Improving the expression of recombinant proteins in E. coli BL21 (DE3) under acetate stress: an alkaline pH shift approach.
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.
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.
Hengwei
H
Wang
New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Fengqing
F
Wang
New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Wei
W
Wang
New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Xueling
X
Yao
New World Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Dongzhi
D
Wei
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Hairong
H
Cheng
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Zixin
Z
Deng
D000085:Acetates / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D050296:Microbial Viability / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013312:Stress, Physiological
D000085:Acetates; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins