An improved bioproduction system for proteins and small molecules

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

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.

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.