Plants produce chemicals to communicate, protect from radiation or defend from pathogens and predators in a hostile environment. Some of these chemicals have medicinal activity, but we are still unable to use the molecular machines of plants for our benefit.Are we getting any closer to produce these precious natural medicines?

An invisible chemical warfare between plants and animals

Plants and animals colonized land around 443 and 417 million years ago respectively. As plants pioneered this evolutionary leap, they had to adapt to a new and very challenging environment full of pathogenic microorganisms, dehydration conditions or high solar radiation. Animals followed the same path colonizing land, finding in plants a vast source of food in this new environment. The new coexistence between plant and animals on land triggered the evolution of different biological strategies to attract or defend from each other. While plants developed a whole arsenal of biologically toxic chemicals to prevent predators from feeding on them, animals responded by evolving mechanisms to avoid the toxicity of such chemicals. An invisible “chemical warfare” had just been set off and could represent an abundant source of future medicines for the human kind.

 colonizing-land

 A highly diverse weapon arsenal

An essential element that enables both organisms to engage in this chemical warfare is the so-called Cytochrome P450 enzyme family (P450s). These are molecular machines located in membranes of inner cell compartments and are responsible for the biosynthesis of natural chemicals in plants. The fact of being bound to a membrane makes P450s particularly difficult to produce and study in the laboratory. Humans have a small set of around 57 genes that encode for these enzymes, which are highly present in the liver and work to oxidize toxic chemicals such as drugs in an unspecific manner. This feature allows humans to detoxify a larger number of potentially poisonous chemicals to which we are exposed in nature. In contrast, plants are provided with a larger of up to 250 different P450 genes. This fact partly reflects the enormous diversity of natural chemicals that plants can synthesise. Most importantly, these chemicals have shown potent anti-cancer activity such as the compound called Taxol compound naturally produced by the bark of the Pacific yew tree. The compound Ingenol found in the plant Euphorbia peplus, is another illustrative example of a natural compound to prevent cancer, in this case, actinic keratosis.

Plant chemicals difficult to replicate in a sustainable way

Plant-derived medicines are complex chemical entities very difficult to replicate in the lab using traditional chemistry methods. This is one of the reasons why direct extraction from plants has been the norm to provide a cost-effective supply. Furthermore, often the precious compounds are produced in very tiny amounts in plants, which may represent a threat to endangered species. For example, the Pacific yew tree is a species on the brink of extinction as a result of overharvesting by poachers seeking the precious taxol. Synthetic biology could be more sustainable and cost-effective solution to these problems because it provides fast-growing microbes with the capabilities to produce natural compounds using DNA elements (biobircks) from different organisms. However, this is not free of controversy since many natural products are currently employing thousands of farmers in developing countries, whose jobs could be threatened by the deployment of synthetic biology.

Smarter bacteria for producing natural compounds

Plants offer a powerful P450 arsenal, however, bacteria do not always recognize the genetic code and signals of plant P450 genes. Research is needed to improve bacterial “decoding” of P450 and screening technologies accelerate the development of new microbial cell factories. During the last three years, I participated in a very exciting research led by Morten Nørholm at DTU Biosustain in the framework of an EU project, called BacTory  (Bacterial Cell Factories). The goal was to tackle the problem of optimizing the production of multiple P450s in the context of an immense plant biodiversity.

In order to figure out whether tweaking the P450’s genetic sequence would have a good or bad effect on protein production levels and membrane targeting, we needed an alternative to the existing time-consuming methods. To solve this bottleneck we used a well-known molecular beacon called Green Fluorescence Protein (GFP) that shines when the protein has been produced and folded correctly. Thereby genetically stitching GFP to the last end of all our P450 genes, we could assess genetic modifications with a simple, inexpensive and rapid manner. The more GFP fluorescence, the more protein is being expressed. Once we could follow production with the GFP, we expanded the genetic modification toolbox available for P450s. We inserted small “tags” as genetic bricks in front of all P450 genes, ina region that encodes for what is known as N-terminal, so that bacteria can easily recognize them. In fact, most of these tags belong to endogenous bacterial proteins that are highly abundant in their membrane.

p450s-modifications

Thanks to both the GFP technology and the new tag toolbox, an unprecedented number of P450 could be produced at high levels in bacteria. Interestingly, some of the P450s expressed at high levels, were part of the ingenol biosynthetic pathway from Euphorbia peplus. To proof that these new tags could be used to produce natural compounds we reconstructed a plant metabolic pathway using individually tagged P450s. Luckily we were able to produce a plant-derived chemical in bacteria to different levels depending on the tag used. Taking together these findings, it would be possible to optimize the production of complex natural compounds using a streamlined P450 optimization strategy. And most importantly, it could make easier to produce complex chemicals in a sustainable manner, providing a natural medicine without overexploiting natural resources.

 

References

  • Chang MC, Eachus RA, Trieu W, Ro DK, Keasling JD: Engineering Escherichia coli for production of functionalized terpenoids using plant P450s. Nat Chem Biol 2007, 3:274-277.
  • Christensen, U., Vazquez-Albacete, D., Sogaard, K. M., Hobel, T., Nielsen, M. T., Harrison, S. J., Hansen, A. H., Moller, B. L., Seppala, S., Norholm, M. H., 2017. De-bugging and maximizing plant cytochrome P450 production in Escherichia coli with C-terminal GFP fusions. Applied microbiology and biotechnology.
  • Drew D, Lerch M, Kunji E, Slotboom DJ, de Gier JW: Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 2006, 3:303-313.
  • Nielsen JS, Moller BL: Cloning and expression of cytochrome P450 enzymes catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glucosides in Triglochin maritima. Plant Physiol 2000, 122:1311-1321.
  • Paddon, C. J. and J. D. Keasling (2014). “Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development.” Nat Rev Microbiol 12(5): 355-367.
  • Podust L, Sherman D: Diversity of P450 enzymes in the biosynthesis of natural products. Natural product reports 2012, 29:1251-1266.
  • Vazquez-Albacete, D., Cavaleiro, A. M., Christensen, U., Seppala, S., Moller, B. L., Norholm, M. H., 2017. An expression tag toolbox for microbial production of membrane bound plant cytochromes P450. Biotechnology and bioengineering 114, 751-760.

 

Links

https://www.theguardian.com/environment/2011/nov/10/iucn-red-list-tree-chemotherapy

http://www.nature.com/news/malaria-drug-made-in-yeast-causes-market-ferment-1.12417

http://orbit.dtu.dk/en/publications/protein-and-dna-technologies-for-functional-expression-of-membraneassociated-cytochromes-p450-in-bacterial-cell-factories(4e0f3cf2-cc1b-410f-99c3-554ae9bc24c0).html

 

 

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