Plant biotechnology for health and nutrition

29 April 2018 - By: Caroline Wood

PLANT BIOTECHNOLOGY FOR HEALTH AND NUTRITION

By Caroline Wood

Plants have always played a traditional role in medicine, as the beautifully illustrated and richly descriptive herbal books of ancient cultures show. In modern times, however, healthcare has become more synthetic, with pills and tablets more common than tinctures and infusions. But new advances in biotechnology will see plants taking centre stage again as a prime source of health promoting agents. These exciting developments will have an entire session - Plant biotechnology for health and nutrition – dedicated to them at the 2018 SEB Annual Meeting in Florence. Here is a sneak advance peek of what’s in store…

Super Tomatoes

‘Superfoods’ is a controversial term, but it is generally accepted that dark coloured fruits and vegetables, such as aubergines, cherries, blackcurrants and blueberries, are particularly associated with good health. This is due to their high content of anthocyanins and flavonoids: compounds which appear to protect against various chronic diseases, including cancer, cardiovascular disease and obesity. However, these foods are typically expensive and seasonal, and the amounts required for these benefits would be beyond the budget of most. Genetic engineering, however, has opened the possibility of developing more commercially viable crops with a high anthocyanin content. “We decided to use tomatoes because they are a very versatile food, can be grown in many places and are relatively easy to transform” says Cathie Martin (John Innes Centre, UK), the driving force behind the ‘purple tomato’ and a keynote speaker at the session in Florence.

Using Agrobacterium-mediated transformation, Cathie and her colleagues engineered tomato plants to express two anthocyanin biosynthesis genes that normally produce purple flowers in Garden Snapdragon (Antirrhinum majus). These were driven by a fruit-specific promoter from tomato, to limit their expression to the tomato fruits. As a result, the tomatoes accumulate up to 3mg purple anthocyanins per gram of fresh weight, a level comparable with cherries and raspberries. “When we supplemented the diet of a cancer-prone mouse model with 10% purple tomatoes, their average lifespan increased by 30% - from 142 days to 182 days” says Cathie1. “But the mice didn’t live significantly longer if they were fed a diet supplemented with 10% normal red tomatoes, showing that the benefit came from the anthocyanins in the genetically modified fruits”.

This success prompted Cathie to set up her own spin-off company, Norfolk Plant Sciences, which is currently in the process of commercialising a purple-tomato juice product in the USA. “We are about to submit an application to the Food and Drug Administration (FDA) – we just need to confirm the precise sequence of the insertion site so we can confirm that no major gene rearrangements have occurred” Cathie says. In the meantime, her latest work has focused on the power of anthocyanin-fortified tomatoes to combat a rapidly growing ‘modern disease’: intestinal inflammation. “We tested a mouse model where intestinal inflammation is induced by chemical treatment (Dextran Sulfate Sodium - DSS). Normally this causes dramatic weight loss and bleeding from the colon but this was decreased in mice fed tomatoes high in anthocyanins, flavonols or resveratrol” she says. Interestingly, the results were even more impressive when the different polyphenols were combined in a single tomato, suggesting a synergy of action. “These effects were seen when we only supplemented 1% of the mice’s diet with the high anthocyanin, flavonol and resveratrol tomatoes, which is a very low level. But you would have to eat a tonne of red grape skins or blueberries to get the same amount of these compounds from a non-GM source”. In her talk at the meeting in Florence, Cathie will describe how her latest results suggest a mechanism whereby anthocyanins and flavonols act on the epithelial cells to reduce the secretion of pro-inflammatory cytokines3. But for these exciting results to truly make a difference, public acceptance is key and this will require much collaboration outside of the lab. As Cathie says, “We absolutely need to have medics and nutritionists on board - they have to understand you can really make a difference with plant science. We are not just engineering for the sake of engineering, but for a purpose, so we can actually have an impact on chronic diseases”.

