Human Practices

Nowadays, the environment is deeply affected by high levels of pollution. Chemical processes are one of the main sources of pollution. Since synthetic biology only uses renewable resources, it seems to be a promising way to produce all kinds of molecules. However, for an interesting application of synthetic biology, a large amount of sugar would be necessary to feed all the microorganisms used to produce a large range of chemicals such as biofuels and drugs. In fact, more than 145 million tons of sugar are produced every year in about 120 countries. They are used to fulfill the various needs in food or biofuel industries for example. Around 60% to 70% of the sugar currently produced comes from sugar canes with the remainder coming from sugar beets. Like all massive monocultures, massive production of sugar has dramatic impacts on the environment (deforestation, loss of biodiversity, soil and water pollution). What would become this need in sugar if all the cars use biofuels? Would there be enough lands? What will be the impact on the environment?

To meet the considerable growing needs of sugar for synthetic biology while being respectful for the environment, we needed to find sustainable alternatives to the use of arable lands for sugar production. Given the important marine surface on Earth, producing sugar in a marine environment seemed to be a relevant solution. This could bring back synthetic biology to its contribution to sustainable development. To produce sugar in a marine environment we chose a green microalgae, Chlamydomonas reinhardtii, aka, Claude.

Impact of the monocultures on the environment

For purposes of clarity, some important impacts that monocultures have on environment are listed hereafter:

• Soil erosion due to irrigation, wind and harvest: Where irrigation application is inefficient or rainfall is high, water withdrawal is generally coupled with the loss of valuable soil from the farm.
• Soil salinization and acidification: Salinization of soils typically results from over irrigation or inadequate drainage. Acidification is largely due to the use of inorganic nitrogenous fertilizers such as urea and ammonium sulfate. Under high rainfall conditions nitrate leaching occurs, which also promotes acidification.
• Water use: Sugar cane ranks among a group of crops noted for their significant water consumption (along with rice and cotton). High water withdrawal is generally coupled with the run-off of polluted irrigation water containing sediment, pesticides and nutrients.
• Use of chemicals: The use of herbicides in sugar beet cultures is among the highest compared to other crops. Inorganic fertilizers typically supply nitrogen, phosphorus and/or potassium in the mineral form. Environmental impacts generally arise because the nutrients in the fertilizers are not entirely taken up by the crop but move into the environment. The overuse of fertilizers on cane or beet crops is typical of farming in general.
• Discharge of mill effluents: In some countries with weak environmental laws, a tremendous amount of matter is released straight into the streams. Cane mill effluents tend to include heavy metals, oil, grease and cleaning agents.

A sugar factory that mills 1,250 tonnes of sugarcane daily uses 151,000 liters of water per hour and releases 30,000 to 75,000 liters of liquid, solid and gaseous waste.

Production of sugar in marine environments by our green microalgae, Chlamydomonas reinhardtii

Why an algae?

As a photoautotrophic organism, Chlamydomonas reinhardtii has the ability to fix carbon through carbon dioxide uptake and a source of light. Its metabolism can also be autotrophic, heterotrophic or mixotrophic. All of these characteristics explain why it is an interesting chassis in comparison to other classical model (bacteria or yeasts) which always need an input of energy such as glucose. This microalgae is well-known as a model organism in the research field on photosynthesis and its genome has been characterized. Moreover, growing range of tools in molecular biology have been developed for use in Chlamydomonas, for example a brand new modular cloning tool was created and published in 2018, the MoClo toolkit set by Pierre Crozet et al. This tool offers a new field of possibilities for the use of Chlamydomonas as it is a very interesting microorganism whose biosynthesis and metabolic pathways can be exploited in synthetic biology. Indeed it was established that this microalgae could be used in the future to produce high value compounds for biotechnology industries.

How to make this algae survive in marine environment?

Furthermore, to make use of the marine environment to produce sugar, we needed to have a microalgae that can survive in salty water. So we contacted Josianne Lachapelle who worked with Chlamydomonas reinhardtii and adapted some strains to live in high salt concentration medium. And she kindly accepted to provide us with some of these strains that we could use them to make sugar without competing with arable land.

Which relevant sugar to produce in this algae?

The sugar we chose to produce in this microalgae is the trehalose C12H22O11. It is a disaccharide of glucose, that can be metabolized by Escherichia coli and Saccharomyces cerevisiae. While, it is also present in Chlamydomonas, however, it is mainly involved in stress response to H₂0₂ as an antioxidant and not as a source of energy. Without a self-production consumption, Chlamydomonas and trehalose are becoming an interesting combination for energy production. As a result, this source of energy would potentially be fully extracted and used by chassis such as E. coli or S. cerevisiae for the production of molecules of interest by synthetic biology.

Our discussions with professionals and scientists who work with Chlamydomonas or other microalgae, on directed evolution or on regulation played a major role in the further perspective of our project.

One main conclusion we got from our meetings was that algae did not bring an added value for the production of sugar or biofuels because of its high production costs. Indeed, the cost of production will highly increase because of the energy to bring to the photobioreactor, the extraction and the collection of sugar.

Thus, the amount of sugar expected by the use of marine surfaces could not be sufficient to meet the needs of synthetic biology development but would not enable a profitable production

Yield improvement

In order to improve the yield of trehalose production, we thought about directed evolution. It aims to identify beneficial mutations on a gene or on a biosynthetic pathway. It is a major tool for synthetic biology in eukaryotes. This relies on in vitro generation of diversity. However, since C. reinhardtii can not replicate plasmids, this tool would be limited in this microalgae. Also, C. reinhardtii has a significantly lower speed of replication than the classical chassis of SynBio (Escherichia coli, Saccharomyces cerevisiae). These particularities make the in vitro generation of a large-sized library of mutants by the classical "error-prone PCR" used in directed evolution, more complicated.
This is why we have been interested in the team BOKU-Vienna 2017 project . To make in vivo evolution in yeast and bacteria, they proposed another solution, a model of retrotransposon. As there is no tool to make directed evolution in Chlamydomonas yet, we worked on adapting this strategy to this organism.

Since directed evolution is an important part in the development of a powerful synthetic biology chassis, we met professionals to get expertise on the subject.

The main returns we had were that it was by itself an extensive and complex subject to work on and develop. As the needs of such a chassis in the industrial environment for sustainable development were clearly explained by the experts, it seemed more relevant to focus on our directed evolution tool instead of the sugar production itself.

GMOs, environment and legislation

Finally, an important part of our discussions focused on the legislation about GMOs in offshore platforms. The current legislation compels us to use a confined environment. Very interesting alternative systems for the photobioreactor design such as biocontainment measures were discussed with all of the experts we met.

From the low added value of the genetically modified microalgae for the production of sugar, we knew how to use the compiled knowledge for a better valuation of our project through our retrotransposon. Indeed, our meetings with professionals brought out the interest of our retrotransposon development for fundamental research and for production of various molecules of interest in biotechnology.

In this case, the engineered microalgae would be an added value to improve the quality and quantity of molecules through a sustainable way of production at industrial scales. Therefore, it would not undergo the same regulations as GMOs used in offshore platforms