The Development of a Density Based Screen to Identify

Transcription

The Development of a Density Based Screen to Identify
The Development of a Density Based Screen to Identify
Yeast with Higher Lipid Content
2 Center
Abstract
Kate Larson1, Marna Yandeau-Nelson2, Basil Nikolau2
1 Des Moines Public Schools, Des Moines IA 50316
for Biorenewable Chemicals (CBiRC), Iowa State University, Ames, IA 50011
Figure 1. Synchronization Experiments
The goal of this project is to create a new density-based centrifugation protocol to screen
for yeast cells that have a high lipid content and, possibly, high fatty acid production. This
test is based on the fact that cells with more lipids will be less dense and should, therefore,
migrate higher on a centrifugation-generated density gradient. Prior to optimizing
centrifugation, we synchronized the cells to a single stage in the cell cycle using factor.
synchronization has been optimized and is effective but further testing needs to be done to
see if synchronization is necessary. Preliminary tests suggest that the lipid accumulating
strain, regardless of whether it is synchronized, is less dense than the wildtype strain.
Therefore, the feasibility of this screen is promising.
Wildtype
Results & Discussion
The goal of this research is to develop a screen for the identification of yeast with high lipid content. To
develop the method, we used a high lipid yeast strain (tgl3; tgl4, which lacks lipases and accumulates lipids)
and a wildtype strain. Cells with higher lipid content (e.g., tgl3; tgl4) should have lower buoyant densities
than cells with normal lipid content and should, therefore, be separable over a density gradient. However,
other factors, such as stage in the cell cycle, could also affect buoyant density of cells. Therefore, we wanted
to first synchronize our cells to a single stage using the mating pheromone, " factor. " factor is secreted by
yeast cells of mating type " to attract cells of the opposite mating type (a). This pheromone induces type a
cells to adopt shmoo morphology (Figure 2) and arrests them at the G1 phase of the cell cycle. To optimize "
factor synchronization, 1 x 108 yeast cells were treated with different concentrations (0 µM, 30 µM, 50 µM,
and 100 µM) for different lengths of time (90 min, 120 min, 150 min and 180 min; figure 1). Greater than 95%
of cells treated with 50 and 100 µM " factor were synchronized and had formed shmoos at the 120-180 min
time points.
tgl3; tgl4
No α Factor
Upon optimizing " factor synchronization, we tested synchronized and unsynchronized wildtype and tgl3;
tgl4 cells for differences in cell density using density-based centrifugation. Cell strains with increased lipid
content (in this case, tgl3; tgl4) should “float” higher in a density gradient than wildtype. Cells either treated
(synchronous) or not treated (asynchronous) with " factor were layered on top of different concentrations of
Percoll, a medium that forms a density gradient during centrifugation, and centrifuged at different speeds
and lengths of time. In addition, we tested whether cells needed to “recover” from the " factor treatment by
washing the cells and then growing them for one cell cycle prior to density gradient centrifugation.
Introduction
It is becoming increasingly important to transition the chemical industry from nonrenewable, petroleum-based carbon sources to renewable, biological sources of carbon.
The goal of Thrust 1 within the Center for Bio-Renewable Chemicals (CBiRC) is to
manipulate the fatty acid biosynthesis pathway to generate short alkyl chains that will
serve as chemical precursors for biorenewable chemicals. These chemical precursors
will be produced in yeast “bio-factories” and will then be transformed by chemical
catalysis into biorenewable chemicals. To create bio-factories that produce large
amounts of chemical precursors our aim is to identify yeast strains that have increased
fatty acid production. The goal of this project is to create high-throughput methods to
identify yeast cells that have a high lipid content and, ideally, high fatty
acid production. Two methods are being developed: 1. Staining of yeast cells with a
dye that preferentially stains lipids; and 2. Density-gradient centrifugation, which is
based on the fact that cells containing more lipids should have a lighter density than
cells with normal lipid content and should therefore be separable across a density
gradient.
To develop these methods it was necessary to identify a yeast strain with
increased lipid content as compared to wildtype. Yeast genes Tgl3 and Tgl4 encode
lipases, which degrade lipids. In the tgl3; tgl4 double mutant these lipases are absent
and, therefore, lipids accumulate within the cell (Kurat et al., 2006). Ultimately, the
optimized staining and centrifugation methods will be used in tandem on mutated
yeast cells to identify novel mutants with increased lipid content. These mutants will be
further characterized and strains with increased flux through fatty acid biosynthesis
will be good candidate strains for production of biorenewable chemical precursors.
This poster focuses on the density-based centrifugation portion of the two-step method.
Kurat CF, Natter K, Petschnigg J, Wolinski H, Scheuringer K, Scholz H, Zimmerman R, Leber R, Zechner R, and
Kohlwein SD. (2005). Obese Yeast: Triglyceride Lipolysis Is Functionally Conserved from Mammals to Yeast. J of Biol
Chem. 281:491-500.
tgl3; tgl4 with no " factor
tgl3; tgl4 with no " factor
Cultures are
centrifuged for 3 hours
at 30,000x g at 4°.
Cells are then
centrifuged for 3
minutes at 3,000x g and
resuspended in fresh
media to wash cells of
factor pheromone.
Less
dense
Burke D, Dawson D, and Stearns T. (2000). Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Wildtype with no " factor
Cultures are
incubated in a
shaker at 30° for
120 minutes.
Test density gradient
centrifugation
Wildtype with no " factor
Cells are layered
onto different
concentrations of
Percoll gradient
Optimize cell cycle
synchronization using α
mating pheromone and
mating type a strains
(incubation time and α
concentrations tested)
tgl3; tgl4 with " factor
No " factor
added
REFERENCES
Anthenstaedt K, and Daum G. (2005). Two Triacyglycerol Lipases of the Yeast Saccharomyces cervisiae Are Localized
to Lipid Particles. J of Biological Chemistry. 280:37301-37309
tgl3; tgl4 with " factor
50 µM of α factor
added
100 !M α Factor
Wildtype with " factor
Cultures are
grown to an OD
of ~ 0.600
50 !M α Factor
Wildtype with " factor
Identify mating type a
double mutants
Our results suggest that " synchronization does not necessarily improve the separation of the tgl; tgl4 lipid
accumulating mutant from wildtype because synchronized vs. asynchronized cells of the same genotype
migrate to similar positions in the Percoll gradient (Figure 3). However, the tgl3; tgl4 mutant does migrate to
a higher position than wildtype. This observation is consistent with our expectation that this lipidaccumulator would have a lower buoyant density. The difference in density between tgl3; tgl4 and wildtype
is not as large as expected. This could be due to the fact that yeast cells in this experiment were in early log
phase, which is ideal for synchronization, whereas fatty acid accumulation has been shown to be highest
during the stationary phase of yeast growth. This study demonstrates that 1) the stage of the cell cycle does
not seem to greatly affect buoyant density of the cells and 2) that yeast cells of varying lipid contents can be
separated based on buoyant density. In the future, we will perform density-based centrifugation on cells in
stationary phase to hopefully improve the separation of wildtype from mutant cells that accumulate larger
amounts of lipids. Although the method is not totally optimized, the current data suggests that a densitybased screen for yeast strains with increased lipid content is feasible.
Figure 1. Yeast wild type BY4741 and tlg3; tgl4 were incubated with increasing amounts of " factor
pheromone for 120 minutes. Yeast were looked at under a microscope at 40x. For both the wildtype and
tlg3; tgl4 having no " factor, cells are in various stages of the life cycle. Cells that were incubated with
30 !M showed ~ 50% cells in shmoo stage. At 50#M and 100 #M ~95% cells were arrested in the
shmoo stage. Not shown- incubation at 90 minutes, 150 minutes and 180 minutes. At 90 minutes, less
than 50% cells were in the shmoo stage at 100 #M. For 150 and 180 minutes, cells showed same results
as the 120 minute pictures above.
Methods
Identify tgl3; tgl4
double mutant by
PCR genotyping
30 !M α Factor
ACKNOWLEDGEMENTS
The NSF Engineering Research Center for Biorenewable
Chemicals
This material is based upon work supported by the National Science
Foundation under Grant No. EEC-0813570
Dr. Adah Leshem-Ackerman and RET program for providing
this internship opportunity.
Dr. Basil Nikolau for opening his lab to this internship program
and his oversight.
Figure 2. "factor reacts with
mating type a cells. " factor
pheromone arrests cell in G1
phase and changes the
morphology of cell structure
into the shmoo shape
Without
With factor
108)
factor
Figure 3. Yeast cells (1 x
were centrifuged in 90% Percoll after 6 hours at 10,000x g at 4°. These pictures show that the
tgl3; tg4 is less dense than the wildtype cells when both are incubated with " factor, as well as when they have no "
factor. Differences in cell density over the cell cycle do not seem to impact our results.
Dr. Marna Yandeau-Nelson for her teaching and allowing a
teacher to participate in her research.
Amy Jacobson for all of her help in the lab.
Ph.D. candidates for their friendly support.

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