2026/06/12 by Yinhui Yu, Melissa Tumen-Velasquez, Michael Melesse Vergara +5 · 1 voice
Biochemistry, Genetics and Molecular Biology · Energy · Engineering · #Microbial metabolism and enzyme function #Metalloenzymes and iron-sulfur proteins #Anaerobic Digestion and Biogas Production
paper · doi:10.1128/aem.00538-26
openalex publication_date 2026/06/12 · openalex created_date 2026/06/13 · openalex updated_date 2026/07/31
ABSTRACT Methylococcus capsulatus Bath is a unique methanotrophic bacterium that uses methane (CH 4 ) as a carbon and energy source and encodes the RubisCO enzyme for inorganic carbon assimilation, positioning it as an excellent candidate for biotechnology to capture atmospheric CH 4 and carbon dioxide (CO 2 ) for chemical production. However, a limited genetic toolbox and minimal high-throughput methodologies for genetic engineering hinder the development of efficient M. capsulatus biocatalysts. In this work, we developed and optimized an electroporation workflow to transfer circular and linear DNA fragments into M. capsulatus with high efficiency by evading the host restriction-modification (RM) systems that recognize and degrade foreign DNA. To achieve this, we determined the M. capsulatus methylome to identify RM system methyltransferases and express them in Escherichia coli , resulting in DNA methylation in E. coli that mirrors that of M. capsulatus , which improved methanotroph electroporation efficiency. Iterative site-directed mutagenesis of plasmid methylation sites identified a m 6 A motif necessary for the increase in electroporation efficiency. An M. capsulatus knock-out strain with a disrupted Type I RM system linked to the m 6 A motif showed improved transformation efficiency compared to wild type, underscoring the importance of proper m 6 A methylation in successful DNA transfer to M. capsulatus . Notably, plasmid methylation coupled to an optimized electroporation workflow enabled the facile generation of an M. capsulatus genome-wide CRISPR interference library for functional genomic screening. Collectively, our results identify an RM system that has, up to now, limited M. capsulatus transformation and establish a methodology to bypass the RM system and improve transformation efficiency, which will accelerate the development of engineered methanotrophs for CH 4 bioconversion technologies. IMPORTANCE Aerobic CH 4 -oxidizing bacteria (methanotrophs) utilize CH 4 as a carbon and energy source in nature, also making them promising biocatalysts for converting CH 4 to valuable products. However, the development of these non-model microbes for use in biotechnology has been slowed by limitations in genetic tools and methodologies. Here, we established DNA electroporation in the methanotroph, Methylococcus capsulatus Bath, which had poor baseline DNA transfer efficiency. By determining the M. capsulatus methylome and identifying its restriction-modification systems that digest foreign DNA, thereby limiting DNA transformation, we developed an Escherichia coli cloning strain expressing M. capsulatus methyltransferases that modify plasmid DNA mirroring the host, significantly improving DNA transfer to M. capsulatus . These improvements enabled the proof-of-concept construction of a genetic library via electroporation. The significant advancements herein bring us closer to realizing the use of methanotrophs for biomanufacturing and atmospheric CH 4 -mitigating biotechnologies.