A MICROBIAL BREATHALYZER: DESIGN OF A COLORIMETRIC ASSAY FOR THE DETECTION AND QUANTIFICATION OF ETHANOL PRODUCTION IN MICROBES
A Major Qualifying Project Report:
submitted to the Faculty
of the
WORCESTER POLYTECHNIC INSTITUTE
in partial fulfillment of the requirements for the
Degree of Bachelor of Science
by
______
Rachel Robillard
Date: April 26, 2007
Approved:
______
Professor Reeta Prusty Rao, Advisor
ABSTRACT
The bio-ethanol industry is in its beginning phases, and not without many problems œ the most troublesome being cost and energy inefficiencies. This project aims to address the issues of efficiency with the design of a three step assay that allows for the rapid screening of large numbers of microbial colonies for ability to produce ethanol from a variety of feedstocks. First, the microbes are grown on agar plates that are adjusted to have a basic pH. The plates are blue in color because they also contain bromothymol blue, a pH indicator. Ethanol production also results in carbon dioxide production, changing the pH of the agar around the colonies to acidic and the color to shades of yellow and orange. Based on their color, colonies that appeared to be producing more ethanol were picked and cultured. These colonies are then treated with pyridinium chlorochromate, an oxidizer that changes color in the presence of primary and secondary alcohols, from orange to brown. This rate of color change is measured spectrophotometrically, and is proportional to the concentration of ethanol in each culture.
To confirm our observations we performed a gas chromatograph, which verified that the assay successfully determines ethanol concentration with almost always at least 80 to 100 percent accuracy. Implementation of this assay would allow researchers to quickly identify mutants or scan genetically modified microbes that over produce ethanol. It would also permit scientists to assess the microbes‘ ability to produce ethanol from unusual carbon sources, cellulosic or otherwise.
2 ACKNOW LEDGEM ENTS
The student would like to acknowledge Worcester Polytechnic Institute
Professors Reeta Prusty Rao, Michael Buckholt, JoAnn Whitefleet-Smith, and Ted
Crusberg for extensive use of laboratory equipment, graduate student Ally Hunter for her assistance with research, and Professor Lee Lynd of Dartmouth College for his donation of a Clostridium Thermocellum culture.
3 LIST OF ILLUSTRATIONS
Figure 2.1 œ Energy balance studies of ethanol production from corn………………….8 Figure 2.2 œ Fermentation reaction for a molecule of glucose………………………….9 Figure 4.1 œ Assay process flow chart………………………………………………..…11 Figure 4.2 œ Bromothymol blue color change indicated pH……………………………12 Figure 4.3 œ The formation of carbonic acid……………………………………………12 Figure 4.4 œ PCC reaction with ethanol………………………………………………….16 Figure 4.5 œ Ethanol standards color change……………………………………………16 Figure 4.6 œ Ethanol standard curve……………………………………………………..17 Figure 4.7 œ PCC and media interactions……………………………………………..…20 Figure 4.8 œ Assay to detect PCC interactions with alternate sugars……………………21 Figure 5.1 œ BTB plate before streaking…………………………………………………21 Figure 5.2 œ BTB plate with Y103 and Y101……………………………………………21 Figure 5.3 œ Close view of BTB plates colonies………………………………………..22 Figure 5.4 œ BTB plate with a possible high ethanol producing mutant………………..23 Figure 5.5 œ Comparison of PCC estimated ethanol content from glucose and gas chromatograph data……………………………………………………….27 Figure 5.6 œ Comparison of PCC estimated ethanol content from maltose and gas chromatograph data………………………………………………………27 Figure 5.7 œ Comparison of PCC estimated ethanol content from sucrose and gas chromatograph data………………………………………………………29 Figure 5.8 œ Comparison of PCC estimated ethanol content for raffinose and gas chromatograph data ………………………………………………………30 Figure 7.1 œ BTB plate 1……………………………………………………………….34 Figure 7.2 œ BTB plate 2……………………………………………………………….35 Figure 7.3 œ a Y101 genetic screen BTB plate with singular large dark colony and its control plate ………………………………………………………………35 Figure 7.4 œ BTB plate 4, a light and dark colony……………………………..………36
LIST OF TABLES
Table 1 œ Structures of the alternate sugars…………………………………………..…13 Table 2 œ A570 data and rate of color change values for ethanol standards………….…16 Table 3 œ A570 data and rate of color change values for medias……………………..…19 Table 4 œ A570 data and calculations for yeast strains grown in glucose…………….…23 Table 5 œ A570 data and calculations for yeast strains grown in maltose……………….25 Table 6 œ A570 data and calculations for yeast strains grown in sucrose……………….28 Table 7 œ A570 data and calculations for yeast strains grown in raffinose……………...29
4
TABLE OF CONTENTS
1. Introduction………………………………………………………………………….…6
2. The Basics of the Bio-ethanol Industry………………………………………………...7 2.1 Ethanol as a Fuel……………………………………………………………...8 2.2 Ethanol Production and Fermentation…………………………………….…..9
3. Current Topics in Bio-Ethanol Research……………………………………………...10
4. Colorimetric Assay Design……………………………………………………………11 4.1 Bromothymol Blue…………………………………………………………...12 4.1.1 pH Sensitive Agar Plates…………………………………………..12 4.2 Microbial Cultures and Carbon Sources……………………………………..13 4.2.1 Know your sugars………………………………………………….13 4.3 Pyridinium Chlorochromate………………………………………………….15 4.3.1 Spectrophotometric Analysis of Alcohol Oxidation…………….…16
5. Results 5.1 Bromothymol Blue plate……………………………………………………..20 5.2 Pyridinium chlorochromate assay……………………………………………23 5.2.1 PCC data for strains grown in glucose…………………………..…26 5.2.2 PCC data for strains grown in maltose………………………….…28 5.2.3 PCC data for strains grown in sucrose……………………………..30 5.2.4 PCC data for strains grown in raffinose…………………………...30
6. Discussion……………………………………………………………………………..31
7. Suggestions for future research 7.1 Wine strains………………………………………………………………….32 7.2 Colstridium Thermocellum…………………………………………………..33 7.3 Genetic Screen……………………………………………………………….33
Appendices A œ Bromothymol blue pH sensitive agar plates for yeast protocol………….…37 B œ Bromothymol blue pH sensitive agar plates for bacteria protocol …………38 C œ Spectrophotometric ethanol content data for yeast strains grown in lactose..39 D œ Spectrophotometric ethanol content data for yeast strains grown in dextran.40 E œ Spectrophotometric ethanol content data for yeast strains grown in xylose ..41 F œ Y101 screen for dark colonies……………………………………………….42 G œ Y101 screen for light colonies………………………………………………48
References………………………………………………………………………………..49
5 1. INTRODUCTION
The earth today is faced with the challenges of global warming, pollution,
resource depletion, and the resulting anxiety surrounding energy security as the direct
consequences of fossil fuel over consumption. The bio-ethanol industry is in the
beginning stages of developing a promising replacement for some of these petroleum-
based fuels. As a fuel source, ethanol burns cleaner than its fossil fuel counterparts. When
ethanol is made biologically œ via microbial fermentation œ under certain conditions its
use and production can be a renewable, carbon-neutral process, making ethanol a
sustainable and long-term supplement to the worlds‘ energy demands.
