Human Practices

1 - The response to the Covid 19 pandemic has highlighted the global inequality of access to new medicines and vaccinations, particularly in developing countries.

Antibiotics are a telling example of this inequality. The three largest exporters of antibiotics are China, Switzerland and Italy, i.e. industrialized countries. Similarly, the largest importers of such drugs are India, Italy and Germany, again industrialized countries.

Of the estimated 17 million annual deaths caused by bacterial infections, more than 50% of the 5.7 million treatable by antibiotics occur in LMICs (Low- and Middle-income countries) (CDDEP, 2019), (Emerson de Lima Procopio et al., The Brazilian Journal of Infectious Diseases, 2012). In these countries, the lack of access to medicines is mainly related to:

• Their price for the patient. In most of these countries, financial constraints severely limit government investment in the health system, placing the cost of health products, including antibiotics, largely on the shoulders of patients.
• Quality. LMIC markets are less regulated than those in developed countries, allowing the distribution of poor quality or even falsified medicines. Antibiotics account for 17% of these medicines (reported to WHO) and their use is responsible for at least 169,000 deaths from childhood pneumonia each year.
• Availability. Lack of supply chain and willingness to stockpile (which may be subject to competition between countries) limits the spread of the drug. In remote/isolated areas, the cost of transporting patients can reach 20% of medical costs.

We believe that health is a fundamental right and want to improve equity in access to medicines. Our project aims at a faster and cheaper access to antibiotics for developing countries by allowing a local production and requiring a low input of carbon resources.

2 - Anthropogenic activities, especially industrial ones, are the cause of an increase in the atmospheric concentration of greenhouse gases.

From 2011 to 2020 human use of fossil fuels was responsible for the emission of 35 GtCO₂ per year on average. A figure three times higher than the average absorption achieved in the same time by the oceans or by the biosphere. In the last decade, this imbalance in the carbon cycle has led to an average negative balance of 1GtCO₂ net released per year (Global Carbon Project, 2021).

In 2011 the atmospheric concentrations of carbon dioxide(CO₂), methane(CH₄) and nitrous oxide (N₂O) were already 391 ppm, 1803 ppb and 324 ppb. They were thus respectively 40%, 150% and 20% above pre-industrial levels (IPCC, 2013). Such an increase is accompanied by a positive radiative forcing, leading to changes in the climate system with serious consequences. The net absorption of energy by the Earth system results in a global increase in temperature (the 30 years prior to the 2013 IPCC study were probably the warmest 30-year period in the Northern Hemisphere in 1400 years) which induces, among other things, deregulations of the water cycle.

In France, the average temperature has increased by 1.7 C° since 1900. That is 0.7 C° more than the global average estimated by the IPCC (IPCC, 2013). The consequences of this increase on a national scale are numerous, but one of them, the increase in intensity and frequency of heat waves and droughts, is particularly heavy for society. In 2021, France Assureurs (French Insurance Federation) estimated that the cost of drought from 1989 to 2019 was 13.8 billion euros and that this cost would increase to 43 billion euros for the period 2020-2050 (French Insurance Federation report, 2021).

Our Solution

In a world where climate change has become a major concern, we believe that technological innovation must be built around increasing efforts to minimize carbon emissions and close its cycle in all industries.

This is what we did with our technology. We decided to implement a CO₂ fixation module (here, the Calvin cycle) into a Streptomyces strain. Streptomyces are a genre of bacteria widely used for the production of antibiotics: “[They] are prolific producers of antibiotics and are responsible for producing more than 50% of our clinically relevant antibiotics” (Zhang et al., Sciences Advances, 2020).

By building an autotrophic strain of Streptomyces that can be used for industrial production of antibiotics, we would close the carbon cycle and help make antibiotic production carbon neutral. The impact of our project is all the more important as the pharmaceutical industry is significantly more greenhouse gas emitting than the automotive industry. In the United Kingdom, the carbon footprint of the NHS (National Health Sector), Public Health and Social Care system represented 38% of public sector emissions in 2012. The largest contributor to this footprint was pharmaceuticals which accounted for 16.25 % (Bekir & Elmeligri, Journal of Cleaner Production , 2019). With the help of our technology, we could reduce this number and increase access to care by lowering the cost of antibiotics. That's why we named our project CO₂Cure.

We hope that this project will be the first step in a generalized movement towards sustainable industrialized production. Here is our contribution to carbon neutrality.

