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.
 
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 The plant species, Nicotiana benthamiana has enormous potential as a platform for producing high-value small molecules and proteins used in medicine and industry. In this process, the leaves of young plants are infiltrated with strains of bacteria carrying genetic instructions to produce proteins and molecules of interest. The leaves of the plant are harvested after 2-6 days and the target products are purified. Several companies have developed commercial-scale facilities for this process producing vaccines and immunotherapies that are in advanced clinical trials. However, little has been done to optimise the genetics of this species to improve yield. Four problems that are encountered during production are:
(i) Expression of some proteins, particularly those with trans-membrane and membrane-associated domains results in the rapid necrosis (cell death) of leaf tissue, significantly impacting yields
(ii) Some types of target molecules, as well as pathway intermediates, are derivatised by the products of endogenous genes.
(iii) The plant recognises the bacterium used to deliver new genetic information as a pathogen and launches a defensive response, resulting in changes to gene expression, including the expression of gene products that are predicted to limit yields
(iv) This species naturally accumulates high levels of some classes of alkaloids. These can make the purification of related molecules of interest more challenging.
The main goal of this project was to address these issues and improve the plant, N. benthamiana as a chassis for the bioproduction of proteins and small molecules.
Our first objective was to identify N. benthamiana genes products likely to interfere with the heterologous expression of certain proteins and to produce plants with mutations in these genes. Preliminary evidence suggested that high levels of peroxidases accumulate when certain types of protein were expressed. Using protein sequencing, target peroxidases were identified and four genes encoding isoforms of this protein were identified in the genome. Constructs were designed to introduce CRISPR-mediated loss-of-function mutations into all four genes. Plants were recovered and the presence of mutations was confirmed. The plants were tested by expressing proteins previously shown to introduce necrosis, however, the levels of necrosis were not reduced. Due to the success of other parts of the project, we did not pursue the identification of alternative targets.
Our second objective was to advance the production of a group of valuable molecules known as monoterpene indole alkaloids in N. benthamiana. Previously, we had observed that the early intermediates of this pathway are derivatised by endogenous gene products. We first designed an experiment in which we increased the number of enzymes in a stepwise manner, rebuilding the pathway. We then detected the presence of each pathway intermediate. We investigated which cellular compartment each enzyme should be expressed in and which combinations of enzymes gave the best yields. We discovered that the successful accumulation of our target product requires a specific helper enzyme and that the best yields were obtained when the pathway was split across the chloroplast and the cytosol. We also identified which intermediates were derivatised. To identify endogenous enzymes that might be responsible for this unwanted activity, we used comparative transcriptomics to identify genes that were upregulated in the presence of these intermediates. In many cases, we observed correlations in the types of genes upregulated and in the chemical modifications observed. To investigate the specificity of the enzymes encoded by these candidate genes, we developed a biofoundry-enabled workflow for automated, nanoscale high-throughput cell-free protein synthesis and characterisation of plant enzymes cell-free protein synthesis. A manuscript describing the molecular toolbox and automated workflow for the cell-free expression of plant proteins has been published. To address our main aim of improving the genetics of the plants, we built and delivered DNA constructs to introduce targeted mutations into candidate genes. We produced 15 lines with mutations in up to four different genes. We then tested these gene-edited plant lines by expressing the pathway and investigating if the levels of any of the unwanted derivatives had been reduced. We identified several lines of plants in which several derivatives no longer accumulated. A manuscript describing the reconstruction and optimisation of the pathway as well as the creation and testing of mutated lines is in preparation.
Our third objective was to investigate if reducing the plant's ability to perceive the presence of the bacterium used to deliver new DNA would improve protein yield. To do this we developed constructs that introduced loss-of-function mutations into genes previously shown to be involved in the recognition of a microbe-associated molecular pattern (MAMP). We then tested these plants by expressing reporter proteins. In contradiction with data reported by other groups, we did not observe any difference in the ability of the bacteria to infect the plants or in the levels of proteins.
Our final objective was to reduce the accumulation of endogenous metabolites that interfere with purification. To do this, we developed constructs that introduced loss-of-function mutations into genes in an alkaloid biosynthesis pathway. We then analysed these plants and found that levels of the target metabolites had been reduced by over 90%. We are currently conducting a final analysis of these plants to determine if the intermediates in this pathway remain available for use in new synthetic pathways. A manuscript is in preparation.
Exploitation Route The pathway reconstructed in this project produces a key intermediate necessary for the production of several medically-important monoterpene indole alkaloids. Our work is being advanced by research groups who are seeking to reconstruct these longer biosynthetic pathways.
Molecular tools and workflows for high-throughput, synthesis, quantification and characterisation of eukaryotic proteins can be used for numerous applications in research and development. We established a collaboration with an industrial partner to explore this.
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 Synthetic Biology Expert Roundtable, Department for Business, Energy and Industrial Strategy
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description 21EBTA Engineering specialised metabolism and new cellular architectures in plants
Amount £1,517,514 (GBP)
Funding ID BB/W014173/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2022 
End 01/2024
 
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 05/2018 
End 06/2019
 
Description Biosynthesis of monoterpene indole alkaloids 
Organisation Max Planck Society
Department Max Planck Institute for Chemical Ecology
Country Germany 
Sector Academic/University 
PI Contribution Reconstruction of a biosynthetic pathway for a plant monoterpene indole alkaloid.
Collaborator Contribution Chemical analyses of plant samples in which monoterpene indole alkaloid biosynthesis has been reconstructed.
Impact A paper is in preparation. https://doi.org/10.1101/2021.08.12.456143
Start Year 2019
 
Description Cell-free expression of plant proteins 
Organisation National Institute of Biology
Country Slovenia 
Sector Charity/Non Profit 
PI Contribution We optimised a method for cell-free expression of plant proteins.
Collaborator Contribution Partner received an ERASMUS training fellowship to visit our lab for 4 weeks. They subsequently transferred the new protocol and tools to their own lab. A publication resulting from this collaborative work has been submitted (February 2022).
Impact Scientific publication.
Start Year 2020
 
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'. 69% of students atteneding the whole summer school responded that they were more interested in plant science as a result of attending and one third of students reported in their feedback that they are now thinking of doing a PhD with some plant science, an increase in 12% from when asked before attending. 93% of students reported that they are interested in or are considering a career in or with plant science as a result of attending.
Year(s) Of Engagement Activity 2018
URL http://intobiology.org.uk/dr-nicola-patron-engineering-plants-for-farming-and-pharming/
 
Description Interview with iNews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with iNews Science and Environment correspondent. This resulted in the article: "Scientists welcome Government plans to lift ban on gene editing in agriculture" published March 16, 2021.
Year(s) Of Engagement Activity 2021
URL https://inews.co.uk/news/science/scientists-welcome-government-plans-to-lift-ban-on-gene-editing-in-...
 
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