Research

In our lab, we develop and apply engineering approaches to understand biological complexity and advance plant biotechnology.

Our research is focused on:

(i) Understanding how phenotypes emerge from network functions. Quantitative traits are phenotypic traits that are determined by many genes and impacted by the environment. In plants, these include many agricultural traits such as biomass, seed yield, resistance to abiotic stress and resistance against generalist pathogens. These traits result from the actions of suites of genes working combinatorially within complex gene regulatory networks (GRNs) that often contain partially redundant nodes and numerous network motifs, such as feedforward and feedback loops, that define and fine-tune network dynamics. The complexity of GRNs has made them challenging to investigate using traditional genetic approaches. Similarly, predicting the effects of perturbations has been a significant barrier to applying genetic engineering to the improvement of quantitative traits. The process of rewiring or reconstructing regulatory networks using synthetic biology approaches can aid the understanding of how phenotypes emerge from network functions and inform rational engineering. As GRNs are underpinned by interactions between transcription factors and regulatory elements, one focus of our work is to investigate these interactions and their impact on the intrinsic and emergent properties of cis-regulatory elements. Our long-term goals are to develop the knowledge and technologies required to optimise crop performance through the rational engineering of regulatory networks.

(ii) Exploring and utilising metabolic diversity. Plants produce a vast array of biologically active metabolites that help them adapt and survive in their ecological niches. Many of these specialised metabolites are specific to lineages or even species and, while they may not be required for growth and development in optimal conditions, contribute to survival and persistence by providing protection against pests and pathogens or acting as agents of communication. Metabolic novelty can arise by several different mechanisms including gene duplication followed by neofunctionalisation (including after polyploidisation events), metabolic pleiotropy, as well as by changes to enzyme specificity. Some plant lineages lose the ability to make specific metabolites while others evolve new compounds. Understanding the origin and evolution of plant metabolites is fundamental to explaining both the distribution of natural products among plant families and their biological roles. The richness of bioactive molecules found in plants also provides a wealth of potential pharmaceuticals, insecticides, flavours and fragrances and molecules of medicinal and industrial value. However, although bioactive plant extracts are frequently reported, the specific molecule(s) responsible for bioactivity is often unknown. Even when activity is connected to a specific compound, it may be present in tiny quantities or in a rare or difficult-to-cultivate species. Chemical synthesis has provided easy and cheap access to a few plant natural products but remains challenging or uneconomical for many molecules. In the past decade, aided by advances in genomics, biotechnology and metabolic engineering, pathways for high-value natural products used in medicine and industry have been elucidated and rebuilt in so-called ‘chassis organisms’, engineered to maximise yields. In our lab we integrate genomics, metabolomics, and transcriptomics to investigate the genetic basis of plant natural products, enabling us to understand mechanisms of metabolic diversification and explore methods for biomanufacturing. We also develop plants as photosynthetic biomanufacturing platforms, applying engineering approaches to improve purity and yield. This includes utilising our knowledge of cis-regulation to engineer synthetic genetic circuits to optimise heterologous expression and engineering plants to improve their suitability and photosynthetic production chassis.

Past and ongoing projects include:

People: Yaomin Cai, Sam Witham, Mahima
Funding: BBSRC (BBS/E/T/000PR9819, 2116919 , BB/W010933/1)