Something Fishy

Successes like the purple tomato can give the impression that we are on the brink of a new plant breeding era where, with the addition of one or two genes, we can re-engineer plants to become factories for any key nutrient of our choosing. However, the process is rarely so straightforward. More often, high-value compounds have complex biosynthetic pathways involving countless different enzymes, requiring an entire metabolic pathway to be reconstructed in the host plant. But it can be done, as researchers at Rothamsted Research have demonstrated with Omega-3 Polyunsaturated Fatty Acids (PUFAs). These compounds are crucial for human health, particularly for maintaining brain function and combating cardiovascular disease. Two of these, Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) are not naturally synthesised by plants; they are actually produced by marine phytoplankton and accumulate up the food chain when these are eaten by fish. Consequently, the World Health Organisation recommends eating at least 2 portions of oily fish such as herring, sardines and mackerel each week, equivalent to 250-500 mg daily of EPA and DHA.

But that could soon change, thanks to the development of transgenic plants that produce and accumulate omega-3 fatty acids in their seeds. Having started the ambitious project in the late 1990s, it has been quite a journey for Johnathan Napier and his team, but one that is now bearing fruit. “Engineering plants to accumulate omega-3 fish oils is a complicated multi-gene trait, requiring advanced metabolic engineering” says Johnathan. “During my talk in Florence, I will describe how my research has gone from fundamental studies in model systems to translating these to a crop species, carrying out GM field trials in the UK and North America, and finally fish and animal feeding studies.”

Johnathan’s crop of choice was Camelina sativa (False Flax), a relative of oilseed rape. “We chose Camelina because it is very easy to transform, with no tissue culture steps involved; it has a rapid seed-to-seed generation time and it is not a commodity crop, which means that important things like identity preservation and stewardship of the GM crop are easier” Johnathan says. Crucially, Camelina also has a naturally high level of a-linolenic acid; a shorter chain omega-3 fatty acid that acts as a precursor for EPA and DHA.

Camelina
Genetically engineered seeds of Camelina sativa Photo: Rothamsted Research


With the addition of seven synthetic genes based on those present in marine phytoplankton, Johnathan and his team were able to divert the a-linolenic acid already present in Camelina into EPA and DHA production. Subsequent field studies demonstrated that the trait is stable in field-grown plants; that the seed lipid profile resembles that of marine microalgae rather than higher plants and that farmed salmon can successfully accumulate omega-3s from feed pellets made from the transgenic crop4. “The potential impact of this is huge – instead of having to harvest and extract fish oils out of the oceans, we can produce them on the land” says Johnathan. “This means we can have a larger and more secure supply of these important fatty acids, useable in both aquaculture and direct human nutrition”.

At the session in Florence, Johnathan also hopes to explore how synthetic biology approaches and computer modelling could make future genetic transformations more predictable, rather than slow, iterative processes of refinement. “We would like to be able to use computational approaches to model the alterations to endogenous metabolism needed to generate a given novel oil profile” he says. “This is currently very hard, as our understanding of plant lipid biochemistry is only partial, but it is an exciting “moon-shot” type of goal to aim for.”

RECONSTRUCTING PATHWAYS

Besides producing nutrients essential for health, reconstructing metabolic pathways in plants could be a source of valuable compounds that fight disease. Vinblastine and vincristine, for instance, potent alkaloids isolated from the Madagascar Periwinkle Catharanthus roseus, are used in chemotherapy treatments against a range of cancers, including breast cancer, Hodgkin’s disease and acute leukaemia. “These chemicals are extracted at a high price because only very low levels accumulate in the plant tissues” says Kirsi-Marja Oksman-Caldentey (VTT Technical Research Centre of Finland). “However, chemical synthesis is not an economical alternative either due to their highly complex structures and specific stereochemical features”. Between 2009-2013 Kirsi-Marja worked as part of the SmartCell consortium, an international effort involving 18 different partners, to develop a more accessible and cost-effective source of these drugs. 

Given the complexity of the enzymatic pathways that produce these alkaloids, Kirsi-Marja decided to focus on the first part of 35 step reaction pathway that finally produces vincristine and vinblastine. “It was like making a puzzle little by little” she explains. “Using next-generation sequencing, we isolated candidate genes, combined these in yeast and in bacteria then used biochemical analysis to confirm the products of each individual step”. Having identified the enzymes and their order in the process, they were able to reconstruct the pathway in tobacco (Nicotiana benthamiana), using Agrobacterium transformation. “While there remain ‘missing steps’ in the downstream pathway, we have demonstrated a real proof of principle that has brought the whole work in metabolic engineering of secondary metabolites forward” Kirsi-Marja says. “If the project had lasted longer, I am sure we could have kept going to the end”.

