Team:RUBochum/Description
Table 1: List of Proteins
Our Inspiration
With a growing world population which has to be fed, limited resources, and global warming on the rise, humanity needs to face many problems.
One of them is farming and the impact it has on the aforementioned problems.
Besides the usage of fertilizer and antibiotics, conventional farming is characterized by its huge land and water use as well as emerging greenhouse gases.
We as the first iGEM team from the Ruhr University Bochum asked ourselves how a sustainable food supply without animal suffering could be achieved.
During our research on this possible project, we found the inspirational iGEM projects Real Vegan Cheese (SF Bay Area 2014) and Syn Mylk (Düsseldorf, 2019).
Both showed the capability of synthetic food, in particular the synthetic production of milk and its products.
Many people do not want to live without animal products. By taking the dairy industry with its big impact on the environment into account, we wanted to create synthetic milk for the production of platypus yogurt.
We chose the platypus because it is not only one of the (subjectively) best mammals out there, but also because it is a threatened species that is under constant pressure by the dairy industry in Tasmania. Through that, we therefore also draw attention to species threatened or endangered by climate change.
The Project
Milk in general is composed of water, sugar, minerals, fatty acids, and proteins (caseins and whey proteins).
For the production of fermented milk products like yogurt and even cheese, mainly water, fatty acids, and casein proteins are needed. Fatty acids in milk vary in their length between 4 and 24 C atoms.
The casein proteins can be divided into four different subgroups (α-s1-, α-s2-, β and κ-casein) and occur in all mammals.
Figure 1: Ingredients of milk.
The production of synthetic milk has the benefits including reduced agricultural land use, water and thereby emerging fewer greenhouse gases, saving resources. To enable a cheap, optimized industrial process, a model organism is needed.
We decided to work with Pichia pastoris as an expression organism. P. pastoris is able to perform post-translational modifications such as glycosylations and is also able to secrete proteins. The secreted proteins allow for an easier purification of those non-tagged food proteins.
The improved process is characterized by a joint synthesis of all products in one fermentor.
To simplify the production the number of genotypes needs to be reduced in an optimal manner to one.
Figure 2: Principle of the heterologous fermentation process. Starting from multiple genotypes producing the whole product, the amount of genotypes is reduced inside the fermentation due to genetic optimization.
For the genetic optimization, a chromosomal integration was planned with the GoldenPiCS system. Loci for the integration would have been RGI2, ENO1, NTS, and AOX1tt. Those could have been further optimized with promoter shuffling.
As a donor for our genes, platypus and bovine data was used.
The milk of the platypus has many astonishing features, such as an antimicrobial
peptide which is needed to protect their young (platypuses” sweat” out their milk, which is why there is a need to protect the milk to be contaminated easily)
An antimicrobial agent like the monotreme lactation protein could make the pasteurization step redundant and create food with a longer shelf life.
Figure 3: Experimental setup.
Additionally, the taste of the yogurt is refined with vanillin and brazzein. Vanillin was chosen as the coveted taste addition for yogurt. The brazzein is a polypeptide with a taste 500 to 2000 times sweeter than a common sugar molecule.
Besides the optimization towards an industrial process, our goal will be to create more varieties of taste. Besides vanillin, chocolate and fruit flavours were the most requested tastes, which will create synthetic platypus yogurt with an unexpected taste.
Platypus image credit: [Clive]/Adobe stock