Sungkyunkwan University, led by President Yoo Ji-beom, has made a significant stride in biotechnology. Professor Woo Han Min's research team in the Department of Food and Biotechnology has achieved a breakthrough by developing a novel RNA interference system. This cutting-edge system utilizes dead Cas13a RNA CRISPR scissors, previously nonexistent in bacteria. Automated biofoundry technology was employed to enhance the efficiency of developing microbial cell factories. The groundbreaking results of this research were officially published on January 11 through 'Nucleic Acids Research,' a globally recognized academic journal (DOI: 10.1093/nar/gkad1130), with the official announcement made on November 30, 2023.

Microbial cell factories, designed for the production of sustainable biofuels, pharmaceuticals, food materials, and chemicals, represent industrial bacteria. These specialized bacteria leverage synthetic biology tools to regulate gene expression and control metabolic processes, aiming to maximize material productivity. This innovative approach holds promise for advancing the production of crucial bio-based products.

In details, the study introduces a technology aimed at suppressing the expression of diverse small RNAs within bacterial cells. Leveraging the unique capabilities of dead Cas13a (dCas13a) RNA CRISPR scissors, the researchers successfully developed a method to control both trans-small RNA and cis-small RNA in bacteria. This achievement fills a gap in existing technologies, providing control over small RNAs that were previously challenging to manipulate. Remarkably, this newly developed technology shares similarities with RNA interference mechanisms found in higher organisms, offering the potential to regulate high-level gene expression in bacteria.

Moreover, the advanced development of modular loop guide RNA has yielded a technology capable of suppressing target RNA expression across a spectrum ranging from 66% to 92%. This application technology effectively inhibits the expression of polycistronic genes prevalent in bacteria. Unlike existing CRISPRi gene inhibition technology, this approach allows for the targeted suppression of individual polycistronic genes, laying the foundation for the efficient development of cell factories.

The RNA CRISPR scissors' bacterial RNA interference technology was applied to create a microbial cell factory dedicated to producing lycopene, known for its antioxidant properties. Biofoundry technology, facilitated by a robot, produced 93 known E. coli sRNAs. A screening process within these libraries identified novel target sRNAs capable of enhancing lycopene productivity. This innovative approach surpasses traditional metabolic engineering, focusing on directly controlling the expression of enzyme genes involved in metabolic reactions. Instead, it introduces a novel metabolic engineering method that regulates downstream gene expression through target sRNA manipulation.

Professor Woo Han Min, Director of the Biofoundry Research Center at Sungkyunkwan University, emphasized, "With novel technologies of bacterial RNA interference and biofoundry, we are well-positioned to address diverse challenges using cutting-edge synthetic biology. Our goal is to extend the application of these technologies to new frontiers in medicine, food production, and the manufacturing of high value-added materials. Taking the lead in this endeavor, we are committed to developing advanced cell factories to fulfill these objectives."

This research has successfully generated large-scale guide RNA through the integration of bacterial RNA interference technology and biofoundry technology—a pivotal component in biomanufacturing. This transformative combination allows for the reprogramming of bacteria to serve as efficient cell factories, facilitating the screening of target materials and automating the entire Design-Build-Test-Learn (DBTL) cycle, a fundamental philosophy in synthetic biology research. Consequently, the development of cell factories is expedited, marking a significant advancement in the field.

Meanwhile, the research results were published online through the renowned academic journal 'Nucleic Acids Research' on November 28th. This research achievement was supported by the National Research Foundation of Korea’s Senior Researchers, Basic Research Laboratory Support Program, Microbial Control and Application Core Technology Development Project, and the Ministry of Science and ICT-supported Petrochemical Alternative Eco-friendly Chemical Technology Development Project, aimed at leading the biochemistry industry through the development of next-generation biorefinery core technologies. The underlying technology was domestically patented, and completed in 2022 (patent registration number 10-2422842).

※ Journal: Nucleic Acids Research (2023), Impact factor 14.9 (2022), Ranked in the top 3.3% in the field of JCR Biochemistry and Molecular Biology

※ Title: CRISPR-dCas13a system for programmable small RNAs and polycistronic mRNA repression in bacteria

※ DOI: 10.1093/nar/gkad1130

※ First Author: Ph.D. Ko Sung-chun (SKKU, Department of Food and Biotechnology)

※ Corresponding Author: . Professor Woo Han Min(SKKU, Department of Food and Biotechnology, the Biofoundry Research Center, Metabolic Engineering)

▲RNA CRISPR scissors' bacterial RNA interference technology was applied to create a microbial cell factory