Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth...

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Escherichia coli (E. coli) offers advantages for extracellular protein expression, including lower pyrogen levels and simpler purification.
• Current extracellular expression systems in E. coli, particularly leaky mutants, suffer from low protein yields compared to intracellular systems, necessitating improvement.

Purpose of the Study:

• To construct, characterize, and elucidate the mechanism of enhanced extracellular expression in Escherichia coli.
• To improve extracellular protein yields for heterologous protein production in E. coli.

Main Methods:

• Systematic elucidation of cell envelope protein effects on cell growth and extracellular expression using modified CRISPR/Cas9 genome editing.
• Utilized a FlAsH labeling assay to assess protein localization and secretion.
• Screened multiple gene deletions to identify optimal strains for enhanced extracellular expression.

Main Results:

• Achieved high extracellular yields of heterologous proteins of various sizes through targeted gene mutations.
• Demonstrated that enhanced extracellular secretion is linked to the derepression of translation and translocation processes.
• Developed universal methods applicable to designing extracellular expression systems for diverse genes.

Conclusions:

• The study presents a successful strategy for significantly enhancing extracellular protein expression in Escherichia coli.
• The findings provide insights into the mechanisms governing protein secretion and offer a foundation for engineering more efficient prokaryotic cell factories.
• This work facilitates the construction of engineered E. coli strains for the efficient extracellular production of valuable heterologous proteins.