"Human Practices is the study of how your work affects the world, and how the world affects your work."
Peter Carr, Director of Judging
Human practices establish the link between an iGEM scientific project and the people and society around us. The goal is to introduce the project to the public, who will be potential users of the product, will be affected by the product (through their environment, for instance), or simply have an opinion on the product.
Research projects can have an influence on many aspects of the world around. As responsible scientists, we must carefully think and take into account the impact our project in synthetic biology will have on the society.
Our project focuses on the production of Conjugated Linolenic Acids (CLnAs) using the oleaginous yeast Yarrowia lipolytica as a chassis organism.
CLnAs are unusual 18 carbons fatty acids that are known to be effective in the treatment of diabetes, obesity and certain cancers in the mouse. These abilities are mainly due to their antioxidant and anti-inflammatory properties, but also due to their capacity to enter cell signaling pathways that can induce important cellular responses.
For the iGEM 2019 season, we focused our work on two CLnAs: punicic and jacaric acids. We have succeeded in expressing two heterologous enzymes in Yarrowia lipolytica allowing the production of punicic acid (Pg-FadX and Tk-FadX). Moreover, we carried out the Jacaranda mimosifolia exome sequencing, that permitted us to identify the enzymes allowing the production of jacaric acid (Jm-FadX).
Our metabolic engineering project rises a series of societal questions: economical, environmental, legal, and ethical. We aimed to answer these in our Humans Practices endeavor.
Marketing and economic analysis
One of the first questions that comes to one’s mind is related to the market and economical implications of such a ‘Manufacturing’ project. Indeed, the final aim of our project is to develop a yeast strain capable of producing CLnAs at relevant industrial levels.
But, to which type of market are these CLnA destined for? What would be the financial and environmental benefits of our product compared to the production of these fatty acids in a conventional way such as the exploitation of agricultural lands?
Description of business
Punicic acid has many interesting properties in the cosmetics and medical field, related to its anti-inflammatory and anti-cancer activities .
Pomegranate is a plant that naturally produces punicic acid, which is particularly found in the seeds of its fruit . The production of punicic acid is very expensive and restrictive because it totally depends on the harvesting of pomegranate, which is a seasonal plant. This makes the trade cost of punicic acid really high. By producing this fatty acid through synthetic biology, we could modify the rules of the market and get rid of the seasonality of production, the negative impact on the environment, the high production/ selling prices, and increase the supply that can meet the high demand. This could also increase the demand and enable high sales volumes to be achieved.
We intended to study the global pomegranate market, but it was quite difficult to estimate due to lack of information and low rate of updates. We decided to focus on France where the import of pomegranate is around 10000 tons per year.
Using a provisional calculation we have estimated the amount of punicic acid imported into France. Only 55% of the 10000 tons corresponds to the fleshy seeds.
In the nutritional values we can observe that it contains approximately
0.078 g of polyunsaturated fatty acid per 100 g of pomegranate. Punicic acid represents approximately 80% of the 0.078 g of polyunsaturated fatty acids contained in the pomegranate .
Then we can estimate that of the 10000 tons of pomegranate only 5500 tons are edible and contains punicic acid.
For each 100 g of edible pomegranate we have 80% of punicic acid * 0.078 g of polyunsaturated fatty acid = 0.0624 g of punicic acid per 100 g. We report this value in grams to obtain 0.000624 g of punicic acid.
If we multiply the 5500 tons of edible pomegranate by the 0.000624 g of punicic acid we have = 343200000 g of punicic acid in the 10000 tons of imported pomegranate.
If all punicic acid were extracted from imported pomegranate in France there would be 4.4 tons of punicic acid each year. The reality of the market is that pomegranate is mainly consumed in the form of whole fruit, juice and essential oil. This allows us to see that there is a huge loss of punicic acid which reinforces us in the utility of a bio-production method.
We intended to produce this fatty acid in a host strain in order to limit the exploitation of pomegranate plant production. We decided to use the Yarrowia lipolytica chassis to produce our molecule of interest. This yeast strain is known to have high yield and storage capacity of fatty acids [3, 4]. The production of punicic acid has not yet been realised using synthetic biology in Yarrowia lipolytica chassis.
