Investigation of the impact of dissolved oxygen on the protein profiles of hyaluronic acidproducing Streptococcus zooepidemicus by gel-based proteomics
1,2
Wu ,
3
Huang ,
1
Chen ,
1
Huang
4
Tsay *
Ting-Feng
Wei-Chih
Yi-Chun
Li-Chen
Yeou-Guang
and
1,2
Chun-Sheng Chang *
1Department of Biotechnology and 2Research Center of Biotechnology, Southern Taiwan University,
Tainan, Taiwan
3Department of Chemical Engineering, National Cheng-Kung University, Tainan, Taiwan
4Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan
Abstract
Hyaluronic acid (HA) is a linear and negatively charged polysaccharide regularly used in medicine
and cosmetics. Recently Streptococcus zooepidemicus has been exploited in the fermentation industry kDa
to produce HA. Many studies showed that higher amounts of HA were produced under aerobic
condition compared anaerobic environment. In order to explore the effect of oxygen on the HA75
synthesis in S. zooepidemicus, two-dimensional gel electrophoresis (2-DE) was used to compare the 50
protein profiles of aerobically and anaerobically fermented bacteria to identify proteins, which might
be associated with the influence of oxygen on the HA synthesis. Totally nine pairs of 2-DE gels 37
collected from three batches were compared and nine over-expressed proteins were observed in
aerobically fermented bacteria. These proteins were identified by liquid chromatography/tandem mass
25
spectrometry as five proteins were involved in acetoin dissimilation, the central carbon metabolism
and the HA anabolic pathway, implicating that oxygen might augment the expression of genes that are 20
involved in energy metabolism to enhance the amount of acetyl-CoA as such more acetyl-CoA can be
divergent from the central carbon metabolism to replenish acetyl-CoA for the HA synthesis.
4
4.5
5.1
6.6
7
pH
4
4.5
5.1
6.6
7
kDa
75
4
3
5
21
3 21
50
5 4
9
9
7 6
37
76
25
20
8
8
Introduction
In recent years, HA from microbial resources is gaining more attention due to the risk of crossspecies viral infection caused by animal HA resources. HA fermentation has been mostly performed
Figure 2. The proteome maps of aerobically and anaerobically cultivated S. zooepidemicus. (A)
with Streptococci spp. where HA is a capsular biopolymer shedding to the medium [1]. It has been
Anaerobically cultured S. zooepidemicus. (B) Aerobically grown bacteria. Proteins were
known that the HA yield can be greatly enhanced under aeration condition relative to anaerobic culture. separated by IEF as first dimension, using 18 cm pH 4–7 gel strip, and by 12.5% SDSPAGE as
Using the HA fermentation by S. zooepidemicus ATCC39920 strain. Huang et al. (2006) [2] found
second dimension. After electrophoresis, the gels were fixed and the proteins were detected by
that under homogeneous agitation, aeration has no effect on cell growth but markedly enhances the HA silver staining. The gel pair is the representative gel of nine replica gels collected from three
yield with the threshold for HA synthesis set at the dissolved oxygen (DO) level of 5% air saturation.
independent fermentations. The differentially expressed protein spots were recorded which were
Although many studies reported that the HA productivity is increased under aerobic conditions, the
2-fold or above in magnitude as observed in all nine replica gels and statistically significant (P
exact mechanism of this stimulant effect has not been elucidated. In this study, gel-based proteomics
0.05) and were shown by the arrows and numbers. 9 up-regulated proteins were observed at 10
was exploited to explore the possible mechanism underlying the augmentation of HA productivity by
% DO. Average normalized volumes (% vol.), fold-variations, the statistical results, experimental
oxygen.
pIs, molecular weights and protein identity were presented in table 1.
Materials and Methods
Table 1. Summary of the differentially expressed proteins in S. zooepidemicus cultivated under aerobic condition
Protein Extraction
Protein I. D.
Experimental
Swiss-Prot
Coverage (%)
O2 (-) (%vol) O2 (+) (%vol) P-value
Fold
Samples were milled with liquid nitrogen and lysed in lysis buffer. Lysis was allowed to proceed Spot number
database
mean ± S.D. mean ± S.D.
difference Mr(kDa)/pI
accession no.
for 1 h at room temperature under continuous shaking. The lysates were then centrifuged at 12,000
Mr / pI
rpm for 30 min and supernatants were stored at -80℃ until use.
