Improving extracellular protein production in Escherichia coli by overexpressing D,D-carboxypeptidase to perturb peptidoglycan network synthesis and structure

Abstract

Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli, for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB, and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB, respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA, and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB. Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli. This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli.

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Funding

This work was funded by National Natural Science Foundation of China (21406089), Natural Science Foundation of Jiangsu Province (BK20140152), the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (KLIB-KF201509), the Open Project Program of the Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, China (KLCCB-KF201607, KLCCB-KF201802), 111 Project (111-2-06), and Postgraduate Education Research and Practice Project of Jiangnan University (YJSJG2017004).

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H.Y. designed the research; H.Y., J.H., X.L., L.W., and F.W. performed the research; X.L., W.S., F.W., and X.C. analysed the data; L.L., W.H., and H.Y. wrote the paper.

Corresponding author

Correspondence to Haiquan Yang.

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This article is in compliance with ethical standards, and does not contain any studies with animals performed or human participants.

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The authors declare that they have no competing interest.

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Yang, H., Hu, J., Lu, X. et al. Improving extracellular protein production in Escherichia coli by overexpressing D,D-carboxypeptidase to perturb peptidoglycan network synthesis and structure. Appl Microbiol Biotechnol 103, 793–806 (2019). https://doi.org/10.1007/s00253-018-9510-7

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Keywords

  • Extracellular protein production
  • Overexpression
  • D,D-carboxypeptidase
  • Peptidoglycan structure
  • Membrane permeability
  • Escherichia coli