Purpose and values of our project
The main reason we want to produce rare fatty acids is to make them more accessible on the market. Despite their very interesting properties, the fatty acids have an excessively high price which restricts their uses in the medical and research fields.
They are available on the market:
We also wish to prevent the direct extraction of punicic acid from pomegranate seed in order to avoid environmental problems such as overexploitation of grenadiers that could lead to deforestation.
The synthetic biology production system that we use is not limited to the production of punicic acid. Indeed, using the Yarrowia lipolytica chassis we built and fine-tuned, we can easily produce different fatty acids.
One of these acids is jacaric acid, which also is a rare fatty acid and has similar properties to punicic acid . This acid is extracted from the plant Jacaranda mimosifolia .
Jacaranda mimosifolia is a tree classified as vulnerable by IUCN Redlist . Its extinction would lead to the loss of the jacaric acid pathway.
By using the Yarrowia lipolytica chassis we built for the production of punicic acid for the synthesis of jacaric acid, we could prevent and stop a possible overexploitation of Jacaranda mimosifolia for production of jacarid acid and thus help prevent an environmental catastrophe.
We performed a PEST analysis in order to acknowledge the different factors that could affect our project (political, economic, social and technological). This approach also gave us an overview on the influence the outside world has on our project, and vice versa.
Fruits and vegetables import market is very important in France. Indeed, France imports 40% of its fruits and vegetables.
The pomegranate is a fruit produced mainly in Spain. Its import into France is facilitated since both countries are part of the European Union (free movement of goods between the members of the European Union).
Commercialized essential oils should be presented with their specific function. The provider of the product is responsible for the notification of consumers on the methods and precautions for the use of the product.
Essential oils are highly concentrated in active chemical elements, such as fatty acids, and may present dangers. European Union classifies some of them as dangerous substances and requires, as such, the presence of clear statements intended to inform the consumer. Punicic acid and fatty acids in general are allowed in Europe since they do not present any known danger .
However, any claim that a non-prescription essential oil could prevent or treat a disease is otherwise prohibited. It would make the essential oil a drug by definition (Code of Public Health). Some essential oils are part of the composition of medicines.
Punicic acid is a fatty acid contained and therefore sold especially in pomegranate fruit based products, in which it is found in greatest abundance . Over the years, popularity of products based on pomegranate extracts such as essential oils increased in a growing market . These essential oils draw their properties from the punicic acid they contain. Also, the price of pure punicic acid costs about 60 € / mg, making it a very expensive product on the market.
Over the last years, studies showed the impact different diets can have on health and environment. People tend to change their eating habits for more eco-friendly and healthy products. This is why the organic market is becoming more and more important. In this context, pomegranate is an increasingly popular fruit, due to its anti-inflammatory and anti-oxidant properties. In France, many farmers have started growing organic pomegranates for their anti-cancer properties in particular.
Using synthetic biology, we have been able to show that the production of punicic acid was possible in the lab. Even if the production yields are not high in our initial attempt it should be possible to reach industrial yield levels by optimizing the production pathway. By looking at the example of semi-synthetic artemisinin synthesis in budding yeast Saccharomyces cerevisiae (therapeutic molecule used in vaccines against malaria and produced using synthetic biology ) we can predict very high production levels. The synthesis technique can therefore be adapted to several types of rare fatty acids and allow production in large quantities.
We conducted a complementary SWOT study to determine the strengths, weaknesses, opportunities, and threats of our fatty acids production technique compared to what is already in the market. This work helped us understand the financial risks taken, the possible lack of innovativity, and the time it would take to commercialize such products.
Meeting with Florian Jabbour
Environment is an essential subject in the current context of the ecological crisis that our planet is undergoing. Global warming, the disappearance of certain species, as well as deforestation that are mainly caused by human actions.
