Introduction

Human practices establish the link between an iGEM scientific project and people (ex. the industrialists who would like to exploit it). The goal is to introduce the project to the public, which can be potential users of the product or simply curious people or people that can be affected by it (through their environment for instance).
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. CLnA 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 activities, but also to their capacity to enter cell signaling pathways that can induce important cellular responses.
For the iGEM 2019 season, we focused our work on punicic and jacaric acids. We have succeeded in expressing two heterologous enzymes in Yarrowia lipolytica allowing the production of punicic acid (PgFadX and TkFadX). Moreover, we carried out the Jacaranda mimosifolia exome sequencing, that permitted us to identify the enzymes allowing the production of jacaric acid (JmFadX).

Our metabolic engineering project rises a series of societal questions : economical, environmental, legal, and ethical. We aimed to answer to theses in our Humans Practices.

Marketing and economic analysis

One of the first questions that comes to one’s mind is related to the marketing 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-cancerous activities [1].

Pomegranate is a plant that naturally produces punicic acid, which is particularly found in the seeds of the fruits [4]. The production of punicic acid is very expensive and restrictive because it totally depends on the harvest of pomegranates, 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 10,000 tons per year [5]. Using a provisional calculation we have estimated the amount of punicic acid imported into France. Only 55% of the 10,000 tons corresponds to the fleshy seeds.

In the nutritional values we can observe that it contains approximately 0. 78gram of polyunsaturated fatty acid per 100grams of pomegranate [6]. Punic acid represents approximately 80% of the 0.10gram of polyunsaturated fatty acids contained in the pomegranate [4]. 5 500 000 000grams of imported pomegranate / 100grams = 55 000 000grams of pomegranate 55 000 000 * 0.078g polyunsaturated fatty acid = 4 290 000grams of polyunsaturated acid 5 500 000 * 80% punicic acid = 3 432 000grams of punicic acid in 5,500 tons of pomegranates

If all punicic acid was 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 production.
We intended to produce this fatty acid in a host strain in order to limit the exploitation of pomegranate. 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 [2] [3]. The production of punicic acid has not 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. Punicic acid on the market
Jacaric acid 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 [7], 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 prevent environmental issues.

PEST

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 our project has on the outside world.

Political

Fruits and vegetables importation 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 importation in France is facilitated since both countries are part of the European Union (free movement of goods between the member of the European Union)

Commercialized essential oils should be presented with their specific function. The provider of the product is responsible of the notification of consumers on the methods and precautions for the use of the product.
It is therefore the destination (cosmetics, food, etc.) mentioned by the manufacturer that determines the applicable regulations and consequently the requirements that the product must reply. We do not choose an essential oil at random and you should pay attention to its instructions. 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 [8].
However, any allegation 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

Economical :

Punicic acid is a fatty acid contained and therefore sold especially in pomegranate fruit based products, in which it is found in greatest abundance [4].
Over the years, popularity of products based on pomegranate extracts such as essential oils increased in a growing market [9].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.

Social:

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-oxydant properties. In France, many farmers have started growing organic pomegranates for their anti-cancer properties in particular.

Technological:

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 it could be possible to reach industrial yield levels by optimizing the production pathway. By looking at the example of semi-synthetic artemisinin synthesis in Yarrowia Lipolytica (therapeutic molecule used in vaccines against malaria and produced using synthetic biology [10]) we can predict very high production shots.
The synthesis technique can therefore be adapted to several types of rare fatty acid and allow production in large quantities.

SWOT

We conducted a complementary SWOT study to determine the strengths and weaknesses of our fatty acids production technique compared to what is already done 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

Environmental preoccupations

Environment is an essential subject in the current context of the ecological crisis that our planet undergoes. Global warming, the disappearance of certain species, as well as deforestation 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 iGEM.
We explained to him in detail our project of cloning and expressing in yeast FadX genes allowing the production of fatty acid specific of vegetal species and how we would proceed because for the jacaric acid the corresponding gene is not described in the literature and the Jacaranda mimosifolia does not have its genome sequenced. He was very enthusiastic that young scientists conduct research in his fields of plant biology and found our project innovative and ambitious.
He helped us a lot in the search for information at the level of genus Jacaranda by letting us one of the only publications about the habitat of Jacaranda and the great diversity of species that make up this genus [1]. 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 Argentina and Bolivia 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, rot and 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 their pinnate compound leaves, the shape of their trunk and their fruit 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 taken for a tree that is not contributed to its vulnerability.

Dr. Florian Jabbour has also pointed us to a more recent reference [2] that stresses that Jacaranda mimosifolia is in a vulnerable state in its natural habitat because its ecosystem is converted into cultivable area for annual & non-timber crops, livestock farming and ranching.
Meeting Dr. Florian Jabbour has allowed us to realize the current conditions of deforestation around the world and especially in Amazonia and that a huge diversity of animal and plant species are at risk because the farm and minerals are prioritized over the well being of the ecosystem.
With this reflection, our project is in line with those environmental concerns. Indeed we want to make rare fatty acids accessible by producing them in large quantities, but not by chemistry or extraction of the plant directly because those conventional production ways are expensive and have a negative impact on the environment as, in order to have a good yield per hectare of cultivated land, it is necessary to grow a large number of trees and exploit the soil resources (and dope 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 the farm). We have indeed explained to 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 punicic acid. To remedy this, he recommended that we try to perform tBLASTn to align the protein sequences of the FADX family found on Uniprot (Trichosanthes kirilowii Q84UC0, Punica granatum Q84UB8, Calendula officinalis Q9FPP7 & Q9FPP8 and Vernicia fordii Q8GZC2) on the genome of a tree in the family Bignoniaceae than Jacaranda mimosifolia; 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 has therefore recommended the genome of this tree for good reason, it is the first member of the Bignoniaceae family whose genome has been sequenced for the purpose of identifying genes responsible for the formation of metabolites of therapeutic interest such as than quinones in the case of H.impetiginosus.

This could have allowed us to find a nearby enzyme allowing the production of jacaric acid and thus avoid useless sequencing to find a gene that also exists in another tree species. After making dozens of tBLASTn without success. We were therefore forced to make an Exome sequencing of J.mimosifolia by extracting the mRNAs from seeds, leaves and flowers using Genoscope. To analyze the raw data resulting from the sequencing we therefore use the genome of H.impetiginosus to align the exome.
See our Model Page
We thank 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, never 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. For this, we decided to organize 2 vox pop’s sessions to collect the opinion of various people.

We went to the Garden of Plants (https://www.jardindesplantesdeparis.fr/en) in Paris on Thursday, July 11th 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 people’s majority 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 with a diversified public.
See the full analysis of Vox-Pop

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 [3] protected the sequence of PgFadX, 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.

Inspired by these patents, we investigated on how to adapt them to the discovery of the gene coding the JmFadX, 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 6 July 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 27 July 1994 on Community plant variety rights (EUR-Lex - 31994R2100).
Article 2 of Law No. 2004-1338 of 8 December 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 JmFadX in modified yeast Yarrowia lipolytica, our culture and other media containing cells will be protected by this law.