we have prioritised a number of targets according to their expression profiles (upregulated in agroinfiltrated plants and/or highly expressed in leaves), we also planned to identify individual enzymes capable of acting on our substrates of interest. We had originally planned to do this by performing Virus-Induced Gene Silencing of target genes, followed by heterologous expression of the molecule of interest. However, advances in the ability to produce and characterise plant proteins using cell-free-protein synthesis have made characterisation of individual proteins more attractive as the resulting data is less ambiguous. We are therefore currently testing substrate specificity of specific enzyme candidates. In addition, we are investigating if different degrees or types of product derivatisation are found if we express our heterologous pathway in different cellular compartments.

Finally, we have performed Virus-Induced Gene Silencing of genes involved in pathogen recognition in order to compromise the plant's innate immune responses to A. tumefaciens. We are currently testing if this has any impact on the heterologous production of proteins and small molecules.

Although ongoing, our experiments are increasing our knowledge of the rich endogenous metabolism of N. benthamiana as well as helping us to understand how it responds to perturbation.
Exploitation Route Knowledge of enzyme specificity can be used to elucidate biosynthetic pathways as well to identify genes to use in biosynthesis pathways.
Knowledge of plant responses to foreign molecules can be used to improve plants are production chassis for proteins and high-value molecules used in industry and healthcare. Metabolically engineered plants with improved yield and purity of target products will demonstrate the potential of sustainable biomanufacturing in photosynthetic chassis.
Sectors Manufacturing, including Industrial Biotechology

 
Description Invited submission to the ad hoc technical expert group (AHTEG) on digital sequence information on genetic resources of the the Convention on Biological Diversity
Geographic Reach Multiple continents/international 
Policy Influence Type Gave evidence to a government review
URL https://www.cbd.int/abs/dsi-gr/ahteg.shtml#submissions
 
Description Policy Round Table on Synthetic Biology, UK Cabinet Office
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description Science and Technology Committee, House of Commons - implications to the UK of the 2018 Euporean Court of Justice ruling on Genome Editing
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Synthetic Biology Expert Roundtable, Department for Business, Energy and Industrial Strategy
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description OpenPlant Fund - Cell-free protein synthesis as a resource for generating plant proteins
Amount £5,000 (GBP)
Organisation University of Cambridge 
Sector Academic/University
Country United Kingdom
Start 06/2018 
End 06/2019
 
Description Nicotine-free benthamiana 
Organisation University of Copenhagen
Country Denmark 
Sector Academic/University 
PI Contribution Leading joint experiment to develop nicotine free plants
Collaborator Contribution Post-doctoral reserach associate to work on progressing the project in our laboratory for one month.
Impact New plant lines
Start Year 2017
 
Description Sustainable Bioproduction of Pheromones for Insect Pest Control in Agriculture 
Organisation National Institute of Biology
Country Slovenia 
Sector Charity/Non Profit 
PI Contribution Co-applicant on funding application
Collaborator Contribution Co-applicant on funding application
Impact Sucessful funding application (ERA-CoBioTech).
Start Year 2017
 
Description Sustainable Bioproduction of Pheromones for Insect Pest Control in Agriculture 
Organisation Spanish National Research Council (CSIC)
Department Institute for Plant Molecular and Cellular Biology
Country European Union (EU) 
Sector Public 
PI Contribution Co-applicant on funding application
Collaborator Contribution Co-applicant on funding application
Impact Sucessful funding application (ERA-CoBioTech).
Start Year 2017
 
Description Sustainable Bioproduction of Pheromones for Insect Pest Control in Agriculture 
Organisation Technical University of Darmstadt
Country Germany 
Sector Academic/University 
PI Contribution Co-applicant on funding application
Collaborator Contribution Co-applicant on funding application
Impact Sucessful funding application (ERA-CoBioTech).
Start Year 2017
 