We wanted to deepen our investigation about environment, to have a more precise idea of the state of cultivation of the plants producing CLnAs (pomegranate tree, jacaranda tree, ....). We started with Jacaranda mimosifolia and, taking it as a kind of adventure towards the unknown, we decided to meet and discuss with a professional in this field. We therefore contacted and met Dr. Florian Jabbour, senior lecturer and collection manager in the field of morpho-anatomy and plant development at the Institute of Systematics, Evolution, Biodiversity of the National Museum of Natural History of Paris.
Thanks to him, we were able to discover the research work conducted in the field of plant anatomy and development. It was very rewarding for us, and for Dr. Florian Jabbour also, who did not know about the iGEM competition.
We explained to him in detail our project of cloning and expressing FadX genes in yeast, allowing the production of fatty acid specific of vegetal species. He was curious on how we would proceed since Jacaranda mimosifolia does not have its genome sequenced and the gene permitting synthesis of jacaric acid is not described in the literature yet.
He was very enthusiastic seeing that young scientists conduct a research project in his expertise field and found our project innovative and ambitious.
He helped us a lot by sharing his knowledge on the Jacaranda genus, and provided us with one of the only publications about the habitat of the great diversity of Jacaranda species . We have studied this PhD thesis, written in German, and discovered that Jacaranda species are natively found throughout South America. More specifically, Jacaranda mimosifolia is native from the lower Andean slopes of northwestern Argentinean-Bolivian border.
The author Wilfried Morawetz writes that Jacaranda mimosifolia is used as an ornamental wood because often confused with other wood species such as Dalbergia nigra and Machaerium villosum also called RoseWood, these trees are vulnerable because of their interesting properties of their woods at the physicochemical and aesthetic level. Indeed their wood is light, soft, and has a color ranging from purple to brown. These trees are complete with Jacaranda mimosifolia because on the one hand there is an amalgam with their morphology (indeed, with their pinnate compound leaves), the shape of their trunk and even their fruits are similar. They have a common localization and in the brazilian culture the term Jacaranda can describe several tree species such as Machaerium villosum also called Jacarandá-do-Cerrado and Jacarandá-da-Bahia which designates Dalbergia nigra. This confusion threatens more and more the real Jacaranda mimosifolia, since to be mistaken for a tree that is not threatened contributes to its vulnerability.
A more recent reference suggested by Dr. Florian Jabbour revealed that Jacaranda mimosifolia’s natural habitat is in a vulnerable state due to the conversion of its ecosystem into cultivable area for annual and non-timber crops, livestock farming and ranching .
Meeting Dr. Florian Jabbour reminded us of the current conditions of deforestation around the world, and specifically in the Amazonia. Several animal and plant species’ ecosystems are at risk because of the priority given to agriculture and mineral extraction.
We tried to think about our project with a view on those environmental concerns. Indeed, we intend to produce large quantities of rare fatty acids using a more accessible and more environment-friendly method. Current methods are expensive, way too disastrous for the environment: in order to have a good yield per hectare of cultivated land, it is necessary to grow a large number of trees, exploit the soil resources, and potentially mix them with chemical supplements for a large scale production.
Integrated Human Practices
In addition to this valuable information about Jacaranda mimosifolia, its morphology, its distribution and the reasons for its vulnerability (mostly deforestation for farming), we have indeed explained to Dr. Florian Jabbour that the genome of this tree was not present on the databases and that it was therefore difficult for us to overcome this obstacle in our project to produce jacaric acid. To remedy this, he recommended that we use tBLASTn to align the protein sequences of the FadX family found on Uniprot (Trichosanthes kirilowii Q84UC0, Punica granatum Q84UB8, Calendula officinalis Q9FPP7 and Q9FPP8 and Vernicia fordii Q8GZC2) on the genome of a different tree in the family Bignoniaceae: Handroanthus impetiginosus. This tree is under the same pressure as the previously mentioned trees (Dalbergia nigra and Machaerium villosum) because Handroanthus impetiginosus is the most expensive tree on the world market and the most exploited in Brazil. Florian recommended the genome of this tree for good reason, as it is the first member of the Bignoniaceae family whose full genome has been sequenced for the purposes of identifying genes responsible for the formation of metabolites of therapeutic interest such as quinones .
