System biology approach to determine differences between acetic acid tolerant
S. cerevisiae 424A(LNH-ST) – AAR and original S. cerevisiae 424A(LNH-ST)
during glucose/xylose fermentation
Chia-Ling Wu1, 3, Nathan S. Mosier1, 3, Jiri Adamec2, Nancy Ho1, 4, Miroslav Sedlak1, 3
1 Laboratory
of Renewable Resources Engineering, 2Bindley Bioscience Center, 3 Department of Agricultural and Biological Engineering, 4 School of Chemical Engineering,
Purdue University, West Lafayette, IN 47907
Introduction
424A (LNH-ST) - AAR
424A (LNH-ST)
Stage 1
Stage 1
Glucose
Xylose
Xylitol
Glycerol
Acetic Acid
Ethanol
120
Bio-ethanol has gained much attention due to its economical benefits as
a renewable energy for replacing petroleum, as well as its potential to
reduce green house gas emissions. A significant resource for producing
renewable biofuels is lignocellulosic biomass. Lignocellulose is
composed of cellulose, hemicelluloses, and lignin. Several compounds
that can inhibit the fermentation step are released from the biomass
during pretreatment and hydrolysis, such as furan derivates, some weak
acids, and phenolic compounds. Developing an industrial, robust strain
that can be resistant to the inhibitors can improve the ethanol yield and
make the bio-ethanol industry more economical.
110
Glucose
Xylose
Xylitol
Glycerol
Acetic Acid
Ethanol
120
110
Stage 2
100
100
Stage 2
90
90
Stage 3
80
Stage 5
Stage 6
70
60
50
80
Concentration (g/L)
Concentration (g/L)
Figure 2.
Microarray signal intensities
of various gene that are
involved in glycolysis and
pentose phosphate pathway
from 424A (LNH-ST) – AAR
and 424A (LNH-ST) are
compared in this figure.
Results from stage 3 to 6
during fermentation are
shown. As expected, most of
the genes analyzed are not
changed significantly.
However, a few genes, such
as GND2, TKL2, ADH2, and
PDC6 seem to have
expression higher levels in
the original strain. On the
contrary, HXK1, GMP3, and
RKS1 are shown having
higher expression level in the
acetic acid-resistant strain.
130
130
70
60
Stage 5
50
Stage 3
Stage 6
40
40
Stage 4
20
10
10
0
0
0
6
12
18
24
30
36
42
48
54
60
66
72
78
84
90
0
96
6
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
Time (hr)
Time (hr)
Figure 1. Fermentation Profile of Saccharomyces Cerevisiae 424A (LNH-ST) and 424A
(LNH-ST) – AAR. Stage 1 to 6 represent six sugar fermentation phases. Stage 1:
beginning. Stage 2 and 3: middle of glucose consumption. Stage 4: transition from
glucose to xylose consumption. Stage 5: middle of xylose consumption. Stage 6: end.