Aside from a slew of political and economic factors, the largest scientific barrier
that is preventing the ethanol fuel industry from major success is cost efficiency. Cost
encompasses all parameters of production including time, labor, materials used, and
energy expenses. In most cases, the energy input required to produce the ethanol actually
exceeds the energy output obtained from its combustion œ which is obviously
counterproductive and defeats the purpose of making fuel. Dramatic improvements on
process efficiency are needed before ethanol can be realized as an alternative fuel.
There are several ways to go about addressing these inefficiencies, and many
scientific hurdles to be jumped. The first is finding the best microbe for the job. Yeast is
typically used but some kinds of bacteria are also capable of fermentation. Identifying a
microbe that can yield high amounts of ethanol is the major discovery waiting to happen
in bio-fuels research. From here, genetic analysis should be used to determine exactly
which genes are responsible for œ and also which are inhibitory to œ the increased ethanol
production, so the microbe could be modified to produce the maximum amount possible.
6 In addition to gene characterization, another exceedingly important advancement is the
identification of inexpensive and abundant carbon sources that are compatible with the
microbes‘ fermentation processes. Linking all of these developments would lead to
process optimization and certainly a more successful approach.
The assay designed in this project was made with many of these issues in mind. It
was also important that the assay go about addressing those issues in a manner that is
itself, environmentally friendly. The goals were to make it simple, inexpensive, able to
assess several parameters, and require low energy input. Improving efficiency in ethanol
production is the only way to realize the goals of wide spread bio-fuels use.
2. INDUSTRY BASICS
Aside from the obvious environmental benefits of using ethanol as a fuel,
increasing its use would support US agriculture, boost the economy by creating jobs,
reduce fuel imports, and actually be a cost effective alternative to consumers by
decentralizing the control of the fuel supply from corrupt oil profiteers [2]. The ethanol
industry has seen dramatic increase in size and production capabilities over only the past
eight years. There are currently 115 ethanol plants in the US, up from only 50 in 1999.
These 115 plants produce more than 5.5 billion gallons of ethanol per year mainly from
corn [2]. Although the data and production procedures vary, most energy balance studies
suggest that the production of ethanol from corn actually has a negative energy balance,
and that innovative technologies, improvement of sustainable farming practices, and the
development of using cellulosic materials are crucial in increasing fuel efficiency and
viability [4].
7
Figure 2.1 œ Energy balance studies of ethanol production from corn [7]
2.1 Ethanol as a Fuel
Methyl Butyl Tertiary Ether (MBTE) is a chemical added to gasoline to boost the
octane level. This of course improves the fuels‘ performance, but it has come at a heavy
environmental cost. MBTE has become a plaguing pollutant that has found its way into
ground water supplies, and unfortunately it is nearly impossible to extract. Many US
states have begun banning the use of MBTE and instead use ethanol for the same purpose
œ in fact, most commercially available gasoline today contains at least ten percent ethanol,
but it can be mixed in varying amounts. Ethanol/gasoline mixtures have a lot of
comparative advantages over straight gasoline, in that they reduce the levels of pollution
emitted, reduce the dependence on oil by supplementing the fuel supply, and increase
engine performance. Currently, ethanol constitutes about 99% of all biofuels produced in
the United States. In 2004, 3.4 billion gallons of ethanol was blended into gasoline and
8 contributed to 2% of the total fuel supply, but that is expected to increase to 7.5 billion
gallons per year by 2012, as mandated by the Energy Policy Act of 2006 [3].
2.2 Ethanol Production and Fermentation
Ethanol fermentation has not greatly changed since the invention of beer and wine
thousands of years ago. While the technology and capacity has changed, the process is
basically the same. Corn is delivered to the refinery and steeped to extract its sugar. The
sugars extracted from corn are generally very simple, soluble sugars. In fermentation,
microbes enzymatically convert sugar into ethanol and carbon dioxide. Typically the
reaction occurs on only a six carbon sugar, whereby it is converted into two molecules of
ethanol and two molecules of carbon dioxide. The ethanol is then extracted from the
watery fermentation tank by distillation, and distributed [5].
Figure 2.2 œ Fermentation reaction of a molecule of glucose
3. CURRENT TOPICS IN BIO-ETHANOL RESEARCH
As the bio-ethanol industry grows, its problems and inefficiencies have grown
increasingly apparent. Biologists, chemists, and engineers have all been racing to fix the
problems, and bio-fuels have become a popular research topic at biotechnology firms and
universities alike.
As mentioned, the energy balance from producing ethanol from corn as a carbon
source has been highly debated and is not a very energy efficient process. So, why not
9 use something besides corn? The Iogen corporation in Ontario Canada is making that
possible. This biotechnology company has engineered a variety of enzymes that can
break cellulosic materials down into their component simpler sugars. Cellulose, along
with lignin, is the most abundant carbohydrate in the world, and is responsible for giving
plants their rigid structure. The problem with using cellulose and other complex
carbohydrates, is that they are usually not soluble, and microbes can not make the
enzymes necessary to break them down. Iogen has developed a line of synthetic enzymes
that can digest paper, textiles, and plant fibers, which look to have a promising future.
Scientists across the world œ from California to Japan œ are working to discover a
microbe that can already produce ethanol from cellulose, and in the mean time they are
trying to engineer one to do the same thing. Researchers are trying to identify a variety of
genes that would allow for the creation of an extremely efficient ethanol producing
microbe. The genes they are looking out for are those responsible for directing the
synthesis of cellulose-digesting enzymes, and those that would allow the microbes to
survive in higher ethanol concentrations. Characterizing the genes of all those microbes is
no easy task however, and only time and improved technologies will allow that to happen.
4. COLORIM ETRIC ASSAY DESIGN
In an attempt to increase the prospects of identifying an organism that can
produce high amounts of ethanol and identifying which genes may be involved (while
also keeping things simple and cost effective), we designed a multi-step colorimetric
assay. The assay allows for the screening of large numbers of microbial colonies based
on their ability to produce ethanol.
10
Figure 4.1 œ Assay process flow chart
4.1 Bromothymol Blue
The first step in the assay involves Bromothymol blue. Bromothymol blue (BTB)
is a color indicator for weak acids and bases that works in the pH range from about 6 to 8.
In basic conditions BTB appears blue/green, but becomes yellow as conditions become
more acidic.
11 Figure 4.2 œ BTB color change indicates pH
pH 6 (acidic) pH 7 (neutral) pH 8 (basic)
4.1.1 pH Sensitive Agar Plates
Bromothymol blue was added to the agar plates to make them pH sensitive. As
mentioned in section 2.2, the production of ethanol also produces carbon dioxide. When
presented with water, carbon dioxide will form carbonic acid, making the pH more acidic.
