Team:RUBochum/Engineering
Brazzein
Brazzein is a protein which originates from the fruit of the climbing plant pentadiplandra brazzeana.
It’s 500 to 2000 times sweeter than a common sugar molecule.
As an ingredient of our yogurt it could be a tasty and healthy alternative to other sugar substitutes due to its different metabolic pathway and the high demand for amino acids in the human body.
"Fact: Brazzein can be up to thousand times sweeter, is extremely stable, is suitable as a balanced food."
Dr. Michael Krohn, executive Vice President as well as head of Research & Development at BrainAG - translated from German
Video 1: Brazzeins molecular structure
Design
The sequence of the brazzein was applied into the MCS with the alpha secretion signal to allow for an easy purification.
Caseine
Milk contains different proteins like caseins and whey proteins.
We are producing the caseins of the milk of the platypus and bovine.
Besides having the ability to carry calcium and phosphate, they are crucial for the production of cheese and yoghurt.
The casein proteins can be divided into four different subgroups (α-s1-, α-s2-, β and κ-casein) and occur in all mammals.
Proteins
αs1 casein Bos taurus
αs2 casein Bos taurus
β casein Bos taurus
κ casein Bos taurus
αcasein Ornithorhynchus anatinus
β casein Ornithorhynchus anatinus
κ casein Ornithorhynchus anatinus
Design
All proteins were cloned with the alpha secretion signal behind the promoter region. The secretion should allow an easy purification.
Due to reports secretion should be a problem with caseins. An alternative approach would be the coupling of the different caseins to hinder the folding of disturbing structures.
Development of an optimized industrial process
Backbone
The variety of proteins were produced in the pPICZαA vector which has the AOX1 promotor and is thus methanol dependent.
Three different types of primers were created to get multiple combinations of integrations into the multiple cloning site. Genes can be inserted with the alpha secretion signal or a polyhistidine-tag through a gibson assembly.
Following to the creation of the plasmids additionally chromosomal integrations were planned.
Therefore, GoldenPiCS system was used with the four integration loci RGI2, ENO1, NTS and AOX1tt.
Figure 1: 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.
The goal was to create a homolog fermentation process with multiple genotypes.
In further work the amount of genotypes should be reduced to one. Plasmids and integrated genes get combined during this process.
Purification of food proteins
Cells were centrifuged and the supernatant was filtered size dependent. The copper chelating Phleomycin D1 can be denatured due to pH lowering during yoghurt production.
Due to chromosomal integration a selection marker and thus antibiotic purification will be redundant.
Fatty acid syntheses
Fatty acids define the taste and consistency of milk.
The lengths of the triacylglycerols vary between 4 to 24 C-atoms.
For the overproduction of fatty acids, the enzyme of the rate-determining step needs to be overproduced. In our case, we used the Acetyl-carboxylases complex from E. coli K12 which consists of 4 subunits.
Thioesterases terminate the elongation of fatty acids and thus determine their length.
To get the broad range of fatty acids, the Thioesterase II of E. coli was cloned into our back bone.
Due to the broad range of action of Thioesterase II, it’s also possible to get mostly fatty acids with a length of 6 to 18 C-atoms.
Design
The four subunits were connected to each other and fused with a glycine-rich linker.
Therefore the Gibson assembly was used.
A strain including the gene for the thioesterases was created.
Monotreme Lactation Protein
The Monotreme Lactation Protein (MLP) is a protein that is only found in the milk of platypus and other monotremes. MLP is highly expressed in platypus milk with antimicrobial properties.
The function of MLP is hypothesized to be the protection of the young from pathogenic infections, because monotremes don't possess teats.
After hatching from their eggs, the young drink the milk directly from a mammary pad on the abdomen of the female platypus.
So there is more chance of contamination with pathogens compared to other nursing strategies e.g. drinking the milk directly from teats.
Studies showed that MLP demonstrated antimicrobial activities against Staphylococcus aureus and Enterococcus faecalis, which are classified as pathogens.
It didn't demonstrate any effect on Escherichia coli, Salmonella enterica, Staphylococcus epidermidis, and Pseudomonas aeruginosa.
MLP is also studied as a new candidate for antibiotics, because of this antimicrobial properties.
Figure 2: Molecular structure of MLP
We hypothesized that MLP is an alternative for milk pasteurization. Different tests, with more bacteria types and the most common from human gut flora, should give a clearer picture if MLP could be safe for human consumption.
If so, we could use MLP to skip the milk pasteurization step, or add it to common dairy products to expand their shelf life.
We also wanted to raise awareness for platypus and monotreme animals in general, because these ancient species are threatened by human pollution and climate change. Especially the platypus population in Tasmania is under pressure by the dairy industry.
Design
The sequence of the MLP was applied into the MCS with the no secretion signal and Hexahistidine-Tag for easy purification.
Vanillin
For the production of vanilla yoghurt, the vanillin synthesis needed to be integrated into the fungus.
In our case we started with the last step of the vanilly synthesis: the conversion of vanillyl alcohol to vanillin via the aryl-alcohol oxidase.
We chose the enzyme from Penicillium simplicissimum. The enzyme was optimized for P. pastoris and coupled to a 6X His-tag.
Figure 3: Drawing of a vanillin molecule
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References
Aryl-alcohol oxidase