Synthesis of Ultra-Large Fibrous Proteins from Bacteria via a Looped-Translation System

Area of Science:

• Biotechnology and Synthetic Biology
• Protein Engineering
• Materials Science

Background:

• Recombinant production of high molecular weight fibrous proteins (e.g., silk, elastin, collagens) is difficult due to their large size and repetitive sequences.
• Existing methods struggle to efficiently synthesize these complex biomolecules, limiting their applications.

Purpose of the Study:

• To develop a novel platform for the efficient recombinant production of ultra-high molecular weight fibrous proteins.
• To overcome the limitations of current protein synthesis technologies for large, repetitive protein structures.

Main Methods:

• Utilized a circular RNA-based ribosome translation platform enabling iterative, 'looped' protein synthesis.
• Employed synonymous codon locker sequences and RNA circularization chaperones to enhance transcript circularization.
• Optimized translation using a ribosome traffic model to improve yields of fibrous proteins.

Main Results:

• Demonstrated successful synthesis of fibrous proteins across at least six classes, with products exceeding 3.8 MDa (greater than titin).
• Characterized synthesized polypeptides using electron microscopy, material fabrication, and mechanical analysis, confirming ultra-high molecular weight properties.
• Achieved secretion of fibrous proteins from *Escherichia coli* and *Bacillus subtilis* by coupling looped translation to programmed ribosomal frameshifting.

Conclusions:

• The circular RNA-based looped translation platform effectively enables the production of ultra-high molecular weight fibrous proteins.
• This technology offers a versatile tool for bioplastics and engineered living materials, overcoming previous production barriers.
• The developed genetic tools facilitate the export of synthesized fibrous proteins, expanding their potential applications.