Project Implementation

Figure: CO2CURE bioreactors near industries producing CO₂. This would reduce the emission of greenhouse gases. In addition, on-site production can limit CO₂ emissions due to transportation costs. Illustration by Sokrich Ponndara (iGEM GO Paris-Sacclay 2022)

Concrete Implementations

Firstly, to maximize the profitability of our autotrophic strains, it would be a great idea to put the bioreactors next to polluting infrastructures, such as industries. For example, cement industries are responsible for 3 % of carbon pollution in the world. This would be an implementation close to other carbon sink projects like that of the Fermentalg company. They put CO₂-fixing bioreactors near biofuel producing ones. Therefore, they can recycle microalgae biomass to produce biofuel (methane). With the same idea, we can imagine that autotrophic Streptomyces bioreactors could be used to produce both antibiotics, and biomass that could be recycled for the production of renewable biofuel.

Secondly, using CO₂ to produce antibiotics could also reduce CO₂ emissions in an indirect way. Normally antibiotics are produced in handful of wealthy countries that have the monopoly in the pharmaceutical industry. Afterwards, these molecules must be transported thousands of miles to reach their destination. This means that tones of CO₂ need to be emitted just to transport these tiny molecules from their production-site to the place where they are needed. CO2CURE helps to partially solve this problem. Thanks to CO₂ fixation these bacteria can be grown anywhere more easily. Thus, if an optimal method of extraction is developed, we can imagine that a kit containing these bacteria can allow for on-site production. This would both reduce the CO₂ emissions caused by transport, as well as reducing the costs due to eliminating the middleman.

Moreover, our interview with Dr. Ferreira-Dos-Santos also inspired a new application (See the Human practices page). 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 have a very low carbon content. By making these copper-resistant Streptomyces C-autotrophic, we could allow them to decontaminate soils at a much higher speed.

While conducting our studies on antibiotic production by Streptomyces, we have identified conditions under which antibiotic production is possible from glycerol. It is an industrial waste produced largely by the petroleum industry as it is a by-product of biodiesel production. As much as 0.1 kg of crude glycerol is generated per kg of biodiesel (Vivek et al., Bioresource Technology, 2017). Although glycerol is not toxic, it can deplete the water resources of oxygen which can endanger many aquatic species, thus threatening many valuable ecosystems. That is why, we think it would be also a good idea to create bioreactors containing Streptomyces next to these biodiesel producing plants that could produce highly valuable molecules from this industrial and harmful waste.

Moreover, nitrates also stimulate antibiotic production by our strains (see media composition in the STREPTObook). Nitrates usually accumulate in water due to their excessive use as fertilizers in farming. They can change the pH of the soil (Savci, APCBEE Procedia, 2012), which decrease the yield of the crop. Furthermore, nitrates can also cause eutrophication of standing waters, which is very harmful for the organisms inhabiting them. Thus, our chassis can potentially also recycle glycerol and capture nitrates from polluted waters, which could then be recycled back to the effected ecosystems they came from.

We also thought about a fancy idea to use antibiotics produced by Streptomyces. We know that it’s important for astronauts to have the carbon cycle to be closed in life in space. So, autotrophic Streptomyces are more than interesting. In fact, we could imagine that astronauts will cultivate them and cure themselves with antibiotics from our strains.

Another interesting feature of Streptomyces for the implementation of the project is that they are spore-forming bacteria. Their spores are produced by septation and are not as resistant as the exospores of bacteria such as Bacillus. However, Streptomyces spores are still more resistant than the mycelium-forming cells and widely used in practice to store strains durably. Moreover, Streptomyces can also be preserved and transported in dried form. These properties could facilitate the reduction of costs related to the transport of strains.

Finally, we cloned our biobricks within integrative vectors such as pSET152 (Bierman M. et al., Gene, 1992) or vectors from the pOSV collection (Aubry et al., Appl. Environ. Microbiol., 2013). This type of vectors allows a stable modification of the Streptomyces strains, by irreversible and site specific integration to the chromosome. The targeted sites are frequently found within Streptomyces strains. In the STREPTObook (pages 20 & 21), we also describe how to identify them within the genome of strains of interest. Moreover, we have cloned a new expression cassette (BBa_K4370010 - See the Part page) under the control of kasOP* promoter has been previously reported as one of the strongest promoter for an expression in Streptomyces (Bai et al., PNAS, 2015 ; Wang et al., Appl. Environ. Microbiol., 2013). In this composite part, this promoter is followed by a RBS from of capsid protein obtained from Streptomyces temperate phage ϕC31 that has been previously described as highly efficient to promote translation when combined with the kasOP* promoter (Bai et al., PNAS, 2015). Altogether, the genetic tools generated in the CO2CURE project can be considered as almost ‘universal vectors’ and use to genetically modify a large panel of Streptomyces strains. Thus, the genes encoding the RuBisCO and PRK from S. bottropensis ATCC 25435 could be introduced in other Streptomyces of biotechnological interest. This could contribute to a wide implementation of the CO2CURE project.

Conclusion

In conclusion, from our point of view, the most important idea in our project is the cycle-based production, which is the most responsible and ecological way to build an industrial process. By interacting with other teams and companies, we can promote the use of 'green' strains, and thus have a positive impact on the future of synthetic biology. The same way, by giving the example of the recycling of atmospheric CO₂ and waste-using bacteria, we can bring the ideas to build strains fixing other greenhouse gases such as CH₄. These will be possible if our project is implemented in a way that encourages the spread of our ideas.

Basically, we think that the design and use of autotrophic chassis seems original and/or exceptional today, but that one day this may be the general rule. This would be ideal to ensure a sustainable development of our societies (See the Sustainable development page).