Metabolism

Dr Ingo Appelhagen

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I am a molecular biologist, with a background in plant transcription factors, flavonoid biosynthesis, natural colours and metabolic engineering.

In Cathie Martin’s lab at the John Innes Centre, we have recently developed novel suspension cultures from engineered tobacco plants, to obtain stable sources of natural colourants. These cultures can produce exceptionally high levels of red to purple anthocyanin pigments, and allow a scalable constitutive year-around production under controlled conditions.

Intense blue colours are rare in nature and difficult to reproduce in pigment formulations, which is the main reason why almost all blue food colourants are synthetic dyes. Our project aims to investigate the structural properties of anthocyanin preparations that confer strong and stable blue colours and to select for anthocyanins with improved stability as reliable natural colourants. Our goal is to extend our plant cell culture approach to develop the first production platform for blue anthocyanin colourants, to replace synthetic food dyes.

Dr Lukas Müller

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I’m interested in the circadian clock and its effect on physiological and agricultural performance in plants. In the OpenPlant project I am investigating the circadian clock in Marchantia polymorpha and analyze the regulation of clock behavior and outputs in this relative of early land plants. In particular, I am focusing on the primary metabolism as an excellent proxy for systemic processes and vegetative growth.

I apply fluorescent imaging tools with computational time-lapse analysis to obtain cell-specific read-outs for the whole plant in real-time. This data is intended to set the stage for both physiological engineering and systems biology approaches.

Part of my project is to engineer fluorescent proteins that are standardised and improved reporters for dynamic changes in gene expression.

Dr Noam Chayut

I am interested in the interface between applied plant breeding and plant metabolism. In my master’s thesis we used classical breeding of passionfruit with the goal of releasing new varieties, now used by farmers. In my PhD thesis we studied carotenoid metabolism in melons and established a molecular marker now used routinely by melon breeders. More importantly, we suggested a novel non-transgenic path toward pro-vitamin A carotenoid biofortification of food crops. The objective of the current OpenPlant project is to develop pre-breeding lines of beetroot for the production of L-DOPA.  

L-DOPA is used to treat Parkinson’s symptoms; however, the current costs of chemical synthesis make it unavailable for deprived populations worldwide. In addition, there is a growing demand for ‘natural’ or plant sourced pharmaceutical substances in the first world. L- DOPA, a product of tyrosine hydroxylation, is an intermediate metabolite in biosynthesis of violet and yellow betalain pigments, in Beta vulgaris (table beet). L-DOPA natural steady state levels are very low, usually undetectable. We intend to block the turnover of L-Dopa in beetroot to allow its accumulation to levels that could enable low-tech accessible production in a plant system.

Current data indicate two betalain metabolic genes that, if repressed, may boost L-Dopa accumulation. Therefore, we aim to inhibit the activity of L-DOPA-dioxygenase, and L-DOPA-cyclase in beetroot. Currently, as proof of concept, we silence both genes in hairy roots system. We adopted three complementary strategies to meet the overarching objective of L-DOPA production in beet: a) Classical genetics; b) targeted genetic mutagenesis; and c) random mutagenesis. Yellow beet, mutated in L-DOPA-cyclase exists and can be crossed with “blotchy” red beet, which probably has lower L-DOPA-dioxygenase activity. Impairing L-DOPA-dioxygenase activity in yellow beet is carried out by both the targeted mutagenesis technology CRISPR/Cas9 and the random, yet more assured, EMS mutagenesis approach.

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Dr Benjamin Lichman

Plants are incredible chemical factories, capable of producing a host of complex molecules that synthetic chemists struggle to produce. These compounds are produced by plants to interact with their environment, but they also have great significance for humans, as we use them for fragrances, agrichemicals and medicines. My general research interests are understanding how plants produce these valuable compounds, and how these pathways have evolved. This knowledge can then be used to produce natural products and novel chemicals in microbial or plant based platforms.

I am currently working with catnip and catmint (Nepeta cataria and N. mussinii), plants famous for their intoxicating effect on cats. The origin of this activity is the nepetalactones, a group of volatile compounds from the iridoid family of natural products. Along with their role as feline attractants, nepetalactones have also been reported to have both insect pheromone and insect repellent properties, in some cases having activities superior to DEET. The biosynthetic origin of these compounds is currently unknown. We have been using transcriptomics and proteomics to discover enzymes in the Nepeta nepetalactone biosynthesis pathway.

This work is being performed in the context of a wider chemical and genetic investigation into the mint family (Lamiaceae), a large plant family of economic importance in which Nepeta resides. I am working closely with the Mint Genome Project (funded by the NSF) to understand the evolution and regulation of natural product biosynthesis across the entire plant family. By placing newly discovered Nepeta enzymes in a detailed phylogenetic context we hope to understand the evolutionary origin of nepetalactone biosynthesis in Nepeta, and ultimately use it as a case-study for natural product evolution.

I am currently undertaking training in molecular evolution and phylogenetics with the aim of taking the principles of evolution into synthetic biology. I hope that this will reveal new methods of optimising and editing synthetic biology systems and devices.

Figure 1.    Nepetalactone biosynthesis pathway in  Nepeta . We are attempting to discover the enzymes that catalyse the formation of all different nepetalactone isomers. We are also attempting to understand how these enzymes have evolved. In the background is  Nepeta mussinii .

Figure 1. Nepetalactone biosynthesis pathway in Nepeta. We are attempting to discover the enzymes that catalyse the formation of all different nepetalactone isomers. We are also attempting to understand how these enzymes have evolved. In the background is Nepeta mussinii.

