Metabolic engineering for novel chemicals and therapeutics

Dr Zhenhua Liu

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It has been estimated that plants can produce over 1 million specialized metabolites, but we know less than 0.1 % of their biosynthetic pathways. Creative methods are eminently needed to look under the iceberg of largely untapped biosynthetic pathways. As a post-doc from Anne Osbourn group at John Innes Centre, I am employing multidisciplinary approaches across bioinformatics, genetics, and chemistry, to comprehensively understand how and why plants produce this hallmark of specialized metabolites.

I am currently focusing on plants from the Brassicaceae family and systematically studying the function, evolution and biosynthesis of triterpenes from this family. I am in particular interested in pathways encoded by gene clusters. It holds great potential to mine more and novel biosynthetic pathways efficiently. However, how and why plants have evolved BGCs is still a mystery. We are aiming to gain the first understanding of their assembly, patterns of evolution and common features in a systematic fashion. This knowledge can then be used as a template guiding the research of BGCs in other types of compounds and plant families.         

Figure legend: Multidisciplinary approaches to discover new pathways and novel natural compounds. We are using combination of bioinformatics, genetics and chemistry in attempt to decode and recode the largely untapped plant specialized metabolism

Figure legend: Multidisciplinary approaches to discover new pathways and novel natural compounds. We are using combination of bioinformatics, genetics and chemistry in attempt to decode and recode the largely untapped plant specialized metabolism

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.