Figure 4.3 œ Formation of carbonic acid
SC dropout agar media was made according to protocol detailed in Appendix A. The pH
of normal media is ~ 6. The pH was adjusted to 8 using 5N NaOH. It was essential to use
a highly concentrated base so that its addition would not cause the media to become
watery. Bromothymol blue was added in the amount of 0.13 grams per liter of media,
turning the plates a vibrant blue/green color. It is possible to add more BTB if a deeper
color is desired, as BTB does not appear to affect the health of the yeast in any way. After
mixing, the plates were poured as usual. 10^-4 dilutions of ten S. cerevisiae strains œ Y1,
Y47, Y49, Y52, Y53, Y101, Y103, Y195, Y197, Y717 œ were prepared and spread on the
12 BTB plates so that individual colonies could be isolated. Plates were incubated at 30°C
for about 2-3 days.
4.2 M icrobial Cultures and Carbon Sources
The colonies that grew on the BTB plates varied in color from very pale yellow to
bright orange. Colonies worth investigating were chosen based on their color; the more
orange, the better. Those colonies were picked and cultured in medias containing xylose,
glucose, maltose, sucrose, lactose, raffinose, and dextran. Cultures were grown in 2 mL
of media in air tight tubes (to prevent the ethanol from evaporating) until the glucose
samples tested negative for its presence, according to glucose test strips œ or
approximately two days. The tubes were allowed to briefly equilibrate at least once per
day to prevent oxygen starvation. Cultures were spun down in a centrifuge to isolate the
supernatant, which contained the ethanol, and the samples were stored in the freezer.
Table 1 œ Alternate sugars
Sugar name Chemical Secondary Structure formula
Xylose C5H10O5
Glucose C6H12O6
13
Maltose C12H22O12
Sucrose C12H22O12
Lactose C12H22O12
C18H32O6 ‡ Raffinose 5 H2O
14
Dextran N/A
4.3 Pyridinium Chlorochromate (PCC)
Pyridinium chlorochromate (PCC) is a strong oxidant [6] that changes color in the
presence of ethanol, from a brilliant orange to increasingly dark shades of brown as the
chromium compound precipates. When presented with ethanol the following reaction
occurs:
Figure 4.4 œ PCC reaction with ethanol
1M PCC was prepared in water, and 100 µl of 1M of PCC solution was added to 100 µl
of supernatant of each of the 70 cultures in a 96 well plate. Like all chromium
compounds and strong oxidizers it was important to handle it carefully.
15 4.3.1 Spectrophotometric Analysis of Alcohol Oxidation
The color change in the reaction of PCC and ethanol was measured at 570 nM
using a spectrophotometer, and was determined to be proportional to the amount of
ethanol present. Varying concentrations of ethanol were prepared and the A570 was
taken every ten minutes for one hour. These were created as a standard to which the color
change in the cultures could be compared. The figure below illustrates the color change
of the ethanol standards.
Figure 4.5 œ Ethanol standards color change
0% 0.5% 1% 2% 4% 10% 20%
The differences in spectrophotometric data at each time point was used to calculate the
rate of color change per minute for each sample, by plotting the A570 reading versus time
and finding the slope of the line.
Table 2 œ A570 data for ethanol standards
Percent A570 nm (minutes) Average concentration A570 nm of ethanol 10 20 30 40 50 60 change per minute 0 0.260 0.260 0.260 0.260 0.258 0.260 0 0.5 0.283 0.307 0.325 0.346 0.369 0.388 0.0021 1 0.255 0.296 0.328 0.362 0.399 0.431 0.0035 2 0.334 0.424 0.481 0.540 0.604 0.652 0.0063 4 0.385 0.532 0.628 0.714 0.796 0.856 0.0092 10 0.485 0.838 0.966 1.069 1.142 1.174 0.0127 20 0.051 0.854 1.009 1.145 1.203 1.270 0.0142
The values for the rate of color change per minute was plotted in Figure 4.6, which shows
how the rate of change is proportional to the ethanol content.
16 Figure 4.6 œ Ethanol standard curve
0.016
0.014
0.012 e t u n i 0.01 m
r e p 0.008 e g n a
h 0.006 c
0 7 5
A 0.004
0.002
0 0 5 10 15 20 25
Percent ethanol
The A570 of the 70 cultures in the 96 well plate was measured in the same manner, every ten minutes for one hour. The rate of change of color was determined for each sample, and that rate was compared to the graph in Figure 4.6, to approximate how much ethanol the sample contained.
To ascertain whether other components of media (such as the sugars) affected the
PCC assay, we tested just the media with PCC to observe and measure any color change.
PCC was added to separate Eppendorf tubes containing water (control), YP media (no glucose), and YPD (with Glucose). Using this quick test, a large color changed was observed in the tube containing YPD and a minor color change was observed in the tube containing YP media, as shown below.
17 Figure 4.7 œ PCC media interactions
Left, PCC/water. Middle, PCC/Glucose. Right, PCC/YP media
In the PCC assay the —background“ color changes from the sugars only added to the color
change from ethanol, making it seem as though there were more ethanol in the sample
than there actually was. Since the PCC/sugar interaction suspicions were confirmed, the
exact background color change from the sugars was also determined as a
spectrophotometric value, shown in Table 3 below. These values were determined in the
same manner as the ethanol standards.
18 Figure 4.8 œ Assay to detect PCC interactions with alternate sugars
PCC/ PCC/ PCC/ Media YP Water w/sugar only only
Xylose
Dextran
Raffinose
Lactose
Sucrose
Maltose
Glucose
19 Table 3 œ A570 spectrophotometer data and rate of color change values for medias
Media type A570 nm (minutes) Average A570 nm change per 10 20 30 40 50 60 minute None (water) 0.235 0.234 0.235 0.235 0.234 0.235 0
YP 0.237 0.254 0.265 0.271 0.272 0.275 0.0007
YP with 0.433 0.509 0.53 0.554 0.579 0.625 0.0034 Glucose YP with 0.363 0.368 0.381 0.394 0.408 0.419 0.0007 Maltose YP with 0.336 0.337 0.343 0.346 0.356 0.364 0.0005 Sucrose YP with 0.356 0.376 0.393 0.41 0.427 0.447 0.0018 Lactose YP with 0.503 0.521 0.532 0.534 0.538 0.548 0.0008 Raffinose YP with 0.488 0.504 0.519 0.535 0.548 0.560 0.0015 Dextran YP with 0.505 0.532 0.592 0.651 0.712 0.759 0.0053 Xylose
The results of the PCC and sugar assay in Figure 4.8 and Table 3 showed that generally, the reactivity of the sugar with PCC is related to the complexity of the molecule. After the rate of color change for each of the culture samples was calculated
(shown in the next section), the color change of the media was subtracted from that value to discount for the background color.
20 5. RESULTS
5.1 Bromothymol Blue Plates
Figure 5.1 œ BTB plate before streaking
The figure above shows the desired blue/green color of the basic BTB plate
before streaking. The color change induced by the growing yeast is shown in Figure 5.2.