Description 2017 Youth STEMM Gold Awards 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Presentation on synthetic biology followed by a practical workshop on designing genetic circuits. Sudents then presented their ideas, asked technical questions and discussed the potential impacts of synthetic biology and biotechnology on society.
Year(s) Of Engagement Activity 2017
URL http://ysawards.co.uk
 
Description Gatsby plant science summer school: engineering plants for farming and pharming 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Presented a lecture and particilaped in a discussion session with undergraduates as part of the Gatsby Plant Science Summer School. The summer school is an opportunity for first year undergraduate students in the UK to discover the challenge and opportunities of studying plant science. 76% of students rated this particular talk as good or very good with some very positive comments such as, 'I realised how much I loved to learn about Synthetic aspect of biology after the talk by Dr Nicola Patron
Year(s) Of Engagement Activity 2018
URL http://intobiology.org.uk/dr-nicola-patron-engineering-plants-for-farming-and-pharming/
 
Description Interview, Farming Today, BBC Radio 4 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with BBC Radio 4 Farming Today "Anna Hill visits the Earlham Institute in Norwich where scientists are gene mapping wildflowers to find out which genes govern the creation of molecules with medicinal potential."
Year(s) Of Engagement Activity 2019
URL https://www.bbc.co.uk/programmes/m000c4z4
 
Description Molecular Training Workshop for African Scientists, University of Cambridge 
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 Lectures deleivered as part of a two week training evint for agricultural research scientists, academics and PhD students from across Africa. This was followed by discussion and follow-up questions by email.
Year(s) Of Engagement Activity 2018
URL http://www.jrbiotekfoundation.org/cambridge-lab-training-2018/
 
Description Neo.Life - 25 Visions for the Future of our Species (Book) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Contributed a book section to Neo.Life - 25 Visions for the Future of our Species (Book). Editors: Jane Metcalf and Brian Bergstein
Year(s) Of Engagement Activity 2020
URL https://neo.life/visions/
 
Description Norwich Biomakers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Norwich Biomakers brings together an interdisciplinary network of people from across NorwichCity and the Norwich Science Park that share an interest in the cross-over of biology with design, technology, engineering, electronics and software. This group meets monthly at a variety of local spaces for events including talks, training in the use of technologies and to work on projects.
Year(s) Of Engagement Activity 2017,2018,2019
URL https://www.meetup.com/Norwich-Biomakers/
 
Description Presentation and discussion at Norwich Science Festival 2018 (Engineering Day) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presentation on biomanufacturing to general public including interactive activity with biochemical pathways and debate about the meaning of the word 'natural', biological vs chemical manufacturing methods and whether manufacturing can be sustainable.
Year(s) Of Engagement Activity 2018
URL https://norwichsciencefestival.co.uk/events/building-with-biology/
 
Description Scientific Advisory Board for Cluster of Excellence on Plant Sciences (CEPLAS), a joint initiative of Heinrich Heine University Düsseldorf (HHU), University of Cologne (UoC), Max Planck Institute for Plant Breeding Research Cologne (MPIPZ) and Forschungszentrum Jülich (FZJ). 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Member of the Science Advisory Board for the Cluster of Excellence on Plant Sciences (CEPLAS), a joint initiative of Heinrich Heine University Düsseldorf (HHU), University of Cologne (UoC), Max Planck Institute for Plant Breeding Research Cologne (MPIPZ) and Forschungszentrum Jülich (FZJ).
Year(s) Of Engagement Activity 2019,2020
URL https://www.ceplas.eu/en/home/
 
Description The Essex Synthetic Biology School (ESBS), University of Essex, UK : 
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 Presented lectures on "Engineering Plant Genomes for Farming and Pharming" and "Sharing Biology in the Information Age: Perceived Threats of Dematerialisation and Open Data". These were followed by discussions on the use of biotechnologies in agricultures and a debate on access and benefit sharing of genetic resources. Participants reported changes in their views on the common practices used to share information relating to genetic resources as well as physical resources.
Year(s) Of Engagement Activity 2018
URL https://esbs.essex.ac.uk