Team:Sorbonne U Paris/Human Practices

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PALM OIL, THE CHALLENGE OF AN EXPONENTIAL GROWTH


Palm oil is the most used and produced vegetable oil in the world. Its production is estimated to be more than 60 million tons per year approximately1. For several decades, palm oil has been used in various sectors such as food, cosmetics and biofuels.

Its very high yield per hectare, its low cost of production as well as its physicochemical properties explain its wide use by the industry. Palm oil demand has grown exponentially in the past 30 years, today it is a huge economical source that raises social and environmental issues1.

As for Indonesia and Malaysia (the two main palm oil producing countries, representing 85% of world production), oil palm exploitation is a vector for economic and social development as 40% of production areas are exploited by small producers.

Even if it’s not the only cause of tropical deforestation, palm oil production is associated with the destruction of tropical forests and loss of biodiversity. In Indonesia, between 1990 and 2008, 40% of agricultural deforestation was due to oil palm exploitation, according to data from the Food and Agriculture Organization of the United Nations1.

In 40 years, the island of Borneo has lost 18.7 million hectares of primary forest (out of the 55 million that it had in 1973). 7 million hectares among that are attributable to industrial plantations (oil palm and pulp). The endemic species in Sumatra and Borneo islands are particularly illustrative of the environmental issues associated with the production of palm oil. Today, the major challenge is therefore to find a balance between conserving biodiversity and supporting the economic development of producing countries.

A NEW WAY OF PRODUCTION : SYNTHETIC BIOLOGY


Synthetic biology is an innovative domain that is essential for our future needs. Indeed, it provides solutions for contemporary problems, by targeting and modifying the functions of targeted organisms. Our team’s goal is to develop a new model for lipid compounds production in a fresh water green microalga, Chlamydomonas reinhardtii, capable of producing the main components of palm oil, palmitic acid and oleic acid, to demonstrate the validity and the relevance of our concept.

In an attempt to answer this issue, we have genetically engineered that unicellular alga to modify its lipid metabolism. Indeed, the two main components of palm oil, palmitic acid (C16:0) and oleic acid (C18:1)2, are already produced by C. reinhardtii but in different quantities compared to oil palm seeds3. Taking inspiration from the 3P strategy4, we have added genes coding for enzymes from the lipid metabolism of the african oil palm Elaeis guineensis in the C. reinhardtii genome. We obtained preliminary results that support the idea that our project is feasible, thus enabling the production of a palm oil like oil by C. reinhardtii.

We selected that alga for its two characteristics: it is not only capable of producing the components of palm oil but also of storing them in the form of triacylglycerol. This unique ability can be amplified under conditions of stress or by co-cultivation along with other organisms.
Also, the development of a cellular model for oil production has several benefits. First of all, this organism can be cultivated in both solid and liquid medium, with a generation time of four hours. Moreover, it is a mixotrophic organism so it is capable to produce its own organic material:    • in presence of light energy, via photosynthesis;
                    • in absence of light, using acetate as a carbon source;

Its development doesn’t require any nutritional supplementation. Finally, since it is an in-vitro model, production will not be subjected to environmental, climatic or political hazards, thus ensuring stable and non-polluting production.

CHLAMYDOMONAS REINHARDTII  CULTURE AND ITS BENEFITS


Although it is unlikely that the production yield of oil by C. reinhardtii could compete with the one from the oil palm5, our project could introduce a different way of producing palm oil. For instance, it opens new spaces for the production of palm oil allowing a different distribution of the production areas which is less damaging for the fragile ecosystems that are threatened by palm oil production nowadays. Furthermore, since C. reinhardtii lives in aquatic environments, its culture does not compete with other crops in terms of land usage. To offset the deficiency of yield in comparison with oil palm, the production of palm oil by C. reinhardtii could be included in a larger scale production planning with other benefits. For instance, cultures of C. reinhardtii in wastewater has been described6 allowing the removal of the nitrate and phosphorus from wastewater. Moreover, as the palm oil is extracted from the TAG of C. reinhardtii, the remaining biomass, from the membrane in particular, could be used to produce ethanol7 or biofuels8.

To conclude, even though palm oil production by C. reinhardtii may not be optimal compared to oil palm culture, it has other benefits that could in the end enable a responsible way of producing palm oil.




  1. Quel est le problème avec l’huile de palme ? [Visited on October 18, 2019]. Available here
  2. Dyer, J. M., Stymne, S., Green, A. G., & Carlsson, A. S. (2008). High‐value oils from plants. The Plant Journal, 54(4), 640-655.
  3. Li‐Beisson, Y., Beisson, F., & Riekhof, W. (2015). Metabolism of acyl‐lipids in Chlamydomonas reinhardtii. The Plant Journal, 82(3), 504-522.
  4. Vanhercke, T., El Tahchy, A., Liu, Q., Zhou, X. R., Shrestha, P., Divi, U. K., ... & Horn, P. J. (2014). Metabolic engineering of biomass for high energy density: oilseed‐like triacylglycerol yields from plant leaves. Plant biotechnology journal, 12(2), 231-239.
  5. Lam, M. K., & Lee, K. T. (2012). Microalgae biofuels: a critical review of issues, problems and the way forward. Biotechnology advances, 30(3), 673-690.
  6. Kong, Q. X., Li, L., Martinez, B., Chen, P., & Ruan, R. (2010). Culture of microalgae Chlamydomonas reinhardtii in wastewater for biomass feedstock production. Applied biochemistry and Biotechnology, 160(1), 9.
  7. Choi, S. P., Nguyen, M. T., & Sim, S. J. (2010). Enzymatic pretreatment of Chlamydomonas reinhardtii biomass for ethanol production. Bioresource technology, 101(14), 5330-5336.
  8. Pruvost, J., Van Vooren, G., Le Gouic, B., Couzinet-Mossion, A., & Legrand, J. (2011). Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application. Bioresource technology, 102(1), 150-158.



