# Enhancing Escherichia coli production of material proteins using circular mRNAs

## TL;DR

This paper shows how circular mRNA can improve the production of complex proteins in bacteria, leading to higher yields and better quality materials.

## Contribution

The novel contribution is the development of a circular mRNA system that enhances both mRNA stability and protein yield in Escherichia coli.

## Key findings

- Circular mRNA increased green fluorescent protein expression by 1.5-fold compared to linear mRNA.
- The system improved the expression of various material proteins, including spider silk and amyloid repeats, by up to 2.5-fold.
- The circular mRNA system also enhanced plasmid stability in E. coli.

## Abstract

Industrial bioproduction of proteins, particularly protein-based materials (PBMs) like spider silk and elastin proteins, is rapidly expanding. PBMs often have high molecular weights and are highly repetitive, transcribed from long and repetitive mRNAs that are prone to degradation in microbial hosts. As a result, recombinant expression of PBMs often has low protein yields. In this study, we engineered a circular mRNA expression system to enhance mRNA stability and protein expression. The system uses self-cleaving ribozymes to form circular mRNA structures and a pair of insulation RNA loops to improve protein translation. When tested using a green fluorescent protein (GFP) reporter, the engineered circular mRNA enhanced GFP expression by 1.5-fold compared to expression from a linear construct. mRNA circularization was further confirmed using reverse transcription followed by DNA amplification and sequencing. We also demonstrate the effectiveness of circular mRNA in enhancing the expression of various material proteins, including a 96-mer repeat of Nephila clavipes dragline silk, a titin repeat, a mussel foot protein oligomer, and an silk-amyloid repeat, resulting in up to 2.5-fold increase in protein yield. Additionally, the circular mRNA system also improved the stability of the PBM-encoding plasmid. Overall, the circular RNA expression system enhances both the expression level and plasmid stability and is suitable for various protein production applications.

Industrial bioproduction of complex proteins is limited by unstable expression. Long and repetitive proteins have unstable expression and often yield truncated products that will change the properties of the final materials. We show that by using a self-circularizing mRNA system, the expression is stabilized to not only increase yields but also prevent truncated products. The ability to produce full-length proteins consistently and control their size offers precise control over protein properties, making it highly relevant for products with specific mechanical properties. The study showcases the potential for scaling up protein production in industrial bioreactors under challenging conditions. The findings contribute to synthetic biology tools and offer new avenues for manufacturing bioproducts at an industrial scale.