Promoters play a critical role in establishing baseline transcriptional capacity by recruiting proteins, including transcription factors. However, we lack a clear understanding of how regulatory function is coded into DNA, which limits our ability to design promoters of desired strengths that are activated by specific endogenous signals. We established a quantitative experimental system to investigate transcriptional function, investigating how the identity, density and position of cis-regulatory elements contribute to regulatory function. We used this system to identify permissive architectures for minimal synthetic plant promoters, enabling the computational design of a suite of synthetic promoters for which the strength could be predicted for the primary sequence (Cai et al, 2020). It has previously been observed that DNA sequences flanking transcription factor binding sites can affect DNA structure and modulate binding affinity. We reasoned that manipulating the context of binding sites to alter binding affinity would enable us to produce variants of individual MinSyns that respond to the same endogenous signal. To do this rationally, we required a method to investigate the impact of sequence variations in transcription factor binding motifs (TFBMs) on TF association. We developed a luminescence-based microplate assay for quantifying the interactions of transcription factors with DNA and demonstrated how this data can be used to design synthetic plant promoters of varying strengths that respond to the same transcription factor (Cai et al, 2023). We are now conducting further analyses into the context of functional elements, investigating how nucleosome stability is encoded into DNA and how this can be engineered to fine-tune gene expression.

People: Tufan Oz, Yaomin Cai, Sam Witham, Zhengao Di
Collaborators: Siobhan Brady, UC Davis
Funding: BBSRC (BBS/E/T/000PR9819, NRP DTP, BB/S020853/1)

Nitrogen (N) is essential for plant growth and basic metabolic processes. Root system architecture, vegetative biomass and even flowering time are all regulated in response to levels of environmental N. In this project, we are unpicking interactions within a regulatory sub-circuit of transcription factors predicted to coordinate large-scale transcriptional changes in response to N. We are investigating how well the network is conserved across plant lineages and are using our network models to predict how mutations will affect gene expression and growth. We have designed a library of plants with mutations in cis-regulatory regions as well as synthetic genetic controllers to modulate network functions. We are also using single-cell transcriptomics to investigate how this network functions across cell types and to inform cell-type-specific engineering.

People: Quentin Dudley, Connor Tansley, Tufan Oz, Seohyun Jo,
Collaborators: O’Connor Lab (MPI-Jena), Geu Flores Lab (University of Copenhagen), Osbourn Lab (John Innes Centre)
Funding: BBSRC:BB/P010490/1 ; UKRI:BB/W014173/1

Heterologous biosynthesis of small molecules can be complicated by the endogenous metabolism of the host, which can divert intermediates or perform unwanted modifications of expressed molecules. Much work has been done to tailor specific strains of bacteria and yeasts to increase the production of compounds. However, until recently, little effort has been spent on improving the plant production chassis, partly due to the complexity of their metabolic networks and a lack of available tools. The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, we and others have observed the derivatisation of some intermediate and target modules. Further, N. benthamiana accumulates high levels of pyridine alkaloids, which complicates the downstream purification processes, particularly of small molecules with similar physics-chemical properties. We have successfully reconstituted the biosynthesis of strictosidine, a key intermediate of all monoterpene indole alkaloids (MIAs) by the co-expression of 14 enzymes (Dudley et al, 2022). We used transcriptomic analysis to identify glycosyltransferases that are upregulated in response to biosynthetic intermediates of this pathway and produced plant lines with targeted mutations in the genes encoding them, showing that expression of the early MIA pathway in these lines produces a more favourable product profile (Dudley et al, 2022). We also produced low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure (Vollheyde et al, 2023). We are currently working on engineering new lines of N. benthamiana, tailored to optimise the production of triterpenes.

People: Melissa Salmon, Daria Golubova, Hong Su, Connor Tansley
Collaborators: O’Connell Lab (UEA Pharmacy), Howes and Leitch groups (Royal Botanic Gardens at Kew)
Funding: BBSRC, John Innes Foundation

Plants produce a vast array of biologically active metabolites that help them adapt and survive. This chemical richness also provides a wealth of bioactives with potential in medicine, agriculture and industry. However, although the bioactivities of plant extracts are frequently reported, the specific molecule(s) responsible are often unknown. Further, even when activity is connected to a specific compound, it may be present in tiny quantities or rare or difficult-to-cultivate species. Chemical synthesis has provided easy and cheap access to some molecules but remains challenging or uneconomical for many molecules. In the past decade, aided by advances in genomics and synthetic biology, biosynthetic pathways have been elucidated and rebuilt in so-called ‘chassis organisms’, engineered to maximise yields. Understanding the genetic basis of natural products, therefore, provides new opportunities for the scalable production of useful compounds. In our lab, we combine genomic, transcriptomic, and metabolomic data to uncover the genetic basis of specialised metabolites, with a focus on terpenoids. We are investigating the genetic changes that enabled the production of these molecules. Further, in collaboration with Maria O’Connell’s lab (UEA Pharmacy), we aim to tie the bioactivity of medicinal plant extracts to specific molecules.