This could have allowed us to find a nearby enzyme allowing the production of jacaric acid and thus avoid extensive sequencing to find a gene that also exists in another tree species. After making dozens of tBLASTn without success, we finally did an exome sequencing of Jacaranda mimosifolia by extracting the mRNAs from seeds, leaves and flowers using Genoscope’s sequencing platform. To analyze the raw data resulting from the sequencing we therefore used the genome of Handroanthus impetiginosus to align the exome. The full analysis is available on the dedicated page on this wiki.
We thank Dr. Florian Jabbour for his invaluable help because he gave us one of the keystones of our project of discovery of an enzyme for the production of jacaric acid, so far not realized by synthetic biology.
Vox pop on fatty acids
Our project is centered around lipids and lipids are generally quite frowned upon by society because they’re automatically associated with weight gain or cardiovascular diseases. Knowing that our project focuses on fatty acids, we went to meet the public in order to "de-demonize" them, and to assess if their perception of fatty acids is rather positive or negative. To this purpose, we decided to organize two vox pop’s sessions to collect the opinions of various people.
We went to the Garden of Plants in Paris on Thursday, July 11th 2019 and talked all day to visitors. We realized that the majority of people had knowledge about fatty acids and that they were more and more concerned about their diet. Indeed, currently, many people are trying to eat healthier and have a balanced diet. Fatty acids are not so badly seen by society, as it’s increasingly known that they provide benefits to our body at moderate doses, unlike sugar, which is now recognized by the public as being unhealthy, because it’s addictive and mostly consumed in refined form.
In particular, we asked a question directly related to our project in order to gauge people’s reaction to fatty acids produced from a genetically modified organism, like the yeast in our project. The majority of people said they would probably accept if it’s prescribed for treatment.
We also did a second vox pop session during the Science Festival (“La Fête de la Science”) on Saturday, October 12th 2019 with a diversified public.
The full analysis of Vox-Pop is available on the dedicated page on this wiki.
Juridic and legal aspects
We have established a legal profile of the project and more particularly in the intellectual property framework on the discovery of a gene and techniques for its exploitation.
Several naturally occurring CLnA are known and their production methods and utilisation were patented. For instance, patent n° CA2454372A1  protected the sequence of Pg-FadX, the techniques associated with its discovery and of its expression in yeasts and plants for the purpose of production and extraction of punicic acid contained in the lipids of these organisms:
- RNA isolation from Punica granatum seed
- Isolation and cloning of Punica granatum punicic acid desaturate and Δ-12-desaturate
- Obtaining and sequencing complete cDNA clones (RACE PCR)
- Expression of punicic acid acid desaturase and Δ-12-desaturase in yeast
- Lipid extraction of the fatty acids from transgenic yeast, and GC / MS analysis
- Expression of punicic acid desaturase in plants in Nicotiana tabacum, Brassica napus and Linum usitatissimum via Agrobacterium-mediated DNA transfer.
Similar patents were filed for the production of calendic  and eleostearic  acids.
Inspired by these patents, we investigated on how to adapt them to the discovery of the gene coding the Jm-FadX, the enzyme catalysis the synthesis of jacaric acid.
The French and European laws allow this filing in this type patent as defined by the directive 98/44/EC of the European Parliament and of the Council of July 6th 1998 on the legal protection of biotechnological inventions (EUR-Lex - 31998L0044) and by the French Intellectual Property Code. Moreover, Jacaranda mimosifolia is not part of the protected plant varieties as defined in Article 5 of the Council Regulation (EC) No 2100/94 of July 27th 1994 on Community plant variety rights (EUR-Lex - 31994R2100).
Article 2 of Law No. 2004-1338 of December 8th 2004 on the protection of biotechnological inventions in France is a continuation of the articles previously mentioned since it specifies that any inventions having for object a microbiological or a product obtained by this process as patentable .
In addition, article L613-2-2 of the French Intellectual Property Code states that “the protection conferred by a patent on a product containing or consisting of genetic information shall extend to any material in which the product is incorporated and in which the genetic information is contained and performs its stated function”. This implies that when producing jacaric acid by means of Jm-FadX in modified yeast Yarrowia lipolytica, our culture and other media containing cells will be protected by this law.
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