Enhancing Escherichia coli production of material proteins using circular mRNAs

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.