People: Kalyani Kallam, Connor Tansley
Collaborators: The SUSPHIRE Consortium
Funding: ERA CoBioTech (BB/R021554/1)

Insects are essential to agriculture, including as pollinators and as predators of insect species that feed on crops or spread viruses and other diseases. However, herbivorous insects are responsible for destroying up to 20% of crops. Farmers employ many different strategies to try and limit these losses. Currently, pesticides are one of the primary methods of control but many insecticides are non-specific and affect pollinators and beneficial species as well as pests. Consequently, pesticides are progressively being restricted by legislation due to concerns about negative impacts on biodiversity and non-sustainability. We argue that insect sex pheromones provide environmentally-friendly alternatives to pesticides for pest management in agriculture (Mateos Fernández, Peteck, Gerasymenko et al, 2022). Pheromones are already successfully employed in the control of several lepidopteran and coleopteran species, however, access is limited to species for which the pheromone can be chemically synthesised at low cost. Synthetic biology has demonstrated that biological organisms can be re-programmed to produce complex, high-value chemicals. Our lab was part of the SUSPHIRE consortium, which demonstrated the biological production of pheromone biosynthesis (see list of publications).

People: Quentin Dudley, Oleg Raitskin, Yaomin Cai, Tony West
Collaborators: Haseloff Lab (University of Cambridge), BRACT group (JIC), Sylvestre Marillonnet (IPB-Halle), Alison Smith (JIC), Earlham Biofoundry
Funding: OpenPlant: BB/L014130/1 ; BB/N019466/1; BB/P010490/1

The field of bioengineering is underpinned by our ability to perform experimental workflows for protein production and gene editing. These and many other experiments require the assembly of synthetic DNA molecules accurately and at scale. In 2015, we led the establishment of a common genetic syntax for exchanging DNA parts for plants – the Phytobrick standard (Patron et al 2015). We also co-developed toolboxes of DNA parts (Engler et al, 2014) and open-source tools for the facile, iterative assembly of multigene constructs (Pollack et al 2018). These foundations have enabled us to automate the fabrication of large DNA assemblies in sub-microliter volumes in the Earlham Biofoundry (Cai et al. 2020). We have applied our automated nanoscale experimental pipelines to an expanded toolbox of CRISPR tools for plants (Raitskin et al 2019), which we used for editing food crops (e.g., Tuncel et al. 2019). We also developed workflows for automated DNA assembly and cell-free expression of plant proteins that accelerate optimisation and enable the rapid screening of enzyme activity (Dudley et al, 2021).

People: Everyone
Collaborators: Haseloff Lab and OpenBioeconomy Lab (University of Cambridge), BioBricks Foundation
Funding: OpenPlant: BB/L014130/1), SynBioLEAP

We are among a group of bioengineers who propose that open access to foundational tools and technologies for plant biotechnology is critical to combatting complex global challenges such as providing access to essential medicines and nutritious food. Open science and technology practices allow the inclusion of a diverse range of new actors into research and provide mechanisms to challenge the monopolies of powerful incumbents. This process is analogous to the current global democratisation of information technology brought about by the expansion of open-source software and affordable access to inexpensive computing devices and performant internet connectivity, in particular, in the developing world. We have been involved in the development of new legal tools for the democratic exchange of scientific resources (Kahl et al, 2018), exploring responsible and open innovation with large bioresources, and the benefits of open access to digital sequence information.