1. Investigation of the impact of dissolved oxygen on the protein profiles of hyaluronic acid- producing Streptococcus zooepidemicus by gel-based proteomics Ting-Feng Wu 1,2, Wei-Chih Huang 3, Yi-Chun Chen 1, Li-Chen Huang 1 Yeou-Guang Tsay 4 * and Chun-Sheng Chang 1,2 * 1 Department of Biotechnology and 2 Research Center of Biotechnology, Southern Taiwan University, Tainan, Taiwan 3 Department of Chemical Engineering, National Cheng-Kung University, Tainan, Taiwan 4 Institute 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 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 HA synthesis in S. zooepidemicus, two-dimensional gel electrophoresis (2-DE) was used to compare the 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 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 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 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. Introduction In recent years, HA from microbial resources is gaining more attention due to the risk of cross- species viral infection caused by animal HA resources. HA fermentation has been mostly performed with Streptococci spp. where HA is a capsular biopolymer shedding to the medium [1]. It has been known that the HA yield can be greatly enhanced under aeration condition relative to anaerobic culture. Using the HA fermentation by S. zooepidemicus ATCC39920 strain. Huang et al. (2006) [2] found that under homogeneous agitation, aeration has no effect on cell growth but markedly enhances the HA yield with the threshold for HA synthesis set at the dissolved oxygen (DO) level of 5% air saturation. Although many studies reported that the HA productivity is increased under aerobic conditions, the exact mechanism of this stimulant effect has not been elucidated. In this study, gel-based proteomics was exploited to explore the possible mechanism underlying the augmentation of HA productivity by oxygen. Materials and Methods Protein Extraction Samples were milled with liquid nitrogen and lysed in lysis buffer. Lysis was allowed to proceed for 1 h at room temperature under continuous shaking. The lysates were then centrifuged at 12,000 rpm for 30 min and supernatants were stored at -80 ℃ until use. Two-dimensional Gel Electrophoresis and and Quantitative Analysis The immobiline pH gradient strips (pH4-7, 18cm) were rehydrated for 16h with the rehydration buffer containing 60μg protein. The proteins were then focused at 200V, 500V, 1000V, 5000V, 8000V with a total of 32,000 volt-hours.The gel were loaded onto the top of 12.5% polyacrylamide gel and sealed with 0.5% agarose. To look for proteins showing disparity in expression, the proteomes were analyzed by PDQuest (Bio-Rad) and further analyzed by t-test. Results and discussions 44.55.16.67 kDa 75 50 37 25 20 3 1 2 7 6 9 4 5 8 44.55.16.67 kDa 75 50 37 25 20 4 5 3 2 1 7 6 9 8 pH a) O 2 (-): anaerobic; O 2 (+): aerobic Table 1. Summary of the differentially expressed proteins in S. zooepidemicus cultivated under aerobic condition Figure 2. The proteome maps of aerobically and anaerobically cultivated S. zooepidemicus. (A) Anaerobically cultured S. zooepidemicus. (B) Aerobically grown bacteria. Proteins were separated by IEF as first dimension, using 18 cm pH 4–7 gel strip, and by 12.5% SDSPAGE as second dimension. After electrophoresis, the gels were fixed and the proteins were detected by silver staining. The gel pair is the representative gel of nine replica gels collected from three independent fermentations. The differentially expressed protein spots were recorded which were 2-fold or above in magnitude as observed in all nine replica gels and statistically significant (P  0.05) and were shown by the arrows and numbers. 9 up-regulated proteins were observed at  10 % DO. Average normalized volumes (% vol.), fold-variations, the statistical results, experimental pIs, molecular weights and protein identity were presented in table 1. 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). 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. Spot number Protein I. D. O 2 (-) (%vol) mean ± S.D. O 2 (+) (%vol) mean ± S.D. P-valueFold difference Experimental Mr(kDa)/pI Swiss-Prot database accession no. Mr / pI Coverage (%) 1Putative dihydrolipoamide Dehydrogenase (Streptococcus pyogenes M1 GAS) 0.38±0.051.62±0.08 P  0.01 +4 58/4.98 QX99ZX5 59/4.93 48 2Putative dihydrolipoamide dehydrogenase (Streptococcus pyogenes M1 GAS) 0.23±0.041.77±0.15 P  0.01 +7 58/ 4.93 QX99ZX5 59/4.93 40.7 3Putative dihydrolipoamide dehydrogenase (Streptococcus pyogenes M1 GAS) 0.19±0.031.81±0.16 P  0.01 +9 58/ 4.90 QX99ZX5 59/4.93 43.1 4UDP-N-acetyl- glucosamine pyrophosphorylase (Streptococcusequi subsp. zooepidemicus) 0.25±0.021.75±0.07 P  0.01 +7 47/5.82 Q8GQP7 49/5.55 30.2 5Putative dihydrolipoamide S-acetyltransferase (Streptococcus pyogenes M1 GAS) 0.27±0.081.73±0.12 P  0.01 +6 47/5.62 Q99ZX6 49/5.21 18.9 6Putative acetoin dehydrogenase  chain (Streptococcus pyogenes M1 GAS) 0.34±0.031.66±0.10 P  0.01 +4 35/ 4.87 Q99ZX7 34/4.71 60.8 7Putative acetoin dehydrogenase  chain (Streptococcus pyogenes M1 GAS) 0.24±0.031.76±0.18 P  0.01 +7 35/4.82 Q99ZX7 34/4.71 17.7 8- 0.27±0.081.73±0.16 P  0.01 +6 15 /4.99-- 9Putative acetoin dehydrogenase  chain (Streptococcus pyogenes M1 GAS) 0.34±0.051.66±0.17 P  0.01 +5 37 /5.11 Q99ZX8 35.4/5.12 7.53 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 over- expressed 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 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 that acetoin might provide more acetyl-CoA for HA production. Figure 4. The biosynthetic pathway of hyaluronic acid. The upward arrows represent the up-regulated proteins under aerobic condition. References 1.Mausolf, A., Jungmann, J., Robenek, H., Prehm, P. Biochem. J. 1990, 267,191- 196. 2.Huang, W.C., Chen, S.J., Chen, T. L. Biochem. Eng. J., 2006, 32, 239-243.