Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli

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.