[1S05a] 13. Microbial Engineering for Natural Products Synthesis - Part 2

3:45 PM - 4:00 PM JST(6:45 AM - 7:00 AM UTC)

[1MENP-12] Development of a High-Efficient Methanotroph Platform for Sustainable Isoprene Production

○DONGUK SONG^1,3, Georgii Emelianov^1,2, Seung-Goo Lee^1,2,3, Hyewon Lee^1,2 (1. Synthetic Biology Research Center and the K-Biofoundry, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea (Korea), 2. Department of Biosystems and Bioengineering, University of Science and Technology (UST), Daejeon 34113, Republic of Korea (Korea), 3. Graduate School of Engineering Biology, Korea Advanced Institute of Science & Technology (KAIST), Daejeon 34141, Republic of Korea (Korea))

Keywords:

Isoprene,Methanotroph,Mevalonate pathway,CRISPR-base editor

Isoprene serves as a critical C5 platform chemical for the synthetic rubber industries, but its production depends heavily on petroleum-based processes. While microbial production from sugar offers a renewable alternative, it faces challenges regarding feedstock costs and food security. Methane-based bioconversion using methanotrophs presents a highly sustainable and economically viable solution. In this study, we established a high-titer isoprene production platform by engineering Methylococcus capsulatus Bath. To overcome the inherent complex metabolic regulation, we introduced heterologous mevalonate pathway under control of a robust phenol-inducible P_o promoter system. Another critical barrier to efficient methanotrophic isoprene synthesis is the broad substrate specificity of the sMMO, which oxidizes the isoprene into epoxyisoprene leading to growth inhibition. We addressed this metabolic bottleneck by utilizing a CRISPR-base editor to precisely disrupt the sMMO(mmoX) gene. The CRISPR-BE tool silenced the sMMO activity while maintaining the cell growth through particulate methane monooxygenase activity. Through systematic optimization of the metabolic flux, nitrogen sources and the supplementation of growth-limiting factors, we achieved isoprene titer of 228.1 mg/L. This result represents the highest reported titer for methanotroph-based isoprene synthesis system to date. Our study demonstrates that the synergy between exogenous pathway expression and targeted genomic editing can establish a powerful methanotrophic production platform for the sustainable upcycling of methane into high-value terpenoids.