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The Multifaceted Benefits of Protein Co-expression in Escherichia coli
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Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Zi-Kai Wang1,2, Jin-Song Gong1,3, Chang Su1,3

  • 1Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, P.R. China.

ACS Synthetic Biology
|September 12, 2024
PubMed
Summary

This study optimized an integrated gene expression system in Escherichia coli (E. coli) for industrial applications. The developed system, CEIES_Ecoli, enhances recombinant protein production without antibiotics, offering a stable and efficient biomanufacturing tool.

Keywords:
E. coligenome editingintegrated expressionrecombinant protein expressionsynthetic biology

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Area of Science:

  • Synthetic biology
  • Metabolic engineering
  • Genomics

Background:

  • Plasmid-mediated microbial fermentation presents challenges like growth burden and instability.
  • Genomic integration offers a more stable alternative for heterologous gene expression in industrial strains.
  • Advancements in synthetic biology and genome editing facilitate gene integration.

Purpose of the Study:

  • To systematically optimize an integrated gene expression system in Escherichia coli (E. coli).
  • To enhance the expression of exogenous genes at the genome level for biomanufacturing.
  • To develop a stable and efficient expression platform without reliance on antibiotics or inducers.

Main Methods:

  • Screening of 18 integration sites in the E. coli BL21 (DE3) genome, identifying slmA for highest expression activity.
  • Characterization of 16 endogenous promoters, combining them with the T7 promoter to create Plpp-T7, a stronger constitutive promoter.
  • Overexpression of T7 RNA polymerase at the chassis cell level to further enhance the T7 expression system.

Main Results:

  • The optimized constitutive efficient integrated expression system (CEIES_Ecoli) demonstrated a 2-fold increase in GFP expression compared to the pET3b plasmid.
  • CEIES_Ecoli achieved stable and efficient expression of nitrilase and hyaluronidase, with activities comparable to plasmid levels.
  • The system provides robust gene expression without the need for antibiotics or inducers.

Conclusions:

  • CEIES_Ecoli offers a stable and efficient platform for integrated gene expression in E. coli.
  • This system is a valuable tool for synthetic biology, enzyme engineering, and biomanufacturing.
  • The developed system overcomes limitations associated with plasmid-based expression systems.