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The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene
(total word count: 1244)
Title: The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene
Introduction: Ever since its invention, beer has been the most popular alcoholic
drink around the world. With thousands of factories and millions of private brewers,
the beer brewing industry is one of the biggest food industries. Two types of beer
yeast are generally used to convert fermentable sugars into ethanol and carbonation,
as well as other byproducts in beer. One is ale yeast, Saccharomyces cerevisiae, which
carries out the fermentation at 13–21 ºC; the other is lager yeast, Saccharomyces
uvarum, that routinely ferments at 4–12 ºC. However, most of the enzymes that are
used to breakdown malt starch have higher optimal working temperatures. βglucanases that degrade cell walls work best at 45 ºC, while proteases, β-amylases,
and the α-amylases that breakdown malt starch all work best above 50 ºC
(Sammartino, 2015). The working temperature difference between beer yeasts and
enzymes complicates the brewing process and thus boost the production cost of beer.
Besides the effect of equalizing the working temperature during fermentation,
developing a thermotolerant beer yeast can also help brewers prevent beer spoilage,
since most of the spoilage organisms are inhibited at the temperature above 40 ºC.
To develop thermotolerant beer yeast, the specific genes relating to
thermotolerance need to be identified first. Researchers have found that heat-shock
protein 90 (Hsp90) plays an essential role in preventing protein denaturation in the
presence of stress factors like heat and high ethanol concentration (Uehara et al.,
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The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene
2018). Hsp90 was also detected in many ethanol producing yeasts. In 2017,
researchers from Khon Kaen University in Thailand conducted an experiment to
isolate yeasts that are capable of carrying out high-temperature ethanol production.
Among the yeast strains, P. kudriavzevii RZ8-1 displayed the best fermentation ability
in high ethanol environment within the temperature range from 37 ºC to 40 ºC. The
quantitative RT-PCR result indicates an up-regulation for genes encoding for heat
shock proteins SSQ1 and HSP90 (Chamnipa et al., 2017).
To maximize the gene insertion chance and minimize the negative side effects on
yeast fermentation, Hsp90 gene from the yeast strains of P. kudriavzevii RZ8-1 is the
best experimental subject since P. kudriavzevii RZ8-1 has the best ethanol tolerance
among the thermotolerant yeast species. And since the lager yeast, Saccharomyces
uvarum, has a lower fermentation temperature, the insertion of Hsp90 will most likely
result in a more significant difference in its thermal-tolerance. Thus, I suggest that the
addition of genes encoding SSQ1 and HSP90 to beer yeast strains can significantly
increase the routine fermentation temperature of lager yeast, Saccharomyces uvarum.
Experimental Design: The transformation will be performed based on Hinnen, Hick,
and Fink’s experiment using plasmid ColE1, as this plasmid and the selective marker,
LEU+ gene, have been used countlessly in previous yeast transformation experiments.
Through restriction digest of the plasmid DNA and the DNA ligation afterward, yeast
LEU+ gene that account for leucine production in yeast and Hsp90 gene will be added
to the original ColE1 vector, creating a new pColE1-LEU+-Hsp90 plasmid. The LEU
gene in the Saccharomyces uvarum recipients will be artificially knock-outed and
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The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene
become LEU-. Two mutations instead of one are created during the knockout to lower
the background rate of its reversion to LEU+ (Hinnen, Hicks & Fink, 1978). This
process will be carried out with the CRISPR-Cas9 technique with the KN2.0 CRISPR
Knockout Kit, an efficient tool to knock-out target genes. The transformation of this
vector into LEU- Saccharomyces uvarum yeast recipients will be performed with the
Frozen-EZ Yeast Transformation II Kit. It is very efficient at opening the yeast
envelope for the insertion of gene vectors.
