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Downregulation of T7 RNA polymerase transcription enhances pET-based recombinant protein production via suppression of Escherichia coli BL21 (DE3) programmed cell death
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  • Xiao-Man Sun,
  • Zi-Xu Zhang,
  • Ling-Ru Wang,
  • Jing-Gang Wang,
  • Yan Liang,
  • Hai-Feng Yang,
  • Rongsheng Tao,
  • Yu Jiang,
  • Jun-Jie Yang,
  • Sheng Yang
Xiao-Man Sun
Nanjing Normal University
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Zi-Xu Zhang
Nanjing Normal University
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Ling-Ru Wang
Nanjing Normal University
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Jing-Gang Wang
HuaRui Biotechnology Company
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Yan Liang
HuaRui Biotechnology Company
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Hai-Feng Yang
Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences
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Rongsheng Tao
Shanghai Institutes for Biological Sciences
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Yu Jiang
Shanghai TaoYuSheng Biotechnology Co., Ltd
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Jun-Jie Yang
Shanghai Institutes for Biological Sciences Chinese Academy of Sciences
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Sheng Yang
CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology
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Abstract

E. coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, serious autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. The membrane structure and mRNA expression analysis showed that cells suffered programmed cell death (PCD) during autolysis period. However, blocking three known PCD pathway in BL21 (DE3) cannot alleviate autolysis completely. Furthermore, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in a host of BL21 (DE3-lac1G), which successfully withstand the PCD of the host. The GDH activity at 43h was greatly increased from 37.5 U/mL to 452.0 U/mL. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/mL/h at 43h, compared to the 3 U/mL/h of BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of other 10 enzymes. The engineered E. coli strain in the study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows potential industrial applications.

Peer review status:ACCEPTED

24 Jun 2020Submitted to Biotechnology and Bioengineering
24 Jun 2020Submission Checks Completed
24 Jun 2020Assigned to Editor
28 Jun 2020Reviewer(s) Assigned
16 Jul 2020Editorial Decision: Revise Major
16 Jul 2020Review(s) Completed, Editorial Evaluation Pending
25 Aug 20201st Revision Received
26 Aug 2020Submission Checks Completed
26 Aug 2020Assigned to Editor
28 Aug 2020Reviewer(s) Assigned
30 Aug 2020Review(s) Completed, Editorial Evaluation Pending
30 Aug 2020Editorial Decision: Accept