Figure: CO2CURE, a first step towards carbon neutrality

Meeting with members of civil society (children, relatives) made us realize how important it was to communicate more about the concept of carbon neutrality. We have also seen the extent to which autotrophy projects such as CO2CURE could be a source of hope. Some people told us “It is nice to know that there are researchers who are working hard on this”. Indeed, global warming is a source of anxiety for our societies. Knowing that synthetic biology projects exist to fight against it is a source of hope for our generations.

Interview with Dr Céline Aubry: Inspiration for a new biobrick

At the beginning of the interview, we gave a brief summary of our project to Dr. Céline Aubry, indicating to her that we planned to introduce different biobricks (at least genes encoding the PRK and RuBisCO) into Streptomyces

Figure: Picture of Dr. Aubry and Raphaël from team GO_Paris-Saclay

Dr. Aubry is a researcher who did a thesis on the refactoring of a biosynthetic pathway in Streptomyces in Orsay. She knows the competition very well, having been an iGEM coach herself ! After a post-doc at the Museum of Natural History of Paris, she then joined as a full time researcher the CEA in the microbiology department within Muriel Gaudry's team (I2BC). In this team, she studied different pathways of Streptomyces biosynthesis. In her publication (Aubry et al. AEM, 2019), Dr Aubry describes insertion plasmids with interchangeable modules. It was during our interview that we were able to get all the information about these plasmids (Table 1, Figure 1). To construct the plasmids that will be described immediately after, she took over Gibson's work by gathering several fragments of different plasmids built by the latter.

Tableau 1: List of the Streptomyces vectors of the pOSV collection built by Dr Aubry (Aubry et al. AEM, 2019)

Figure 1: Genetic organization of pOSV805 vector and of its modified version harboring BBa_K4370010 part

The pOSV plasmids have five different modules including two interchangeable modules:

• Module 1: Origin of replication
• Module 2: Antibiotic resistance cassette. There are three interchangeable resistance modules encoding the resistance to apramycin, hygromycin or kanamycin.
• Module 3: Origin of transfer of the plasmid by conjugation from Escherichia coli to Streptomyces
• Module 4: Integration system cassette. There are four possible specific insertion site modules for this part. This allows us to target with very high precision an area of 25 to 50bp in the target genome.
• Module 5: amilCP cassette. The inserts we want to clone into the plasmid will replace the amilCP cassette. Indeed the latter makes the E. coli colonies blue. This means that if the cloning failed (i.e. the gene of interest did not replace the amilCP module), the colonies are blue. Otherwise the colony will be white. This can be useful to screen easily the good clones.

By combining the different interchangeable modules, we can therefore obtain twelve different integrative vectors for Streptomyces engineering!

When Dr Aubry built these plasmids, the idea was to build a plasmid where each module would be perfectly described and known but also that would allow testing a maximum combination of antibiotics and insertion sites in a minimum time. By discussing with her, we realized that this collection of plasmid does not allow the direct cloning of a CDS under the control of Streptomyces promoter and ribosome binding site (RBS), since these latter are not already present in the plasmid. This led us to want to develop a new cloning module for the competition. This is how the idea of the biobrick BBa_K4370010 was born!

Interview with Dr Luisa Ferreira-Dos-Santos: Inspiration for a new application of our project

We met Dr. Ferreira-Dos-Santos a few weeks after the first interview with Dr. Aubry. We began by giving a brief summary of our project Dr. Ferreira-Dos-Santos. We asked her how she would implement our project, either in the industry or elsewhere, because she used the work on the expression of specialized metabolites in Actinobacteria, in relation to industries.

Figure: Picture of Dr. Ferreira-Dos-Santos, Thomas and Raphaël from team GO_Paris-Saclay

She told us that Streptomyces are used, among many other Actinobacteria , to do bioremediation of copper contaminated soils, especially in South America. The strains used to do this were discovered in copper mines, and are naturally highly copper resistant. Though, these strains grow very slowly because these kinds of soils are very low in carbon. By making this type of Streptomyces C-autotrophic, we could allow it to decontaminate soils at a much higher speed. Moreover, this kind of C-autotrophic strains can help a lot of research teams working in bioremediation of soils because on the opposite of industries, the funding they receive is very limited. This interview made us realize that there wasn’t only one way to implement our project, and that making strains C-autotrophic had to become the epicenter of our focus. From this point on, the team focused as much as it could on implementing the C-autotrophy in our strains, and on the engineering of biobricks that would allow us to implement our genes in much more strains than what we previously wanted.

Meeting of Sokrich Ponndara: The artistic inspiration of our project

Sokrich Ponndara is an artist-scientist that we met in the laboratory during our experiments. He is a phD student working on Salmonella in Frédéric Boccard’s team (I2BC). We had the opportunity to discuss industrial sustainability, ecological transition and the role of science, and more particularly synthetic biology, in these processes, and also of the responsibility that we, young scientists, feel in the fight for the climate.

Sokrich was inspired by the values of our project, and its ambition to pave the way for systematic carbon neutrality for all processes. He made an animated video, "CO₂ crisis, synthetic help for a better future", depicting with music the steps that led from the big bang to the human impact on the climate system and our response through the project. This video, without words, is a cry for carbon neutrality and an opening to the potential of synthetic biology to help us. The result of an enriching scientific and artistic interaction, the images speak for themselves and for everyone.

This interaction reminded us that if science has a role to play, it is not alone. Art has a power of diffusion and communication essential to the realization of a project. We realized our shortcomings in this area, and after discussion with Sokrich, he joined the team.

Discussion with medical students: Thoughts on antibiotic resistance

During our discussions on the project and its consequences, we interviewed medical students from Paris Cité University. It seemed important to us to have the opinion of future actors in the field of health, and members of a generation lulled by the climate issues. Beyond their enthusiasm for the sustainability of CO2CURE, they expressed concern that our project could promote antibiotic resistance. Their idea was as follows: while promoting access to antibiotics in certain areas and reducing their cost is an issue in the equity of access to care, their excessive availability (particularly in already deregulated markets) could increase their misuse and thus the development of resistance. All this in an already tense context of the emergence of multiple resistances.

This vision made us think long and hard. We wanted our project to be beneficial to society, not a source of additional risk. Should we therefore change it in depth? Would the project inevitably lead to an increase in antibiotic resistance? And could we put in place measures to limit these possible drifts?

We thought, debated, researched, and found some answers.

First, in LMICs (Low- and Middle-income countries), the increase in antibiotic resistance is, surprisingly, partly due to poor access to antibiotics. Although the use of antibiotics has indeed increased in these countries in recent years, their regulated and reasoned access is only partial. The price of treatment weighs heavily on certain populations, which can only follow it incompletely and shorten the prescription, thus favoring the proliferation of resistant clones. The diffusion of poor quality antibiotics also contributes to the emergence of resistance. Moreover, in some countries, physicians prescribe more according to the availability of the antibiotic than to its necessity.

The CO2CURE project, by allowing the reduction of the cost of care, and the local production of good quality antibiotics would reduce these phenomena. Facilitating access to antibiotics also means reducing the burden of disease and therefore the use of antibiotics.

Secondly, the development of new antibiotics has decreased since the 1960s. Yet, one of the means to fight antibiotic resistance is precisely the development of new molecules. We now know that the Streptomyces genome contains dozens of SMBGCs (see our STREPTObook) even if the expression of only a few of them is controlled under laboratory conditions. Thus, these bacteria possess a hidden metabolism that remains to be discovered in order to eventually find new antibiotics. The study of the production of antibiotics by Streptomyces would allow to fight against the emergence of resistance that we know today.

We hope that our guides and tools on the study of Streptomyces will help young researchers in such research.

Third, as described earlier, antibiotic use is a public health and equity issue in many countries: it is the misuse of antibiotics (and market deregulation) that needs to be addressed, not their availability. One of the weapons against this, perhaps the most powerful, is the prevention for patients and prescribers of the risks linked to these misuses and the dissemination of information on good practices to adopt.

We have therefore decided to make prevention a commitment of our project. This commitment has been translated on the one hand into the creation of a poster dedicated to the popularization of the antibiotic resistance phenomenon, and on the other hand into the creation of a prevention stand by our team at the “Cité des Sciences” during the Science Festival. This event of discovery of sciences attracts a large public from all over the Paris region, the presence of such a stand seemed to us relevant and really impactful.

Figure: Picture of Simon presenting at the “Cité des Sciences”

Here is what CO2CURE has set as its objective: favor access to antibiotics, and make them fully sustainable, to pave the way for their reasoned and controlled use.

We conclude with this sentence from CDDEP : “[A]ll antibiotic use has the potential to select for resistance and must be carefully managed to ensure both that they are deployed when they deliver the best possible value to patients and public health and that they remain effective for the longest period possible.”