1
Putative
58/4.98
QX99ZX5
48
0.38±0.05
1.62±0.08
+4
P 0.01
Two-dimensional Gel Electrophoresis and and Quantitative Analysis
dihydrolipoamide
59/4.93
Dehydrogenase
The immobiline pH gradient strips (pH4-7, 18cm) were rehydrated for 16h with the rehydration
(Streptococcus
buffer containing 60μg protein. The proteins were then focused at 200V, 500V, 1000V, 5000V,
pyogenes M1 GAS)
2
Putative
58/ 4.93
QX99ZX5
40.7
8000V with a total of 32,000 volt-hours.The gel were loaded onto the top of 12.5% polyacrylamide
0.23±0.04
1.77±0.15
+7
P  0.01
dihydrolipoamide
59/4.93
gel and sealed with 0.5% agarose. To look for proteins showing disparity in expression, the
dehydrogenase
(Streptococcus
proteomes were analyzed by PDQuest (Bio-Rad) and further analyzed by t-test.
a) O2 (-): anaerobic; O2 (+): aerobic
pyogenes M1 GAS)
Cell biomass & HA concentration (g/L)
Results and discussions
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10
Cell biomass & HAconcentration (g/L)
Time (hrs)
6
5
Figure 1. The Effect of aeration on the
HA fermentation. The aerobic culture
was conducted with an aeration rate of 1
vvm with saturated dissolved oxygen (DO)
kept over 10 %. Prior to anaerobic
fermentation,
dissolved
oxygen
level was adjusted to 0 % air saturation by
purging nitrogen gas into the fermentor
and during anaerobic fermentation
nitrogen gas was shut down under stirring.
Cell biomass: (●), aerobic and (■),
anaerobic; HA concentration: (○), aerobic
and (□), anaerobic. The biomass grown at
anaerobic cultivation was comparable to
that at aerobic environment (Figure 1).
12 However, the HA yield at aerobic
cultivation was much higher than that at
anaerobic fermentation [Figure 1; 0.76 (g
HA/g biomass) v.s. 0.56 (g HA/g biomass),
P= 0.03 at 11th hour], suggesting that the
anabolic pathway for HA might be
affected by DO content in the media.
4
3
2
1
0
0
2
4
6
Time (hr)
8
10
S. zooepidemicus. The results of acetoin
addition demonstrated that the biomass with
acetoin was comparable to that without
acetoin but the HA yield was increased from
0.76 g HA/g biomass to 0.86 g HA/g biomass
with P= 0.005 (Figure 3). Acetoin was
assimilated with HA production, suggesting
12 that acetoin might provide more acetyl-CoA
for HA production.
Figure 3. The impact of acetoin on the HA fermentation. The aerobic culture was
performed as in fig. 1. The 5 g/L acetoin was added at 6.5th hour. Cell biomass: (●),
no acetoin and (■), 5 g/L acetoin; HA concentration: (○), no acetoin and (□), 5
g/L acetoin; (▼), acetoin. In this study, the proteins associated with the acetoin
dissimilation and the HA anabolic pathway (Figure 4) were observed to be overexpressed in DO-exposed S. zooepidemicus. Therefore, 5 g/L acetoin was
incorporated in the media at mid-log phase (6.5 hours) of aerobically grown
3
Putative
dihydrolipoamide
dehydrogenase
(Streptococcus
pyogenes M1 GAS)
0.19±0.03
1.81±0.16
P  0.01
+9
58/ 4.90
QX99ZX5
59/4.93
43.1
4
UDP-N-acetylglucosamine
pyrophosphorylase
(Streptococcusequi
subsp.
zooepidemicus)
0.25±0.02
1.75±0.07
P  0.01
+7
47/5.82
Q8GQP7
49/5.55
30.2
5
Putative
dihydrolipoamide
S-acetyltransferase
(Streptococcus
pyogenes M1 GAS)
0.27±0.08
1.73±0.12
P  0.01
+6
47/5.62
Q99ZX6
49/5.21
18.9
6
Putative acetoin
dehydrogenase 
chain (Streptococcus
pyogenes M1 GAS)
0.34±0.03
1.66±0.10
P  0.01
+4
35/ 4.87
Q99ZX7
34/4.71
60.8
7
Putative acetoin
dehydrogenase 
chain (Streptococcus
pyogenes M1 GAS)
0.24±0.03
1.76±0.18
P  0.01
+7
35/4.82
Q99ZX7
34/4.71
17.7
8
-
P  0.01
P 0.01
+6
+5
-
-
Putative acetoin
dehydrogenase 
chain (Streptococcus
pyogenes M1 GAS)
1.73±0.16
1.66±0.17
15 /4.99
9
0.27±0.08
0.34±0.05
37 /5.11
Q99ZX8
35.4/5.12
7.53
Figure 4. The biosynthetic
pathway of hyaluronic acid.
The upward arrows represent
the up-regulated proteins
under aerobic condition.
References
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Biochem. J. 1990, 267,191196.
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Chen, T. L. Biochem. Eng.
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