Figure 5.2 œ BTB plate with Y103 on the left and Y101 on the right
21 The plate above shows two strains, the one on the left appears orange while the one on
the right looks more yellow. It was experimentally determined that Y103 (left) produces
more ethanol as per GC analysis than Y101, hence the darker color correlates with the
amount of ethanol.
Figure 5.3 œ BTB plate with Y53 on the left and Y103 on the right
The plate in figure 5.3 shows the orange colonies of strain Y103 on the right and the
yellow color of Y53 colonies on the left. Y53 was determined to be on the lower
spectrum of ethanol production, whereas Y103 was the highest of all the strains tested.
22 Figure 5.4 œ BTB plate with a mutant
Figure 5.4 is a closer view of a BTB plate, but it is apparent that a mutant orange colony
has risen out of the yellow colonies in Y195 (left); the white arrow in the picture points to
the mutant. This is the sort of colony that was termed a —colony of interest“ because it
may be producing more ethanol than the wild type.
5.2 Pyridinium Chlorochromate assay
As time elapses in the PCC assay the color change became more apparent. While
it is difficult to differentiate color change visually, the spectrophotometer data shows
clear differences in rate of color change for each sample.
To verify the accuracy of the PCC assay in determining the ethanol concentration
of the sample, a gas chromatograph was done for each sample œ the results are shown in
the next three graphs, as well as on Appendices C, D, and E.
23 5.2.1 Percent Ethanol as determined using PCC for strains grown in glucose
Table 4 œ Spectrophotometer data and calculations for strains grown in glucose
Strain Change in A570 Change in A570 Percent ethanol Percent per minute per minute, after determined from ethanol baseline PCC standards determined adjustment from GC Y1 0.0042 0.0036 1 0.97 Y47 0.0040 0.0033 0.9 0.94 Y49 0.0047 0.0040 1.2 0.88 Y52 0.0043 0.0036 1 0.19 Y53 0.0040 0.0033 0.9 0.76 Y101 0.0039 0.0032 0.8 0.95 Y103 0.0048 0.0041 1.25 1.06 Y195 0.0045 0.0038 1.1 0.82 Y197 0.0038 0.0031 0.75 0.66 Y717 0.0039 0.0032 0.8 0.83
The rate of orange to brown color change of each strain was calculated in the
same manner as the ethanol standards in section 4.3.1. The spectrophotometer data was
plotted against time and the slope of the line was determined, and is shown in the second
column of Table 4. To adjust for the baseline color change 0.0007 was subtracted from
each of the values in column two. As shown in Table 3, 0.0007 is the color change
induced by YP media. Since it is a well known fact that yeast readily digest glucose,
confirmed by the negative results of glucose presence as indicated by the glucose test
strips, it was not necessary to subtract more than just the value for YP media.
24 Figure 5.5 œ Comparison of PCC estimated ethanol content from glucose and gas chromatograph data
The graph above is a comparison of the calculated ethanol content for each sample using PCC as shown in Table 4, to the ethanol content specified by the gas chromatograph, also shown in Table 4. The value listed above each set of columns is the accuracy of the two values, as determined by calculating a chi squared value using Eq. 1.
Equation 1 œ Chi squared formula
U× = (Observed value œ Expected value)× Expected value
25 5.2.2 Percent Ethanol as determined using PCC for strains grown in maltose
Table 5 œ Spectrophotometer data and calculations for strains grown in maltose
Strain Change in A570 Change in A570 Percent Ethanol, Percent per minute per minute, after as determined ethanol baseline from Graph 1 determined adjustment from GC Y1 0.0009 0.0002 0 0 Y47 0.0030 0.0023 0.65 0.8 Y49 0.0029 0.0022 0.65 0.92 Y52 0.0027 0.0025 0.7 0.95 Y53 0.0030 0.0023 0.6 0.94 Y101 0.0022 0.0015 0.3 0.29 Y103 0.0011 0.0004 0.05 0 Y195 0.0029 0.0022 0.65 0.99 Y197 0.0028 0.0021 0.65 0.83 Y717 0.0027 0.0020 0.65 0.72
In Table 4, for glucose, 0.0007 was subtracted from the A570 rate of change per
minute to discount for the baseline color of the media as determined in Table 3. In Table
3 it is shown that the A570 color change per minute for maltose media is also 0.0007, so
this amount was subtracted from each row of column two in Table 5 to discount for the
baseline color change, giving the adjusted values in column three; these values were the
ones used to predict the ethanol concentration.
26 Figure 5.6 - Comparison of PCC estimated ethanol content from maltose and gas chromatograph data
Like Figure 5.6, the graph above shows the accuracy between the predicted ethanol content using the PCC assay and the actual ethanol content determined from the gas chromatographs, as determined using the chi squared test (Eq.1).
27 5.2.3 Percent Ethanol as determined using PCC for strains grown in sucrose
Table 6œ Spectrophotometer data and calculations for strains grown in sucrose
Strain Change in A570 Change in A570 Percent Ethanol, Percent per minute per minute, after as determined ethanol baseline from Graph 1 determined adjustment from GC Y1 0.0032 0.0027 0.7 1.11 Y47 0.0034 0.0029 0.75 0.84 Y49 0.0036 0.0031 0.75 0.96 Y52 0.0035 0.0030 0.75 0.77 Y53 0.0023 0.0018 0.3 0.89 Y101 0.0026 0.0021 0.5 1.09 Y103 0.0033 0.0028 0.75 1.04 Y195 0.0053 0.0048 1.35 0.96 Y197 0.0025 0.0020 0.65 1.18 Y717 0.0027 0.0022 0.65 1.08
The value for the rate of change per minute of media containing sucrose was
0.0015, so this amount was subtracted from column two for each strain grown in sucrose.
28 Figure 5.7 - Comparison of PCC estimated ethanol content for sucrose and gas chromatograph data
The graph in Figure 5.8 shows the comparison of PCC and gas chromatograph
ethanol concentration data.
5.2.4 Percent Ethanol as determined using PCC for strains grown in raffinose
Table 7 œ Spectrophotometer data and calculations for strains grown in raffinose
Strain Change in A570 Change in A570 Percent Ethanol, Percent per minute per minute, after as determined ethanol baseline from Graph 1 determined adjustment from GC Y1 0.0018 0.0010 0.1 0.06 Y47 0.0007 0 0 0 Y49 0.0010 0.0002 0 0 Y52 0.0010 0.0002 0 0 Y53 0.0016 0.0008 0.1 0 Y101 0.0017 0.0009 0.1 0.05 Y103 0.0018 0.0010 0.1 0.04 Y195 0.0008 0 0 0 Y197 0.0015 0.0007 0.05 0.05 Y717 0.0019 0.0011 0.15 0.07
29
While the gas chromatograph showed that most of the yeast strains grown in raffinose did not produce ethanol, or produced very small fractions of a percent, it is important to keep in mind that the PCC assay detected the ethanol by showing an increase in color change for those with only trace amounts. The value for raffinose media change was 0.0008 per minute, and was subtracted in column three in the same manner as the other sugars.