BERRY GOOD FOR YOU

In the meantime, Kirsi-Marja is already immersing herself into her next project – harnessing the natural power of berries. “Here in Finland, our forests are very rich in edible berries, with over fifty different species, some of which have compounds with very interesting biological activities” she says.

Various studies have reported the health benefits of incorporating berries into the diet. A national project in Finland, for instance,found that a daily addition of 300 grams of berries into the diet was able to reverse symptoms of metabolic syndromes, promote beneficial gut bacteria and improve blood lipid profiles in as little as 3 months5.

This is thought to be due to the action of ellagitannins: polyphenols abundant in strawberries, raspberries and cloudberries which are metabolised by bacteria in the colon. “In addition, phenolic compounds from berries show strong antimicrobial activity against pathogenic bacteria without harming the beneficial bacteria such as Lactobaccillusspecies. Staphylococcus, for instance, is very sensitive to these” says Kirsi-Marja. “We can’t use genetic engineering to increase the production of these compounds however, because these plants have not yet been sequenced and the biosynthetic pathways are largely unknown. Also, it is most likely that the antibacterial effect is not behind one single compound but due to synergistic effects of several phenolic compounds. So we have optimised the plant cells by altering the growth conditions and modulating the culture media as well as using elicitation to get them to produce the correct ratio of these compounds”.

Plant cell cultures on pertidishes
Cultures of berry cells Photo: VTT images


Beside cultivating cells in this way, VTT is bioprocessing berries and their by-products (e.g. seeds) to generate new types of highly active extracts against pathogenic bacteria. The work has already attracted interest from the cosmetic industry, who face increasing pressure to replace synthetic preservatives such as parabens with more natural agents. “We are also seeing an emerging trend in skin care for products that have a balancing effect towards the whole microbiota” says Kirsi-Marja.

Kirsi-Marja also believes that besides being a source of niche chemicals, the future will see plant cell cultures play an increasingly important role in food production. “I believe the whole food production chain is in a disruption phase now and that within ten years, it will certainly be very different with more food being produced locally and in specified laboratories or small factories” she says. Until then, Kirsi-Marja gives her advice on how you can benefit from berries yourself: “I love berry smoothies and prepare one every morning. I would recommend combining fresh or frozen berries with natural yoghurt, a small ripe banana and 4-5 basil leaves in a blender - then simply mix and enjoy!”

CHLOROPLASTS – A PRODUCTION POWERHOUSE

So far, most techniques have concentrated on introducing DNA into the nucleus. But Henry Daniell (University of Pennsylvania, USA) believes that to unlock the full potential of plants as pharmaceutical factories we should be looking elsewhere – the chloroplast. “One of the most abundant proteins on earth – rubisco, a key component for photosynthesis – is made in chloroplasts” he explains. “So I asked the question: if the chloroplast is so effective as a production system, can we use it to make highly expensive biopharmaceutical proteins?” An ideal candidate to test this was insulin, the protein treatment for those with diabetes.

“With current methods of production and delivery, only 10% of the population can afford insulin, even though it was made available 50 years ago” says Henry. But getting the insulin gene into chloroplasts presented a formidable challenge. “Whilst the nucleus has pores through which DNA can enter, the chloroplast is surrounded by a complete double-layered envelope” he explains. “The only successful method so far is to use brute force”. This involves coating razor-sharp gold particles with the DNA cassettes containing the gene of interest and an antibiotic resistance gene. “We then fire millions of these at the chloroplasts, and one or two make it inside” Henry says. The DNA is then precipitated using calcium chloride, which releases it from the gold particles. The chloroplasts that successfully take up the DNA are then selected for using the antibiotic; these then multiply within the cells to replace the others.

“Using this technique in tobacco and lettuce, we managed to make three-quarters of the leaf protein to be insulin” says Henry. Although this caused the level of rubisco to decrease, surprisingly the plants appeared to grow normally. “This suggests that the level of photosynthesis is not directly related to the amount of rubisco and that an overabundance of rubisco may serve as storage for protein” says Henry.