In order to verify the transformation results, 3 experimental groups will be set
up. One group will use the wild-type LEU+ Saccharomyces uvarum yeasts, with no
modification applied. The other 2 groups will use the CRISPR modified LEUSaccharomyces uvarum yeasts, one will undergo the pColE1-LEU+-Hsp90 plasmid
transformation and one will not. The solution volume and the concentration of
floating yeast will be controlled to be identical among all groups. After the same
incubation time, same volumes of solution from each group was transferred to
separated agar plates with basic nutrients for beer yeast to grow. However, Leucine
will not be provided during their growth on agar plates. After 48 hours, colony
amounts will be counted and recorded for each plate. Pictures will be taken with the
same magnification using a professional camera kit. Pictures will then be processed
using ImageJ tool to measure the total colony size in each plate. Colony amounts and
total sizes for each plate will be compared among groups using the one-way ANOVA
test. If the p-value is less than 0.05, the difference will be considered significant. In
that case, individual Welch’s t-test will be performed between the 2 groups using the
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The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene
CRISPR modified LEU- Saccharomyces uvarum yeasts, with the alternative
hypothesis that the plasmid transformation group is greater in size. If the p-value is
less than 0.05, the transformation will be validated at 95% confidence level. In this
case, the experiment will proceed to second phase.
For the second phase, disinfected sealed containers will be set up and separated
into 3 groups. Glucose will be dissolved in water and added to all containers as
resource for fermentation. For the first group, the genetically modified
Saccharomyces uvarum yeast will be added to each container. Same mass of wild-type
Saccharomyces uvarum yeast will be added to the second group. Same mass of P.
kudriavzevii RZ8-1, the yeast that is known to ferment at 40 ºC, will be added to the
third group. All containers will be inserted a CO2 sensor to record the shift of CO2
concentration. They will be sealed tightly and incubated at 40 ºC for 12 hours.
The alcohol concentration of each beaker was measure with alcohol hydrometer
at the end of the experiment and recorded for data analysis. A trendline will be plotted
for CO2 concentration shift. The CO2 concentration trendline slope, as well as the
ethanol concentration, will be compared among groups using the one-way ANOVA
test. If the p-value is less than 0.05, the difference will be considered significant. In
that case, individual Welch’s t-tests will be performed between the genetically
modified Saccharomyces uvarum group and the wild-type Saccharomyces uvarum
group, with the alternative hypotheses that the genetically modified lager yeast will
produce higher amount of ethanol and the rate of CO2 production will be higher. If
the p-value is less than 0.05, the result will be insufficient to reject the hypothesis.
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The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene
The most significant potential pitfall in this experimental design is
contamination. Many microorganisms, like bacteria, also have the ability to ferment
and they may affect the result. This experiment requires a sterile instrument to achieve
the best result.
Reference
Chamnipa, N., Thanonkeo, S., Klanrit, P., Thanonkeo, P. (2017). The potential of
the newly isolated thermotolerant yeast Pichia kudriavzevii RZ8-1 for hightemperature ethanol production. Brazilian Journal of Microbiology. 49(2):378-391.
Hinnen, A., Hicks, J. B., Fink, G. R. (1978). Transformation of yeast. Proceedings
of the National Academy of Sciences of the United States of America. 75(4):19291933.
Sammartino, Mark. (2015). Enzymes In Brewing. Technical Quarterly - Master
Brewers Association. 52(3):156-164
Uehara, Y., Temma, K., Kobayashi, Y., Irie, N., & Yamaguchi, T. (2018).
Reduction of Thermotolerance by Heat Shock Protein 90 Inhibitors in Murine
Erythroleukemia Cells. Biological and Pharmaceutical Bulletin. 41(9):1393-1400.
ZYMO RESEARCH CORP. (2015). Instruction: Frozen-EZ Yeast Transformation
II. Retrieved from
https://www.zymoresearch.com/media/amasty/amfile/attach/_T2001_FrozenEZ_Yeast_Transformation_II.pdf
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The Shift of Saccharomyces uvarum’s Fermentation Efficiency at a Higher
Temperature After the Insertion of the Hsp90 Gene