Figure 5.8 - Comparison of PCC estimated ethanol content for raffinose and gas chromatograph data
In the graph in Figure 5.9 is important to note the sensitivity of the PCC assay in determining trace amounts of ethanol.
30 6. DISCUSSION
The BTB assay is qualitative, and is functional under the assumption that carbon
dioxide as a byproduct of fermentation lowers the pH of the plate. While there is a
positive correlation among strains that lower the pH of a BTB plate and the GC assay, we
can not conclusively say that this is due to ethanol content. Therefore we used the BTB
assay as a primary screen to identify potentially interesting colonies. A variety of factors
could have been responsible for the media becoming more acidic. However, when
comparing the colors of a high ethanol producer next to a low ethanol producer as in
Figures 5.2-5.4, those that were producing more ethanol as verified by the PCC assay and
GC confirmation, were darker. So, this is followed up by a more quantitative PCC assay.
The PCC assay is necessary as the quantitative step to determine just how much
ethanol the microbe is producing. Cultures were grown in a variety of sugars. While some
were sugars such as xylose, lactose, and dextran resulted in no ethanol, others such as
sucrose, raffinose, and maltose did produce ethanol. The highest ethanol production was
in glucose (shown in Figure 5.5) but the accuracy of the PCC tests for the other sugars
(shown in the appendices) was comparable, suggesting that this assay can be used for a
variety of feedstocks to assess viability for fermentation. In fact, the PCC assay appears
to be more accurate in as the complexity of the sugar increases. Furthermore, this assay
would work well with complex sugars because the —background“ color change (discussed
in section 4.3.1. Table 3) is negligible in complex sugars. This provides a more accurate
calculation of the ethanol concentration in the sample. Considering the yeast strains used
were mild lab strains and produced relatively low concentrations of ethanol, the assay
31 was very sensitive and would likely be very accurate in determining the ethanol produce
for high yielding strains.
Because this assay is simple and cheap it is amiable for high throughput screens
of mutants, such as the yeast in the Y101 library. It is also easy to scale up even when
testing thousands of samples. Since the assay can accurately predict ethanol concentration
just by observing a chemical reaction, it should eliminate the need to use a gas
chromatograph to determine ethanol concentration in a sample. Not only is GC tedious on
a large scale (assessing thousands of samples could take several weeks), but also a very
expensive piece of equipment that requires a lot of energy input to run. With a little more
optimization and testing on a range of microbes that produce higher percentages of
ethanol, this assay does, and will offer a simple way to detect and quantify ethanol
production in microbes that is quick and inexpensive.
7. SUGGESTIONS FOR FUTURE RESEARCH
7.1 W ine Strains
In the future this assay should be used to test the yeast strains isolated from wine
vineyards collected by Professor Mortimer to see their capacity for producing ethanol.
Since they were isolated from a vineyard there is a very good chance that they are already
good ethanol producers. Additionally, the strains could be mutagenized to try to isolate
colonies that are very high ethanol producers.
32 7.2 Clostridium Thermocellum
Since yeast are not the only organisms that can produce ethanol, expanding the
scope of the assay to accommodate bacteria is essential, and possible. The assay does not
require oxygen, so it should be able to work well in anaerobic conditions as well. The
most promising microbe right now is C. thermocellum, an obligate anaerobe that can
produce ethanol from a variety of feed stocks, including grass, wood, and other cellulose-
containing materials [1].
7.3 Genetic Screens
Laboratories with existing libraries would be interested in this assay because it
allows for the screening of thousands of strains at a time. Scientists could easily identify
mutants whose gene deletion has resulted in increased ethanol production. Identification
of various genes related to ethanol production would be a major breakthrough in
eventually developing a microbe that can efficiently produce ethanol in high amounts
from a carbon source that is cheap and renewable.
A genetic screen of strain Y101 was done on BTB plates, but because of limited
time the PCC portion has not been done yet. However, color variation in the colonies,
both light and dark, was obvious, and these may be genes worth investigated as they may
be related to ethanol production. The following figures are photographs of some of the
plates created in the genetic screen, and also illustrate the various light and dark colonies.
33
Figure 7.1 œ BTB plate
The variations in orange and yellow color of Y101 are shown in
Figure 7.1 Dark and light colonies are present on the same plate.
34 Figure 7.2 œ a Y101 genetic screen BTB plate with singular large dark colony and its control plate.
Figure 7.3 œ BTB plate 4, a light and dark colony
35 Figure 7.4 œ BTB plate 5, dark colony
As a result of the screen, there were 45 genes identified as possibly related to increased ethanol production, and 8 genes identified as possibly inhibitory to ethanol production. Each gene and a description of its function can be found in Appendices F and
G.
36 APPENDIX A: Bromothymol blue pH sensitive agar plates for yeast
1. SC drop out media:
6.7 g/L Yeast nitrogen base without amino acids 1.4 g/L Yeast synthetic drop out supplement 18 g/L Bactoagar 1 L DI H2O Autoclave for 15 minutes
40 mL of 50% Glucose solution (w/v) Autoclave for 15 minutes
After autoclaving, add the 40 mL of glucose solution to the agar media. Also add the following amino acid supplements if it is necessary:
10 mL/L Leucine 6 mL/L Tryptophan 9 mL/L Uracil 3 mL/L Histidine
2. pH change:
After the SC media is made, the pH of the entire bottle must be adjusted. The pH of the media is normally between 5-6, but must become more basic for the indicator to work. Add:
1.5 mL/L 5 N NaOH
After mixing, check the pH of the media by using a simple pH strip. Addition of this amount of NaOH should result in a pH of around 7.
3. BTB:
Add 0.13 grams of BTB per liter of media.
The media will turn dark blue/green. Mix well and pour into plates immediately.
37 APPENDIX B: Bromothymol blue pH sensitive agar plates for bacteria (suggested recipe)
1. LB media:
20 g/L LB broth mix 18 g/L Bactoagar 1 L DI H2O
Autoclave for 30 minutes
2. pH change:
After the SC media is made, the pH of the entire bottle must be adjusted. The pH of the media is normally between 5-6, but must become more basic for the indicator to work. Add:
1.5 mL/L 5 N NaOH
After mixing, check the pH of the media by using a simple pH strip. Addition of this amount of NaOH should result in a pH of around 7.
3. BTB:
Add 0.13 grams of BTB per liter of media. The media will turn dark blue/green, but more BTB can be added sparingly if a darker color is desired. Mix well and pour into plates immediately.
