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Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.

Wang H, Wang F, Wang W, Yao X, Wei D, Cheng H, Deng Z - PLoS ONE (2014)

Bottom Line: In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0.Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress.The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

View Article: PubMed Central - PubMed

Affiliation: Innovation & Application Institute (IAI), Zhejiang Ocean University, Zhoushan, China; State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

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Effects of pH on the MTT-reducing activity of the E. coli cell membrane under acetate stress.E. coli BL21 (DE3) cells were investigated on 0, 50, 100 and 300 mM NaCl (A) or NaAc (B) in BYT-glycerol medium at pH 6.5, 7.0, 7.5 and 8.0.
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pone-0112777-g008: Effects of pH on the MTT-reducing activity of the E. coli cell membrane under acetate stress.E. coli BL21 (DE3) cells were investigated on 0, 50, 100 and 300 mM NaCl (A) or NaAc (B) in BYT-glycerol medium at pH 6.5, 7.0, 7.5 and 8.0.

Mentions: For BYT-glycerol medium containing no additional NaCl or NaAc, electron production by the E. coli cell membrane remained at a level of 760±35 MRU/(OD600 • mL) when the medium pH decreased from 7.5 to 6.5 (Figure 8A and 8B). For BYT-glycerol medium containing 50–300 mM NaCl, when the pH shifted from 7.5 to 6.5, the electron production decreased slightly, by a drop of less than 10% (Figure 8A). For example, at 300 mM NaCl, the activities were estimated to be 665±36 at pH 7.5 and 638±21 MRU/(OD600 • mL) at pH 6.5. In contrast, for the medium contained 50–300 mM NaAc, the shift from pH 7.5 to 6.5 caused a sharp drop in electron reduction, by approximately 55–75%, depending on acetate concentrations (Figure 8B). In the presence of 300 mM NaAc, the activities were estimated to be 706±30 MRU/(OD600 • mL) at pH 7.5 and 220±23 MRU/(OD600 • mL) at pH 6.5, a drop of 69%. Our results show that in the presence of acetate, a one-unit drop in pH from 7.5 to 6.5 causes a drop in bacterial MTT activity, although pH 7.5 and 6.5 are both suitable for cell growth in the BYT-glycerol medium without any acetate (Figure 2).


Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.

Wang H, Wang F, Wang W, Yao X, Wei D, Cheng H, Deng Z - PLoS ONE (2014)

Effects of pH on the MTT-reducing activity of the E. coli cell membrane under acetate stress.E. coli BL21 (DE3) cells were investigated on 0, 50, 100 and 300 mM NaCl (A) or NaAc (B) in BYT-glycerol medium at pH 6.5, 7.0, 7.5 and 8.0.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4234529&req=5

pone-0112777-g008: Effects of pH on the MTT-reducing activity of the E. coli cell membrane under acetate stress.E. coli BL21 (DE3) cells were investigated on 0, 50, 100 and 300 mM NaCl (A) or NaAc (B) in BYT-glycerol medium at pH 6.5, 7.0, 7.5 and 8.0.
Mentions: For BYT-glycerol medium containing no additional NaCl or NaAc, electron production by the E. coli cell membrane remained at a level of 760±35 MRU/(OD600 • mL) when the medium pH decreased from 7.5 to 6.5 (Figure 8A and 8B). For BYT-glycerol medium containing 50–300 mM NaCl, when the pH shifted from 7.5 to 6.5, the electron production decreased slightly, by a drop of less than 10% (Figure 8A). For example, at 300 mM NaCl, the activities were estimated to be 665±36 at pH 7.5 and 638±21 MRU/(OD600 • mL) at pH 6.5. In contrast, for the medium contained 50–300 mM NaAc, the shift from pH 7.5 to 6.5 caused a sharp drop in electron reduction, by approximately 55–75%, depending on acetate concentrations (Figure 8B). In the presence of 300 mM NaAc, the activities were estimated to be 706±30 MRU/(OD600 • mL) at pH 7.5 and 220±23 MRU/(OD600 • mL) at pH 6.5, a drop of 69%. Our results show that in the presence of acetate, a one-unit drop in pH from 7.5 to 6.5 causes a drop in bacterial MTT activity, although pH 7.5 and 6.5 are both suitable for cell growth in the BYT-glycerol medium without any acetate (Figure 2).

Bottom Line: In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0.Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress.The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

View Article: PubMed Central - PubMed

Affiliation: Innovation & Application Institute (IAI), Zhejiang Ocean University, Zhoushan, China; State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5-8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0 ± 0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.

Show MeSH
Related in: MedlinePlus