VIDEO SEQUENCE


Here we would like to give an overview on the huge production of palm oil: its importance but also its consequences and drawbacks. This subject is quite complex: most of popular beliefs concerning oil palm cultivation, environmental impacts and its alleged harmfulness on health are wrong. We therefore thought that a short video - inspired from the format of the famous videos proposed on social media - would be a simple and accessible way to deny some misconceptions, raise awareness on social media of palm oil production, and even encourage people to get more information. We made this video based on reliable and recent bibliographical sources in order to propose an objective point of view about the subject. The images are from a footage shot by a famous French youtuber, non-governmental association (WWF International) and media (National Geographic).










JEAN-LUC CACAS


Dr. Jean-Luc CACAS is a lecturer at the engineering school AgroParisTech. He is part of the research team “Differentiation and Cell Polarity” at the institute Jean-Pierre Bourgin (UMR1318, AgroParisTech, INRA, CNRS) where he studies the roles of lipids in cellular processes in plants. His teaching courses includes metabolic engineering for lipid production in plants.


Interview

We initially sought to interview Jean-Luc CACAS as its expertise in lipids would help us in our understanding of lipid metabolism in plants and what strategy we could apply to reach our goal. After explaining to us how the lipid metabolism works in general in plants, he gave us several clues on how to engineer our metabolic pathway of interest by giving us many examples of successful designs aimed at producing a specific lipid of interest in plants. He also explained that metabolic engineering requires a lot of knowledge and data on your system in order to be efficient. It requires many cycles of design-build-test to reach the objective. Due to limitations in our current capabilities in modelling systems, we will have to decide at one point of a satisfying design. No matter how much we think and analyze ahead, our design will still be hypothetical so we should not get stuck on this part. Only by implementing our design we will know whether it is good or not.


How it affected our project

His explanations and successful examples of engineered systems helped us identify our strategy of cloning key enzymes to tweak the fatty acid composition of triglycerides: by cloning a specific isoform of fatty acid synthase, we can enhance the production of a specific fatty acid length as every isoform has a different affinity depending on the fatty acid length. By cloning lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase enzymes, which are part of the triglyceride synthesis pathway, it will enhance the incorporation of fatty acid in triglycerides. As the pool of free fatty acid has been tweaked to be higher in a certain specific fatty acid of interest, it will enhance the overall quantity of triglycerides produced with a tweaked fatty acid composition.


JULIETTE PUYAUBERT


Juliette Puyaubert is a lecturer at Sorbonne Université. She is part of the team Seed Biology of the Institut de Biologie Paris-Seine (UMR 7622 Sorbonne Université, CNRS) where she studies seed biology in Arabidopsis thaliana. She has worked in the past on lipids in plants.


Interview

She gave us many papers on metabolic engineering in plants and helped us tackle the issue of fatty acid desaturation in our model. One of the drawbacks of Chlamydomonas reinhardtii is that it produces many mono- and poly-unsaturated C18 fatty acids. She explained to us the different possibilities in preventing these unsaturation and its potential effects on growth and viability of a modified strain that would produce less or no unsaturated fatty acids.


How it affected our project

Thanks to her help we were able to identify the potential targets that need to be modified in order to design a strain of C. reinhardtii that wouldn’t produce these unwanted unsaturated C18 fatty acids. The most important desaturase is CrFAD2, which catalyzes the second unsaturation of the oleic acid. As the unsaturations are done in a defined order, targeting the beginning of the unsaturation process should prevent the other unsaturations. From these findings we decided that the most promising method of preventing unsaturation would be to create an inducible artificial microRNA (amiRNA) targeting the CrFAD2 mRNA. Our MoClo kit contains the elements required to create one. Unfortunately, due to limited time and knowledge, we were able to come up with a amiRNA design but not implement it. We weren’t sure whether this design would work and we didn’t have the time to develop the experimental setup needed to validate the expression and activity of this amiRNA.

ALAIN RIVAL


Alain Rival is a biologist and an agronomist. He is the regional director of CIRAD (Center for International Cooperation in Agricultural Research for Development) for South East Asia and correspondent for the palm oil industry.