38 APPENDIX C: PCC assay data for yeast grown in lactose
Strain Change in A570 Change in A570 Percent Ethanol, Percent per minute per minute, after as determined ethanol baseline from Graph 1 determined adjustment from GC Y1 0.0026 0.0008 0.1 0 Y47 0.0026 0.0008 0.1 0 Y49 0.0027 0.0009 0.1 0 Y52 0.0018 0 0 0 Y53 0.0033 0.0015 0.2 0 Y101 0.0019 0.0001 0 0 Y103 0.0015 0 0 0 Y195 0.0017 0 0 0 Y197 0.0018 0 0 0 Y717 0.0027 0.0009 0.1 0
39 APPENDIX D: PCC assay data for yeast grown in dextran
Strain Change in A570 Change in A570 Percent Ethanol, Percent per minute per minute, after as determined ethanol baseline from Graph 1 determined adjustment from GC Y1 0.0005 0 0 0 Y47 0 0 0 0 Y49 0.0005 0 0 0 Y52 0.0005 0 0 0 Y53 0.0004 0 0 0 Y101 0.0011 0 0 0 Y103 0.0004 0 0 0 Y195 0.0004 0 0 0 Y197 0.0004 0 0 0 Y717 0 0 0 0
40 APPENDIX E: PCC assay data for yeast grown in xylose
Strain Change in A570 Change in A570 Percent Ethanol, Percent per minute per minute, after as determined ethanol baseline from Graph 1 determined adjustment from GC Y1 0.0068 0.0015 0.2 0 Y47 0.0073 0.0020 0.65 0 Y49 0.0085 0.0032 0.85 0 Y52 0.0080 0.0027 0.7 0 Y53 0.0077 0.0024 0.65 0 Y101 0.0066 0.0013 0.15 0 Y103 0.0046 0 0 0 Y195 0.0065 0.0008 0.1 0 Y197 0.0074 0.0021 0.65 0 Y717 0.0074 0.0021 0.65 0
41 APPENDIX F: Y101 screen; dark colonies
Gene ID: ALF1 Info: Microtubules are conserved cytoskeletal elements that form by the polymerization of alpha- and beta- tubulin heterodimers. The formation of polymerization-competent tubulin heterodimers requires that alpha- tubulin and beta-tubulin be properly folded. Specific cofactors are required for the folding of alpha- and beta-tubulin in vitro and homologs of these cofactors have been found in many organisms, including S. cerevisiae. In S. cerevisiae, ALF1 is a non-essential gene that is homologous to mammalian cofactor B 3, 1. In vitro, cofactor B acts in the post-chaperonin folding of alpha-tubulin 3. Consistent with in vitro studies, Alf1p genetically acts upstream of Pac2p/cofactor E 3, 1. ALF1 genetically interacts with the other tubulin cofactors (CIN1/cofactor D, RBL2/cofactor A), and is essential in combination with specific alpha-tubulin mutants 3, 1. alf1 null mutants are super-sensitive to benomyl, a microtubule depolymerizing drug 1. Alf1p interacts with alpha-tubulin in the yeast two-hybrid and immunoprecipitation assays 1. Alf1p and cofactor B both contain a single CLIP-170 domain, which is found in several microtubule-associated proteins and is required for the Alf1p-alpha-tubulin interaction 1. Alf1p binds to a face of alpha-tubulin distinct of that of beta-tubulin binding 1. Alf1p-GFP localizes to cytoplasmic microtubules, suggesting that Alf1p may play an additional role in microtubule maintenance 1.
Gene ID: ALO1 Info: D-Arabinono-1,4-lactone oxidase, catalyzes the final step in biosynthesis of D-erythroascorbic acid, which is protective against oxidative stress. ALO1 encodes D-arabinono-1,4-lactone oxidase (called ALO in other organisms), a mitochondrial protein that converts D-arabinono-1,4-lactone to D-erythro-ascorbate (EASC), the enantiomer of vitamin C. Saccharomyces cerevisiae does not normally synthesize vitamin C (ASC), but Alo1p is sufficiently promiscuous that it can convert a number of related substrates to either EASC or ASC, depending on the chirality of the substrate. Like vitamin C, EASC is an antioxidant. Deletion of ALO1 results in increased sensitivity to oxidative stress and an increased rate of gross chromosomal rearrangements, implying that Alo1p suppresses oxidative damage of DNA. Transcription of ALO1 is not regulated in response to oxidative stress. Alo1p exists as a monomer embedded in the mitochondrial membrane and binds FAD.
Gene ID: BSC2 Info: Protein of unknown function, ORF exhibits genomic organization compatible with a translational read through-dependent mode of expression.
Gene ID: DFG10 Info: Protein of unknown function, involved in filamentous growth.
Gene ID: DOA4 Info: Ubiquitin hydrolase, required for recycling ubiquitin from proteasome-bound ubiquitinated intermediates, acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.
Gene ID: EAF3 Info: Esa1p-associated factor, nonessential component of the NuA4 acetyltransferase complex, homologous to Drosophila dosage compensation protein MSL3.
42
Gene ID: ELP6 Info: Subunit of Elongator complex, which is required for modification of wobble nucleosides in tRNA; required for Elongator structural integrity. Elp6p is part of the six-subunit Elongator complex, which is a major histone acetyltransferase component of the RNA polymerase II holoenzyme responsible for transcriptional elongation. Elongator contains two discrete subcomplexes, one consisting of Iki3p/Elp1p, Elp2p, and Elp3p, and the other consisting of Elp4p, Iki1p/Elp5p, and Elp6p. Elongator binds to both naked and nucleosomal DNA, can acetylate both core histones and nucleosomal substrates, and plays a role in chromatin remodeling. Its activity is directed specifically toward the amino-terminal tails of histone H3 and H4, with the predominant acetylation sites being lysine-14 of histone H3 and lysine-8 of histone H4. Of the six Elongator subunits, only Iki1p/Elp5p is essential for growth, and deletion of the other individual subunits causes significantly altered mRNA expression levels for many genes. Disruption of the Elongator complex confers resistance to the Kluyveromyces lactis zymotoxin, and a reduced sensitivity to the Pichia inositovora toxin.
Gene ID: EPT1 Info: sn-1,2-diacylglycerol ethanolamine- and cholinephosphotranferase; not essential for viability. Gene products are diacylglycerol cholinephosphotransferase, diacylglycerol ethanolaminephosphotransferase.
Gene ID: FM C1 Info: Mitochondrial matrix protein, required for assembly or stability at high temperature of the F1 sector of mitochondrial F1F0 ATP synthase; null mutant temperature sensitive growth on glycerol is suppressed by multicopy expression of Odc1p. Deletion results in decreased metabolite accumulation (glycogen).
Gene ID: GDH1 Info: NADP(+)-dependent glutamate dehydrogenase, synthesizes glutamate from ammonia and alpha- ketoglutarate; rate of alpha-ketoglutarate utilization differs from Gdh3p; expression regulated by nitrogen and carbon sources, Exhibits sensitivity at 15 generations when grown in medium of pH 8. Topoisomerases are highly conserved; yeast Top1p shares 57% identity with human Top1. The Top1 protein, like other type IB topoisomerases, relaxes supercoiled DNA by forming a DNA-enzyme complex and transiently cleaving one strand via a nucleophilic attack that results in a covalent linkage with the 3' end of the cleaved strand. The 5' end can then rotate freely. Top1p is the target of the antitumor drug camptothecin. Camptothecin increases the half-life of the enzyme-DNA complex, which results in double-stranded DNA breaks during DNA replication. Specific amino acid substitutions in Top1p have the same effect as the drug. Suppressors of these mutations were identified that reduced the enzyme's affinity for DNA.