Dr Michael Stephenson

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I am a chemist, with a background in natural product total synthesis, medicinal chemistry, and pharmacy. In the Osbourn group we are interested in plant secondary metabolites, and this places us at the very interface between biology and chemistry. I bring expertise in small organic molecule extraction, purification, and structural characterisation. This strengthens the group’s ability to functionally characterise biosynthetic enzymes; something which is important for many areas of research within the Osbourn lab. As such, I am involved in a number of different projects.

My main focus is on the application of transient expression in Nicotiana benthamiana towards the preparative production of high value triterpenes. I have been heavily involved in platform and method development, improving both the efficiency and scalability of procedures used within the group. I have also demonstrated the preparative utility of this platform by producing triterpenes on the gram scale.

As a medicinal chemist I am interested in applying these techniques to engineer chemical diversity, and to explore the structure activity relationships of bioactive triterpenes. I have been involved in isolating and characterising several novel triterpenes structures arising from co-expression of ‘un-natural’ combinations of biosynthetic enzymes. In addition, I have solved the structure of a number of novel and usual triterpene scaffolds, produced by oxidosqualene cyclases under investigation within the group. It would seem that despite the huge number of unique triterpene scaffolds already reported from many decades of natural product isolation, there is still a wealth of novel chemistry to be discovered, and that its discovery can be accelerated by utilising synergy between bioinformatives, synthetic biology, and chemistry.

In addition to my research, I also take a keen interest in public engagement. I have been involved in several outreach events where we attempt to present concepts in synthetic biology and chemistry in an assessable and ‘hands on’ way.    

Dr Hans-Wilhelm Nützmann

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Plants produce a wide variety of specialised metabolites. These molecules play key roles in the interaction of plants with their biotic and abiotic environment. In addition to their ecological functions, plant-derived specialised metabolites are major sources of pharmaceuticals and other high-value compounds.

Recently, it was discovered that the genes for the biosynthesis of several major classes of these compounds are physically co-localised in so called ‘gene clusters’ in plant genomes. Such clustering of non-homologous genes contrasts the expected arrangement of genes in eukaryotic genomes. The co-localisation of functionally-related genes enables the formation of fundamentally different mechanisms of gene regulation in comparison to the control of dispersed genes. The purpose of this project is to improve our understanding of the transcriptional control of plant metabolic gene clusters. The focus within OpenPlant will be on chromatin related regulatory processes that govern the expression of gene clusters. By chromatin immunoprecipitation, chromosome conformation analyses and genome engineering we aim to characterise the chromatin environment at gene clusters and its impact on cluster regulation. The findings of this project will open up new opportunities for the discovery and engineering of metabolic pathways using genetic and chemical approaches. They will also underpin synthetic biology-based approaches aimed at refactoring of plant metabolic gene clusters and the development of synthetic traits.

 

Dr Thomas Meany

I am jointly hosted by the labs of Lisa Hall (Chemical Engineering and Biotechnology) and Jim Haseloff (Plant Science) as an interdisciplinary fellow part funded through OpenPlant. My background training is as a physicist, with a specific emphasis on optics and microfabrication. I undertook a PhD in Macquarie University (Sydney, Australia) where I developed microphotonic circuits using a 3D laser printing technique. My postdoctoral research continued in Toshiba’s Cambridge Research Labs where I worked on advanced manufacturing techniques for semiconductor quantum dots.

As a part of OpenPlant I am passionate about using optical analytical tools to study the production of secondary metabolites in specialised plant tissues. Specifically, the oil bodies of the Liverwort, Marchantia polymorpha, are potentially rich reservoirs of bio-active compounds. Using Raman microscopy, a label-free, non-destructive spectroscopy technique it is possible to study metabolic processes in real-time. As this is non-destructive it can be performed in situ and therefore both spatial and temporal information can be obtained. My hope is to correlate this data with information available using other approaches such as Matrix Assisted Laser Deposition Ionisation Mass Spectroscopy (MALDI), Gas Chromatography Mass Spectrometry (GC-MS), fluorescence microscopy and other high resolution analytical approaches. In future this could be then adapted to studies of transgenic plant species as an additional tool to study metabolic pathways. Additional model species can also be explored, for instance Nicotiana benthamiana, and potentially crop plants. I am keen to engage with teams operating in the area of natural product chemistry, metabolic engineering or teams focused on alternative analytical approaches.

Photo: Prototype microfluidic rapid 3D printed circuit fabricated during the Bio-Hackathon.

Photo: Prototype microfluidic rapid 3D printed circuit fabricated during the Bio-Hackathon.

Working with the Cambridge University Technology and Enterprise club (CUTEC), I organised the UK’s first Bio-Hackathon, hosted in the Department of Plant Science (Cambridge) during the week of 21-25 June 2016. This was possible with thanks, in part, to a grant provided by the University of Cambridge Synthetic Biology Strategic Research Initiative. This event brought together a diverse interdisciplinary group of 50 participants from across the UK and the world. Teams focused on “bioware” by incorporating hardware, software and wet lab tools. One team developed a 3D printed microfluidic prototyping tool, another built a comparison software tool for DNA synthesis pricing. The winning team built a tool called “Alpha-Brick” which is a drag and drop tool for assembling bio-bricks and plugs directly into Transcriptic (a cloud laboratory) allowing immediate order of an assembled part.