Having perfected the technique, Henry has since turned it to a range of protein drug treatments. One which particularly excites him is the production of blood clotting proteins for haemophilia patients. Currently, the best available treatment involves injecting clotting factors into the bloodstream, however this approach – which costs between $60-200,000 a year – typically provokes the immune system to develop antibodies that render it ineffective. But it seems that producing clotting factor proteins within plants can circumvent this. “Using animal models, we have found that introducing these factors orally does not induce an immune response – a phenomenon we call ‘oral tolerance’” Henry says. “Plant cells are ideal for oral delivery because their cell walls stop the drug from being digested in the stomach, so that it reaches the gut intact for absorption”. Having demonstrated this by feeding dogs with transformed lettuce leaves, this treatment is now ready to for testing in humans. At the meeting in Florence, Henry and Shashi Kumar (International Centre for Genetic Engineering and Biotechnology, India) will be describing how this method is now being applied to produce vaccines against infectious diseases, including malaria and cholera.

Exciting as these projects sound, regulatory procedures mean it could be a while before they gain approval for clinical use. But one of his other ventures could be introduced much sooner: a novel treatment for dental cavities, caused by bacteria that live in dental plaque. Whilst antimicrobial proteins have been identified that kill these bacteria, they are prohibitively expensive to produce using normal methods and their effectiveness is severely limited by the extracellular matrix surrounding the bacteria in the mouth. Using his chloroplast-production system, Henry and his colleagues were able to develop tobacco and lettuce plants that produced both an antimicrobial peptide and a matrix-degrading enzyme. The combination proved deadly effective against bacterial biofilms growing on artificial tooth-like surfaces6. “As this is seen as a topical treatment rather than an internal one, regulatory approval could be very swift” says Henry. “We are already working with Johnson & Johnson Consumer Inc. on the possibility of developing antimicrobial chewing gums that release these peptides when chewed”.

SEE YOU IN FLORENCE?

As these case studies show, the potential applications of plant science are influencing an ever-growing number of different scientific disciplines, acting as a meeting place for researchers from a diverse range of fields. “No individual lab could do this work alone, it really needs a multidisciplinary approach” says Kirsi-Marja. “For instance, my background training is as a pharmacist, but now I am working with food scientists and the cosmetic industry”. Henry agrees: “None of these problems regarding health and nutrition can be solved with a single discipline – they require knowledge of human physiology, animal and plant models, immunology and biochemistry”. As a forum for plant, animal and cell biologists, he hopes the 2018 SEB Annual meeting will provide a chance for a whole cross-section of researchers to learn about the most recent developments in this exciting field. “It’s a wonderful opportunity for researchers to learn how to incorporate this knowledge into their own work, and we hope it will lead to some exciting new collaborations” he says.

1. Butelli, Eugenio, et al. “Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors.” Nature biotechnology 26.11 (2008): 1301.
2. Scarano, A., et al. “Combined dietary anthocyanins, flavonols and stilbenoids alleviate inflammatory bowel disease symptoms in mice”. Frontiers in Nutrition, 4, (2017) 75.
3. Tomlinson, Matthew L., et al. “Flavonoids from engineered Tomatoes inhibit gut Barrier Pro-inflammatory cytokines and chemokines, via saPK/JnK and p38 MaPK Pathways.” Frontiers in nutrition 4 (2017).
4. Betancor MB, Sprague M, Sayanova O, Usher S, Metochis C, Campbell PJ, Napier JA, Tocher DR. (2016) Nutritional Evaluation of an EPA-DHA Oil from Transgenic Camelina Sativa in Feeds for Post-Smolt Atlantic Salmon (Salmo salar L.). PLoS One. 11(7):e0159934.
5. Puupponen-Pimiä, Riitta, et al. “Effects of ellagitannin-rich berries on blood lipids, gut microbiota, and urolithin production in human subjects with symptoms of metabolic syndrome.” Molecular nutrition & food research 57.12 (2013): 2258-2263.
6. Liu, Yuan, et al. “Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells.” Biomaterials 105 (2016): 156-166.
 
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Caroline Wood

Caroline Wood

Caroline Wood was the SEB’s 2014 science communication intern. Since then, Caroline has been a regular contributor the SEB, reporting on events and writing insightful features for our members.
Caroline has an undergraduate degree from Durham University in Cell Biology and is currently a PHD student at Sheffield University studying parasitic Striga weeds that infect food crops. You can read her blog here.