Team:Paris Bettencourt/Description
Project Overview
Socio-economic context
Enzymes are an invaluable resource in production. They significantly reduce the environmental impact of many processes while improving the yield, safety and quality of the products (textile, dairy, detergents…). Which is why the market for enzyme production is a fast-growing one (1). The scale at which proteins and enzymes are produced keeps increasing. The Samsung Biologics plant in South Korea is a great example. It will have a total capacity of more than 600 000L by 2023 and become by far the biggest protein production plant in the world and supply all companies alike.
This results in the production of enzymes to be mainly done through international supply chains, creating a strong and inevitable dependence on international shipping. The phenomenon was particularly highlighted during the covid pandemic where these supplies chains were easily dismantled and broken by the crisis. In fact, the global supply chain network showed poor resilience in 2020 with nearly 35% of the manufacturers reporting its supply chain network failure due to the pandemic (NAM, 2020). This caused, amongst many other consequences, the availability, and production of many essential items such as food, grocery, and pharmaceutical products to be drastically reduced.
Nevertheless, due to the various measures taken to fight the COVID-19 outbreak, people were able to see a clear sky again, free from suspended particles. Air quality considerably increased , biodiversity was permitted to recover a little.
Therefore, there is a need for a transition toward a system where people are still able to see the sky while production can still benefit from the added value brought by enzymes. And to do this, we need a new approach to their production .
Our approach is to propose a new process in its entirety where enzymes are produced locally to ensure resilience of the supply chain and transport reduction, but still safely even without benefitting from the procedures implemented in big production units.
Another reason to go towards local production was brought to light during the covid crisis, the maker movement. Fablabs across France came to the aid of the health system by printing oxygen masks, sewing protective masks, preparing and crafting essential equipment for hospitals and nurses… The crisis forced people to work together, allowing them to take care of their own needs.
Our project is not limited to technical advancement, it aims at showing a systemic approach to the future of enzyme production combining sustainability, safety and social well being (2).
Scientific context
Division is an essential process in bacterial life as it contributes to ensure their phenotypic diversity by giving their chromosome to their daughter cells. In rod-shaped bacteria such as E. coli , one of the key cellular components of cell division is formed between the two replicated chromosomes: the septum - the cell wall between the two dividing cells (3). Most bacterial division processes are managed by the FtsZ and Min systems.
1. Bacterial division ensured by the ftsZ system
The ftsZ (filamenting temperature-sensitive mutant Z) gene is encoding an essential cell division protein: FtsZ (4). FtsZ is a tubulin-like protein that has a crucial role during bacterial division as this is the first cell-division protein to arrive on the division site (the septum of dividing cells). Moreover, its GTPase activity enables the contractile movement to promote cell division. FtsZ expression regulates the formation of a contractile ring structure also called ftsZ ring or Z ring (5) , it consists of the assembly of protofilaments at the future cell division site (6) .
Figure 1. Formation of the FtsZ ring during bacterial cell division
2. Centering of the Z-ring with the Min system
The min operon in E. coli is composed of three genes: minC, minD and minE respectively encoding for the proteins MinC, MinD and MinE. More precisely, MinC is one of the main negative regulators of FtsZ by preserving FtsZ polymerization. And overall, this Min system helps to center the position of the Z-ring by restricting FtsZ assembly to the middle of the cell thanks to a high concentration of FtsZ polymerization inhibitor at the pole of the cell (7).
3. Minicell production
Minicells - nano to micro-sized particles lacking chromosomal DNA - can be formed through two methods. The first lies in the mutation of the Min system genes - responsible for the location of the site for cytokinesis.
Therefore, the cell cannot find the midpoint for partitioning, leading to the formation of a “normal cell” with chromosomal DNA and a smaller one called “minicell”(Figure 2). Because they lack chromosomal DNA, they are unable to grow or proliferate. Despite this, these particles have the ability to carry one or several plasmids and can then produce a protein of interest. They have the same cellular components - cell membrane, RNA, proteins (8) - as their mother cell except some slight differences such as their membrane composition.
Figure 2. Overproduction of FtsZ leading to the formation of minicells in E. coli
Why Minicells?
1. Minicells are not considered living organisms
A living organism is defined by its ability to maintain, grow, reproduce and adapt. (9) But minicells, because they lack chromosomal DNA, are unable to replicate or grow, therefore, they cannot be defined as living organisms.
2. Minicells cannot be defined as GMOs
Moreover, genetically modified organisms (GMOs) are organisms which genetic material has been artificially modified - according to the definition of the European Commission. (10) A part of the system using only minicells and not classical bacterial cells would therefore not be considered at the same level of biosafety. In fact, our hardware device would provide a place for separating mother cells from their minicells. Thus the final product - without engineered bacterial cells - is not considered as a GMO containing system, guaranteeing a higher biosafety level for our hardware.
3. Minicell producing strain can be easily biocontained
The engineered Min-mutantE. coli strain wastes resources producing minicells and then become filamentous. Filamentous cells may grow to 50 to 200 normal cell lengths before growth ceases and lysis occurs (11). Additionally,E. coli K12, so-called a lab strain, is part of the Risk Group 1 (12). In fact, it does not produce toxins and would not be able to proliferate in the human digestive system. is not likely to survive in the outside environment, easing its biocontainment strategy.
4. Minicells to lower the energy cost
We had a meeting with Colorifix, a company dedicated to the bioproduction of pigments for textile. Their technology is based on using microorganisms such as bacteria to considerably reduce the pollution usually emitted by the toxic industry of pigments’ production. At the end of their pigment production process, Colorifix raises the temperature to get rid of the bacteria. However, this method has the important disadvantage of needing a certain amount of energy to raise the temperature and the overall energy cost. By proposing a solution limiting the amount of energy used in our hardware, minicells appear theoretically as a more eco-friendly solution compared to traditional and the bioproduction pigment industry.
Project Description
REFERENCES
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(10) European Commission. Genetically Modified Organisms. URL: https://ec.europa.eu/food/plants/genetically-modified-organisms_en
(11) iGEM. White List. [2021]. URL: https://2021.igem.org/Safety/White_List
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https://doi.org/10.1128/jb.90.1.223-226.1965