Table 1
Stage 3
8-fold
Functional Category
Down
Up
3
1
2
3
Down
Up
3
3
1
7
1
3
1
1
10
1
3
1
1
8
4
13
4
40
4
4
2
1
1
3
3
2
20
1
1
7
3
4
5
1
1
6
1
2
1
3
2
6
2
3
2
28
3
5
2
27
12
3
1
1
7
2
1
1
6
1
2
1
4
1
1
0.8
0.6
0.4
0.2
0.0
2
1
2
3
4
5
6
6
47
3
4
10
2
1
10
4
1
28
20
12
4
1
1
6
3
3
1
3
9
3
2
1
5
10
78
11
24
89
4
21
76
9
17
49
167
125
4
3
2
3
4
5
6
7
4
2
0
4
3
2
1
0
1
2
3
4
5
FermentationStage
2
6
7
3
4
5
6
7
FermentationStage
Xylulose
5.5
424A (LNH-ST)
424A (LNH-ST) - AAR
Cellular Concentration ( umole/g cell dry weight)
5
2
2
11
18
50
10
30
131
9
18
50
158
8
1
6
424A (LNH-ST)
424A (LNH-ST) - AAR
3
2
3
424A (LNH-ST)
424A (LNH-ST) - AAR
Ribose-5-Phosphate
Gluconate-6-Phosphate
6
4
1
9
24
108
5
4
3
2
1
424A (LNH-ST)
424A (LNH-ST) - AAR
5.0
4.5
4.0
3.5
3.0
2.5
2.0
0
1
2
3
4
5
FermentationStage
6
7
1
2
3
4
5
FermentationStage
6
7
48
2
1
3-Phosphoglycerate
6
1
2
18
4
FermentationStage
7
1
2
18
9
2
1
5
2
10
5
22
5
6
1
1
1
1
7
1
10
FermentationStage
1
2
8
29
424A (LNH-ST)
424A (LNH-ST) - AAR
7
1
13
3
1
1
2
2
Cellular Concentration ( umole/g cell dry weight)
1.0
4
1
1
2
2
Down subtotal
1
7
7
424A (LNH-ST)
424A (LNH-ST) - AAR
Stage 6
5-fold
Up
Glycerol-3-Phosphate
Fructose-6-Phosphate
1.4
Stage 5
6-fold
Down
Total
1.2
Stage 4
6-fold
Up
amino acid metabolism
apoptosis
cell cycle
cell wall and membrane
protein
cellular metabolism
detoxification
DNA replication
DNA repair
energy reserve metabolism
fatty acid metabolism
nucleobase\side\tide and
nucleic acid metabolism
protein folding
proteolysis
regulation of transcription
ribosome biogenesis and
assembly
RNA processing
signal transduction
sporulation
steroid and lipid
biosynthesis
stress response
sugar metabolism
transcription
translation
transport genes
unknown
Subtotal
Cellular Concentration ( umole/g cell dry weight)
Yeast strains:
• S. cerevisiae 424A (LNH-ST)
• S. cerevisiae 424A (LNH-ST) – AAR (acetic acid-resistant), developed
and funded by US Department of Energy Biomass Program, Contract
GO17059-16649.
Fermentation:
• Medium: 110ml YEP + 120 g/L glucose + 85 g/L xylose + 13 g/L
galactose + 10 g/L acetic acid, adjusted with ammonium hydroxide to
pH 6.
• Microaerobic fermentation at 28 oC with 200 rpm in 300 ml side-arm
flask.
Fermentation Profile
• HPLC (BioRad HPX 87H and HPX 87P)
Microarray analysis
• 2 ml of fermentation samples with 3 technical replicates were
collected at time = 3 hr, 6 hr, 12 hr (for 424A(LNH-ST) strain), 14 hr
(for 424A(LNH-ST)-RA strain), 30 hr, and 72 hr, naming stage 1 to 6
respectively. The total RNA was extracted using hot-phenol protocol.
• YeastGenome 2.0 chip (Affymatrix) was used for microarray analysis,
and the data was analyzed using GeneSpring software (X11).
Mass spectrometry analysis
• 5 ml of fermentation samples with 2 technical replicates were
collected at time = 3 hr, 6 hr, 12 hr (for 424A(LNH-ST) strain), 14 hr
(for 424A(LNH-ST)-RA strain), 30 hr, and 72 hr (stage 1 – 6). The
samples were immediately cooled to -45 oC and extracted in 5 ml of
75% ethanol in 95 oC.
• The concentration of the metabolites were measured by LC/MS/MS
and GC/MS and analyzed by using Masshunter software (Agilent).
20
Cellular Concentration ( umole/g cell dry weight)
Material and Methods
Stage 4
Cellular Concentration ( umole/g cell dry weight)
Our lab has genetically engineered a Saccharomyces cerevisiae strain
424A (LNH-ST) that can efficiently co-ferment glucose and xylose. Our
previous work has shown that acetic acid under process relevant
conditions does not significantly affect glucose fermentation. However
xylose utilization is significantly affected, especially at low pH
environment (pH < 5.5) and high acetic acid concentration (> 10 g/L).
