Genetically Engineering Yeasts which Express Killer Toxins

Brendan Wood

Introduction
Saccharomyces Cerevisiae is a yeast strain that is found naturally in many environments. This
certain yeast strain is used in baking yeast, beer brewing, and it is also found in opportunistic
infections in immunocompromised individuals. S. Cerevisiae also carries the ability to secrete
killer toxins. These toxins include K1, K2, and K28. These toxins can be either proteins or
glycoproteins that are secreted and ten interact with receptors on surfaces of yeast cells. Each
toxin disrupts the electrochemical gradient that is across the plasma membrane. Each S.
Cerevisiae M virus contains a single ORF (open reading frame) for a preprotoxin (pptox), which
is a precursor of a mature secreted killer toxin, (Schmitt, Frank, 2006). The
genes that are needed to express these toxins are transcribed and extruded
from the virion into the cytoplasm of the yeast cell. This results in an RNA
template which is packaged into new viral particles. Once a yeast cell
becomes infected with a killer toxin virus it first makes pptox eventually
leading to the secretory pathway. In this pathway, maturation, toxin
secretion, and further processing occurs. The toxin K28 can be taken up by
endocytosis after being bound to the surface of another cell. However,
there are certain yeast mutants that are altered in the fluid-phase and
receptor-mediated endocytosis (Pictured in Figure 1 & 2). This alteration
leads to toxin resistance; therefore, it is not capable of reaching its final Figure 1: Receptor Mediated
target within the cell. Endocytosis

In addition, S. Cerevisiae killer toxins can kill yeast cells through a

receptor-mediated process. For the toxins to kill the cell it must go through
specific steps. First, it must find a primary toxin receptor, and then it must go
through toxin translocation. Once the virus achieves these steps, K1 and
Zygocin are released disrupting the cytoplasmic membrane. In addition, the
K28 toxin blocks DNA cells causing them to stop during the cell cycle.
However, the sensitive yeast cells that are susceptible can develop
chromosomal mutations that can give them resistance to the virus and its
toxins.
Figure 2: Fluid Phase
As I mentioned earlier, S. Cerevisiae can cause infections mainly in Endocytosis

immunocompromised individuals. However, it is also found in infections

within healthy individuals. The National Institute of Health says, “S. cerevisiae has been related
to a wide variety of infections, which range from vaginitis in healthy patients and cutaneous
infections, to systemic bloodstream infections and infections of essential organs in
immunocompromised and critically ill patients (Enache-Angoulvant and Hennequin,
2005; Muñoz et al., 2005; de Llanos et al., 2011),” (nhv.gov, 2016). This has not always been the
case and scientists discovered its pathogenicity recently. S. Cerevisiae is used in probiotics and
found to be healthy, however, some pathogenic strains of this yeast can produce proteinase, are
capable of phenotypic switching, and can also show partial or complete resistance to antifungal
medications. Another important characteristic of the pathogenic isolate is that they can grow at
42 degrees Celsius whereas most S. Cerevisiae strains cannot grow at this temperature. This
means that it can still grow under fever conditions in the human body when the other strains
cannot. This allows the pathogenic isolates to continue growing and cause infection. (Figure out
what to add onto right here).

Hypothesis
A genetically engineered S. Cerevisiae strain using CRISPR-Cas9 to isolate K69 and to
translocate it into another model system.

Research Aims

Aim 1: Use PCR to obtain single stranded

DNA from S. Cerevisiae.
Aim 2: Design a guide RNA using CRISPR-
Cas9 and insert the single stranded DNA into
a Cas9 plasmid found in E. Coli.
When using CRISPR-Cas9 technology, we
create gRNAs (guide RNA) that target and
cut the gene of choice. In this case, we will
design a guide RNA to target and cut the K62
gene found in S. Cerevisiae (not found in S.
Cerevisiae, ask Dr. Rowly). First, a guide
Figure 3: Designing gRNA using CRISPR-Cas9 and RNA will be developed that can efficiently
Plasmids. Option one of this diagram is the approach I
will be taking. First, you design a guide RNA and then
target the K62 toxin gene. Obtaining the genes
clone it into a Cas9 plasmid. Once this is achieved, we for this killer toxin will allow us to insert it into
can use this plasmid to target the gene we want. S. Cerevisiae and analyze for K62 killer toxin
activity. Our main focus is looking for the loss of activity of the K62 toxin. If successfully
completing this aim, we will be able to use our CRISPR model and guide RNAs that will target
these genes. This will allow us to conduct for testing and research on the toxin and its traits.
Aim 3: Transform S. Cerevisiae with the K62 containing Cas9 plasmid and look for K62 activity
loss.
Works Cited
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705302/
https://www.sciencedirect.com/science/article/abs/pii/S0141022999000861#:~:text=cerevisiae
%20may%20cause%20disease%20in,are%20capable%20of%20pseudohyphal%20growth.