Cre/lox-Mediated CRISPRi Library Reveals Core Genome of a Type I Methanotroph Methylotuvimicrobium buryatense 5GB1C

Methanotrophs play key roles in global methane cycling and are promising platforms for methane bioconversion. However, major gaps existing in fundamental knowledge undermines understanding of these methane-consuming microorganisms. To associate genes with a phenotype at the genome-wide level, we developed a Cre/ lox-mediated method for constructing a large-scale CRISPRi library in a model methanotroph Methylotuvimicrobium buryatense 5GB1C. The efficiency of this Cre mediated integration method was up to a level of 10⁵ CFU/mg DNA. Targeting 4,100 predicted protein-coding genes, our CRISPRi pooled screening uncovered 788 core genes for the growth of strain 5GB1C using methane. The core genes are highly consistent with the gene knockout results, indicating the reliability of the CRISPRi screen. Insights from the core genes include that annotated isozymes generally exist in metabolic pathways and many core genes are hypothetical genes. This work not only provides functional genomic data for both fundamental research and metabolic engineering of methanotrophs, but also offers a method for CRISPRi library construction.