Therefore, we have developed an acetic acid-resistant yeast strain that
is alternated from original 424A (LNH-ST) strain, 424A (LNH-ST) – AAR
(acetic acid-resistant) Small-scale fermentation (110 ml YEP containing
120 g/L glucose + 85 g/L xylose + 1.3 g/L galactose, with 10 g/L acetic
acid) at starting pH 6.0 has shown that the new strain can utilize more
than double of xylose compared to the original strain (64.4% to 22.5%).
In this study, a systems-biology approach, including transcriptomic and
metabolomic analyses, were completed to understand gene expression
and metabolic fluxes in this improved strain as compared to the original
strain.
30
30
Cellular Concentration ( umole/g cell dry weight)
Acetic acid is one of major inhibitors in lignocellulosic fermentation. Its
concentration can range from 1 – 15 g/L depending on the source of
biomass. It especially has strong inhibitory effect at pH below 5.0. The
acetic acid can enter the cells by diffusion in the form of acetic acid
(protonated) and then disassociates into proton and anion inside the
cell which has neutral pH. This results in decreasing the cytosolic pH and
the accumulation of anions inside the cells. Eventually it becomes toxic
to yeast and inhibits cellular function or even leads to apoptosis.
Figure 3.
Cellular
concentration
of various
metabolites
involved in
glycolysis and
pentose
phosphate
pathway.
20
7
14
1
68
162
181
Table 1. Relative
gene expression
level of 424A (LNHST) – AAR compared
to 424A (LNH-ST)
from stage 3 to 6
during fermentation,
and the functional
categories the genes
belong to. The
categories with
three highest gene
numbers are
highlighted.
Results and Discussion
• The new strain Saccharomyces cerevisiae 424A (LNH-ST) – AAR can ferment xylose more efficiently, both in total xylose consumed and xylose
consumption rate, compared to the original strain Saccharomyces cerevisiae 424A during glucose/xylose co-fermentation. The ethanol yield is also
higher when the new strain is used. However, in both strains, galactose is not utilized (data not shown) under our experimental conditions (Figure 1)
• Transcriptomics:
 Relative gene expression level of 424A (LNH-ST) – AAR compared to 424A (LNH-ST) from the six stages during fermentation are analyzed, and the
genes are categorized according to their functions. Most of these up- or down-regulated genes are still unknown proteins or putative proteins. Also,
many genes that are involved in metabolism or transport and in steroid/lipid/membrane biosynthesis are also up- or down-regulated in 424A (LNHST) – AAR. (Table 1)
 Microarray signal intensities of various gene that are involved in glycolysis and pentose phosphate pathway from 424A (LNH-ST) – AAR and 424A
(LNH-ST) are compared. As expected, most of the genes analyzed are not changed significantly. However, a few genes, such as GND2, TKL2, ADH2,
and PDC6 seem to have higher expression levels in the original strain. On the contrary, HXK1, GMP3, and RKS1 are shown having higher expression
levels in the acetic acid-resistant strain. (Figure 2)
• Metabolomics:
 Cellular concentration of 27 metabolites involved in glycolysis, pentose phosphate pathway, cellular energy and reduction/oxidation are measured
in both 424A (LNH-ST) and 424A (LNH-ST) – AAR strains.
 For most of the metabolites analyzed, their cellular concentrations and the changes in concentration throughout the course of fermentation behave
similarly compare one strain to the other (data not shown).
 However, a few metabolites stood out. The cellular concentrations of gluconate-3-phosphate, ribose-5-phosphate, and xylulose from the pentose
phosphate pathway and glycerol-3-phosphate derived from glycolysis are higher in the acetic acid-resistant strain, whereas the concentrations of
fructose-6-phosphate and 3-phosphoglycerate from glycolysis are higher in the original strain. (Figure 3)
 All metabolites analyzed decrease in concentration once the glucose is depleted from the medium.
** This work is supported by the US Department of Energy Biomass Program, Contract GO17059-16649