A new synthetic biology technology has been developed that transforms linear messenger RNA (mRNA) into a circular form inside microbial cells, significantly boosting protein production. The technology, named CRESEnT (Circular RNA Expression for Stable and Enhanced Translation), was developed by a research team led by Professor Sang Woo Seo from the Department of Chemical and Biological Engineering at Seoul National University. In experiments, CRESEnT increased the production of fluorescent proteins by up to 5.95 times compared to a control group, while also raising intracellular mRNA levels by 3.95 times. Additionally, the amount of protein produced per mRNA molecule increased by 1.51 times. The method has been successfully tested in several microorganisms, including Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum, and has been used to produce valuable compounds like flaviolin, itaconic acid, lycopene, and violacein. The findings were published in the journal Nucleic Acids Research. Microorganisms are often used as "cell factories" to produce a wide range of products, including biofuels, chemicals, and pharmaceuticals. The efficiency of these cell factories depends on the ability of the cells to produce the necessary enzymes in sufficient quantities. Traditionally, efforts to boost gene expression have focused on elements like promoters and ribosome-binding sites (RBSs), which control how genes are read and translated into proteins. However, mRNA—the molecule that carries genetic instructions for protein production—is inherently unstable in bacteria. Unlike in eukaryotic cells, bacterial mRNA lacks protective structures like a cap or poly(A) tail, making it vulnerable to degradation by enzymes called RNases. This instability results in mRNA lifespans of only a few minutes, which is useful for microorganisms in natural environments but can hinder productivity in controlled industrial settings where continuous production is needed. To address this issue, the research team altered the structure of mRNA by converting it from a linear form—resembling a thread with two open ends—into a circular shape. Circular mRNA is less accessible to enzymes that break it down, thereby increasing its stability. The researchers used a naturally occurring RNA structure called an intron, which can self-splice and reconnect segments of itself. By reversing the order of the intron's segments, they designed the mRNA so that its ends would join inside the cell, forming a circular structure without requiring additional enzymes or processing outside the cell. They also identified two key design principles that improve the efficiency of CRESEnT. One involves the length of the untranslated regions (UTRs), which are non-coding parts of mRNA that affect its stability. The second principle involves adding short DNA sequences that help the intron fragments bind more efficiently, like the teeth of a zipper. When applied to the optimized CRESEnT system, the results were striking. Protein production increased by up to 5.95 times, and mRNA levels rose by 3.95 times compared to the control group. In experiments where transcription was stopped, linear mRNA degraded rapidly, while circular mRNA remained largely intact. This confirmed that the improvement in protein expression was due to enhanced RNA stability. The circular structure also improved the efficiency of protein production per mRNA molecule by 1.51 times. CRESEnT is versatile and can be used alongside existing gene-expression control technologies, as the increase in protein production was consistent across various promoter and RBS combinations. The system was tested in multiple strains of E. coli, as well as in Bacillus subtilis and Corynebacterium glutamicum, demonstrating its broad applicability. The researchers also applied CRESEnT to produce useful compounds like flaviolin, itaconic acid, lycopene, and violacein, achieving significant increases in their production. The technology is expected to be used in the future for cell factories producing pharmaceuticals and eco-friendly materials. Professor Seo described the study as a shift from focusing solely on transcription and translation to introducing a new concept called "RNA topology engineering." The team plans to continue research on RNA engineering-based biomanufacturing platforms.