Interview

We sent him a mail with a detailed description of our project and he made some comments about it. Firstly, he said that there is no “overproduction” of palm oil. The demand has been growing exponentially for 30 years, and the supply is following. Public policies in South-East Asia are aimed at stabilizing prices, by regulating the incorporation of palm oil in domestic agrofuel (260 million inhabitants in Indonesia) or for export. Moreover, the world production will exceed 70 million tons this year and oil palm represent only 4% of global deforestation. Secondly, for him it is not possible to consider producing 72 million tons of palm oil annually from algae crops. Indeed, this project raises awareness : what would become the 20 million hectares of tropical agricultural land devoted to the cultivation of the palm? And the 20 million people of planters, transporters and refiners who depend on this resource in countries with declining but still high poverty rates, especially in the countryside? Thirdly, he said that the interest of your project is real, but it is elsewhere. Indeed, for him the aim is to explore lipid biosynthesis pathways in an original, in vitro, perfectly stable controlled model independent of agro-environmental variables (which will never be the case for a sunflower, rapeseed or palm crop). Finally, he said that the "ideal alternative for more environmentally friendly and responsible palm oil production" is certainly not to replace it with a high-tech model in developed countries, but to work on the ground in tropical countries to develop agricultural practices and to integrate sustainable protection of natural biodiversity.


How it affected our project

His comments made us thinking about the social progress dimension and the ethical limits of our project. Moreover, he made us realize that it was impossible to replace the actual production of palm oil by a microalgae production for scientific reasons but most importantly for ethical reasons. For this reason, we decided to modify the main goal of our project.

STÉPHANE DUSSERT


Stéphane Dussert is a researcher at IRD (Development Research Institute). He is part of the research team “Diversity, Adaptation and Development of plants” (UMR DIADE, IRD Montpellier, France) where he studies the evolution of genomes and transcriptomes in relation to the adaptation of plants to environmental constraints, adaptive species divergence, and modes of regulation of specialized metabolism.


Interview

We sent him a mail with a detailed description of our project. He said it was an ambitious project but he made some comments about it.
First, he said that if we want to produce an oil which contains the compounds of palm oil, we have to consider the fatty acid composition of palm oil but also its vitamin E and pro-vitamin A composition. Indeed, these compounds are very present in palm oil and this is why the South population is very interested in the nutritional aspect of red palm oil. But to do so, we would have to modify different metabolic pathways at the same time and it seems very ambitious to Stéphane Dussert.
Second, if we only want to produce the fatty acid compounds of palm oil, it could interest the North agri-food industries because they need concrete fats without hydrogenation of vegetable oils. Indeed, this step create trans fatty acids which have negative effects on health. To do so, we have to stop the 16:0-ACP to 18:0-ACP elongation step. Last, he said that if we want to produce with C. reinhardtii as much oil as oil palm, he is not sure that the push, pull and protect strategy could be applied to our microalga. Moreover, to help us with our project, he gave us scientific papers about his work.


How it affected our project

His comments made our project evolve. Indeed, at the beginning we wanted to engineer C. reinhardtii to produce palm oil. Now, we want to use C. reinhardtii as new chassis for lipid production and as a proof-of-concept, we aim to produce the main fatty acid components of palm oil, for instance palmitic acid and oleic acid. Moreover, he gave us a scientific paper about the transcriptome analysis of palm oil tissues (Dussert et al. 2013) which helps us deciding the final list of the African oil palm enzymes we express in our microalga: FAT-A, FAT-B2, LPAAT-A, DGAT1-2.

IGEM SORBONNE UNIVERSITÉ


As the release of GMO's is a major issue in France, we have searched the French laws about it. Moreover, the iGEM Sorbonne Université 2018 worked on the same chassis - Chlamydomonas reinhardtii, we have drawn our inspiration from their work. Indeed, last year, they interviewed Stéphane Lemaire, a researcher that works at CNRS (National Center of Scientific Research). He is studying the photosynthetic fixation of carbon in Calvin’s cycle in microalgae, particularly C. reinhardtii. He identified several challenges in their project. For example, he raised the issue of what could happen if the GMOs were released without any control in the environment. Indeed, he said that the release of our microalgae into the marine environment could lead to horizontal gene transfer between marine microorganisms. Therefore, the team imagined a bio-containment strategy for their engineered microalgae.


How it affected our project

We decided to include this strategy in the design of our engineered C. reinhardtii. For more informations, see the Safety page of our wiki.

NICOLAS HECK


Nicolas Heck, associate professor at Sorbonne University, carries out research in neurosciences at the Institut de Biologie Paris-Seine (UMR 8246 Sorbonne Université, CNRS). We discussed in depth with Nicolas Heck about the bioethical issues raised by the implementation of our project. Concerning the consequences of our project: we minimize the risks to human health as our manipulators scrupulously respect proper laboratory practices and our GMOs will not be directly consumed. We are also very careful about the risks for the environment, they are also highly limited because our GMOs remain confined in the laboratory and our waste is properly treated. An environmental problem remains the consumption of fresh water necessary for the cultivation of C. reinhardtii. We are therefore considering a water recycling system that is being used already. This question also brings us to the threats to human rights: our consumption must not deprive some communities of freshwater. In addition, we must think about the economic future of countries whose economy is based on palm oil production: we must prevent unemployment and precariousness in these countries.