Gene ID: GPG1 Info: Proposed gamma subunit of the heterotrimeric G protein that interacts with the receptor Grp1p; involved in regulation of pseudohyphal growth; requires Gpb1p or Gpb2p to interact with Gpa2p.
Gene ID: IKI3 Info: Subunit of Elongator complex, which is required for modification of wobble nucleosides in tRNA; maintains structural integrity of Elongator; homolog of human IKAP, mutations that cause familial dysautonomia. Iki3p/Elp1p is part of the six-subunit Elongator complex, which is a major histone acetyltransferase component of the RNA polymerase II holoenzyme responsible for transcriptional elongation. Elongator contains two discrete subcomplexes, one consisting of Iki3p/Elp1p, Elp2p, and Elp3p, and the other consisting of Elp4p, Iki1p/Elp5p, and Elp6p. Elongator binds to both naked and nucleosomal DNA, can acetylate both core histones and nucleosomal substrates, and plays a role in chromatin remodeling. Its activity is directed specifically toward the amino-terminal tails of histone H3 and H4, with the predominant acetylation sites being lysine-14 of histone H3 and lysine-8 of histone H4. Within Elongator, Iki3p/Elp3p interacts specifically with Elp2p. Of the six Elongator subunits, only Iki1p/Elp5p is
43 essential for growth, and deletion of the other individual subunits causes significantly altered mRNA expression levels for many genes. Disruption of the Elongator complex confers resistance to the Kluyveromyces lactis zymotoxin, and a reduced sensitivity to the Pichia inositovora toxin. Mutations in the human Iki3p homolog IKAP are associated with the disease Familial Dysautonomia.
Gene ID: NAS2 Info: Protein with similarity to the p27 subunit of mammalian proteasome modulator; not essential; interacts with Rpn4p.
Gene ID: NAT5 Info: Subunit of the N-terminal acetyltransferase NatA (Nat1p, Ard1p, Nat5p); N-terminally acetylates many proteins, which influences multiple processes such as the cell cycle, heat-shock resistance, mating, sporulation, and telomeric silencing.
Gene ID: NUP2 Info: Protein involved in nucleocytoplasmic transport, binds to either the nucleoplasmic or cytoplasmic faces of the nuclear pore complex depending on Ran-GTP levels; also has a role in chromatin organization. NUP2 encodes a non-essential nuclear pore protein that has a central domain similar to those of Nsp1p and Nup1p. Transport of macromolecules between the nucleus and the cytoplasm of eukaryotic cells occurs through the nuclear pore complex (NPC), a large macromolecular complex that spans the nuclear envelope. The structure of the vertebrate NPC has been studied extensively; The yeast NPC shares several features with the vertebrate NPC, despite being smaller and less elaborate. Many yeast nuclear pore proteins, or nucleoporins, have been identified by a variety of genetic approaches. Nup2 mutants show genetic interactions with nsp1 and nup1 conditional alleles. Nup1p interacts with the nuclear import factor Srp1p and with the small GTPase Ran.
Gene ID: OPI9 Info: Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially overlaps the verified ORF VRP1/YLR337C.
Gene ID: PCL7 Info: Pho85p cyclin of the Pho80p subfamily, forms a functional kinase complex with Pho85p which phosphorylates Mmr1p and is regulated by Pho81p; involved in glycogen metabolism, expression is cell- cycle regulated. Involved in regulation of glycogen biosynthetic process, regulation of glycogen catabolic process.
Gene ID: PDR17 Info: Phosphatidylinositol transfer protein (PITP), downregulates Plb1p-mediated turnover of phosphatidylcholine, found in the cytosol and microsomes, homologous to Pdr16p, deletion affects phospholipid composition.
Gene ID: RHR2 Info: Constitutively expressed isoform of DL-glycerol-3-phosphatase; involved in glycerol biosynthesis, induced in response to both anaerobic and, along with the Hor2p/Gpp2p isoform, osmotic stress.
44 Gene ID: RIM 15 Info: Glucose-repressible protein kinase involved in signal transduction during cell proliferation in response to nutrients, specifically the establishment of stationary phase; identified as a regulator of IME2; substrate of Pho80p-Pho85p kinase.
Gene ID: RVS167 Info: Actin-associated protein, subunit of a complex (Rvs161p-Rvs167p) involved in regulation of actin cytoskeleton, endocytosis, and viability following starvation or osmotic stress; homolog of mammalian amphiphysin.
Gene ID: SDH2 Info: Iron-sulfur protein subunit of succinate dehydrogenase (Sdh1p, Sdh2p, Sdh3p, Sdh4p), which couples the oxidation of succinate to the transfer of electrons to ubiquinone. Involved in TCA cycle, aerobic respiration pathways.
Gene ID: SM I1 Info: Protein involved in the regulation of cell wall synthesis; proposed to be involved in coordinating cell cycle progression with cell wall integrity. Involved with 1,3-beta-glucan biosynthetic process, cell wall organization and biogenesis.
Gene ID: SOP4 Info: ER-membrane protein; suppressor of pma1-7, deletion of SOP4 slows down the export of wild-type Pma1p and Pma1-7 from the ER.
Gene ID: SPO22 Info: Meiosis-specific protein essential for chromosome synapsis, similar to phospholipase A2, involved in completion of nuclear divisions during meiosis; induced early in meiosis.
Gene ID: SQS1 Info: Protein of unknown function; overexpression antagonizes the suppression of splicing defects by spp382 mutants; green fluorescent protein (GFP)-fusion protein localizes to both the cytoplasm and the nucleus.
Gene ID: SRB8 Info: Subunit of the RNA polymerase II mediator complex; associates with core polymerase subunits to form the RNA polymerase II holoenzyme; essential for transcriptional regulation; involved in glucose repression.
Gene ID: STP22 Info: Component of the ESCRT-I complex, which is involved in ubiquitin-dependent sorting of proteins into the endosome; homologous to the mouse and human Tsg101 tumor susceptibility gene; mutants exhibit a Class E Vps phenotype.
Gene ID: TOP1 Info: Topoisomerase I, nuclear enzyme that relieves torsional strain in DNA by cleaving and re-sealing the phosphodiester backbone; relaxes both positively and negatively supercoiled DNA; functions in replication, transcription, and recombination. Topoisomerases catalyze the interconversion between topological states
45 of DNA by breaking and rejoining DNA strands. These changes in DNA topology are required during several cellular processes such as replication, transcription, recombination, and chromosome condensation. There are three classes of topoisomerases that are distinguished by substrate (IA, IB, II). Type I topoisomerases cleave one DNA strand, while Type II enzymes cleave a pair of complementary DNA strands. The type IB topoisomerases relax both positively and negatively supercoiled DNA; TOP1 encodes the type IB enzyme in yeast. Type IA topoisomerase, encoded by TOP3 in yeast, relaxes only negatively supercoiled DNA, and yeast topoisomerase II is encoded by the TOP2 gene.
