[P006] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P03-404] Establishing a CAST-based genome engineering framework in methanotrophs

○Do-Hyeon Kim^1,2, Seong Keun Kim¹, Seung-Goo Lee^1,2,3, Dae-Hee Lee^1,2,3, Hyewon Lee^1,3 (1. Synthetic Biology Research Center and Korea Biofoundry, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea (Korea), 2. Graduate School of Engineering Biology, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea (Korea), 3. Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea (Korea))

Keywords:

Methanotroph,CRISPR-associated transposase (CAST),Genome engineering

Methanotrophic bacteria have attracted increasing attention as promising platforms for sustainable biomanufacturing because they utilize methane as their sole carbon source. Despite this potential, their broader application in metabolic engineering and synthetic biology has been limited by the lack of efficient and reliable genome engineering tools, particularly for stable DNA integration in technically challenging hosts. CRISPR-associated transposase (CAST) systems represent a promising strategy for targeted genome integration, yet their application in methanotrophs has remained largely unexplored. In this study, we developed and optimized the CAST system to enable genome engineering in Methylococcus capsulatus Bath. Molecular analyses confirmed site-specific insertions, indicating that the CAST system operates effectively in methanotrophs. Moreover, the CAST system was further optimized to improve integration efficiency, which is particularly important for slow-growing microorganisms such as methanotrophs. This work provides an initial framework for CAST-based genome engineering in methane-utilizing microorganisms and supports the future development of methanotrophs as genetically tractable platforms for metabolic engineering and synthetic biology.