Gene ID: TPS2 Info: Phosphatase subunit of the trehalose-6-phosphate synthase/phosphatase complex, which synthesizes the storage carbohydrate trehalose; expression is induced by stress conditions and repressed by the Ras- cAMP pathway. In Saccharomyces cerevisiae, trehalose is a major reserve carbohydrate involved in reponses to thermal, osmotic, oxidative, and ethanol stresses, as well as the suppression of denatured protein aggregation. Trehalose biosynthesis is a two-step process in which glucose 6-phosphate and UDP- glucose are converted by trehalose-6-phosphate synthase (TPS), encoded by TPS1, into alpha,alpha- trehalose 6-phosphate, which is then converted with water into trehalose and phosphate by trehalose-6- phosphate phosphatase (TPP), encoded by TPS2. The trehalose biosynthetic pathway can affect glycolysis in that one of its intermediates, trehalose-6-phosphate, inhibits hexokinase activity, which restricts the influx of sugars to glycolysis during the switch to fermentative metabolism. Tps1p and Tps2p are part of the alpha,alpha-trehalose-phosphate synthase complex with Tps3p and Tsl1p, regulatory proteins with partially overlapping functions, though some Tps1p appears to be present in the cell as a monomer. TPS1, TPS2, TPS3 and TSL1 are coinduced under stress conditions, and corepressed by the Ras-cAMP pathway. Deletion of TPS1 results in loss of both TPS activity and trehalose biosynthesis, whereas deletion of TPS2 results in temperature sensitivity and loss of TPP activity.
Gene ID: TRM 10 Info: tRNA methyltransferase, methylates the N-1 position of guanosine in tRNAs.
Gene ID: UBA3 Info: Protein that acts together with Ula1p to activate Rub1p before its conjugation to proteins (neddylation), which may play a role in protein degradation; GFP-fusion protein localizes to the cytoplasm in a punctate pattern.
Gene ID: UBI4 Info: Ubiquitin, becomes conjugated to proteins, marking them for selective degradation via the ubiquitin- 26S proteasome system; essential for the cellular stress response.
Gene ID: UPF3 Info: Component of the nonsense-mediated mRNA decay (NMD) pathway, along with Nam7p and Nmd2p; involved in decay of mRNA containing nonsense codons; involved in telomere maintenance.
Gene ID: VID28 Info: Protein involved in proteasome-dependent catabolite degradation of fructose-1,6-bisphosphatase (FBPase); localized to the nucleus and the cytoplasm.
Gene ID: W SC3 Info: Partially redundant sensor-transducer of the stress-activated PKC1-MPK1 signaling pathway involved in maintenance of cell wall integrity; involved in the response to heat shock and other stressors; regulates 1,3-beta-glucan synthesis.
46 Gene ID: YJL163C Info: Putative protein of unknown function.
Gene ID: YIL028W Info: Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data.
Gene ID: YIL029C Info: Putative protein of unknown function; deletion confers sensitivity to 4-(N-(S- glutathionylacetyl)amino) phenylarsenoxide.
Gene ID: YIL092W Info: Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the cytoplasm and to the nucleus.
Gene ID: YNL305C Info: Putative protein of unknown function; green fluorescent protein (GFP)-fusion protein localizes to the vacuole; YNL305C is not an essential gene.
Gene ID: YOL114C Info: Putative protein of unknown function with similarity to human ICT1 and prokaryotic factors that may function in translation termination; YOL114C is not an essential gene.
Gene ID: YPL066W Info: Hypothetical protein.
Gene ID: YPL102C Info: Dubious open reading frame, not conserved in closely related Saccharomyces species; deletion mutation enhances replication of Brome mosaic virus in S. cerevisiae, but this is likely due to effects on the overlapping gene ELP4.
47 APPENDIX G: Y101 screen; light colonies
Gene ID: BUD20 Info: Protein involved in bud-site selection; diploid mutants display a random budding pattern instead of the wild-type bipolar pattern.
Gene ID: CAF120 Info: Part of the evolutionarily-conserved CCR4-NOT transcriptional regulatory complex involved in controlling mRNA initiation, elongation, and degradation.
Gene ID: DFG5 Info: Putative mannosidase, essential glycosylphosphatidylinositol (GPI)-anchored membrane protein required for cell wall biogenesis in bud formation, involved in filamentous growth, homologous to Dcw1p.
Gene ID: RPS0B Info: Protein component of the small (40S) ribosomal subunit, nearly identical to Rps0Ap; required for maturation of 18S rRNA along with Rps0Ap; deletion of either RPS0 gene reduces growth rate, deletion of both genes is lethal.
Gene ID: RRD2 Info: Activator of the phosphotyrosyl phosphatase activity of PP2A; regulates G1 phase progression, the osmoresponse and microtubule dynamics; implicated in the spindle assembly check; subunit of the Tap42p- Pph21p-Rrd2p complex.
Gene ID: TCO89 Info: Subunit of TORC1 (Tor1p or Tor2p-Kog1p-Lst8p-Tco89p), a complex that regulates growth in response to nutrient availability; cooperates with Ssd1p in the maintenance of cellular integrity; deletion strains are hypersensitive to rapamycin. Involved in chitin- and beta-glucan-containing cell wall organization and biogenesis, glycerol metabolic process, regulation of cell growth, and response to salt stress.
Gene ID: YEH2 Info: Steryl ester hydrolase, catalyzes steryl ester hydrolysis at the plasma membrane; involved in sterol metabolism.
Gene ID: YLR112W Info: Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data.
48 REFERENCES
1. Chinn, M., Nokes, C., and Strobel, H.J. —Screening of Thermophilic Anaerobic Bacteria for Solid Substrate Cultivation on Lignocellulosic Substrates.“ 2006. Biotechnol. Prog. 22 : 53 œ 59.
2. —Ethanol Facts.“ The Renewable Fuels Association. 20 Apr 2007.
3. —Ethanol Fuel Blends,“ U.S. Department of Energy. 8 Sept 2006.
4. Farrell, A., Plevin, R., Turner, B., Jones, A., O‘Hare, M., and Kammen, D. —Ethanol can Contribute to Energy and Environmental Goals.“ 27 Jan 2007. Science 311: 506-508.
5. —How Ethanol is Made.“ The Renewable Fuels Association. 20 Apr 2007.
6. Hunsen, M. —Pyridinium chlorochromate catalyzed oxidation of alcohols to aldehydes and ketones“ 2005. Tetrahedron Letters 46: 1651-1653
7. Shapouri, H., Duffield, J., and Graboski, M. —Estimating the net Energy Balance of Corn Ethanol“ U.S. Department of Agriculture. Jul 1995. < http://www.ethanolrfa.org/objects/documents/83/aer721.pdf>
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