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Abstract Aerobic methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH₄) and have potential to mitigate the climate-active gas. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a methanotroph CRISPR interference (CRISPRi) system to enable functional genomic screening. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph,Methylococcus capsulatus, consisting of ∼45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH₄identified 233 genes whose transcription repression resulted in a fitness defect and repression of 13 genes associated with a fitness advantage. Enrichment analysis of the 233 putative essential genes linked many of the encoded proteins with critical cellular processes like ribosome biosynthesis, translation, transcription, and other central biosynthetic metabolism, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs, including the identification of novel essential genes.M. capsulatusgrowth was inhibited when the CRISPRi system was used to individually target genes identified in the screen, validating their essentiality for methanotrophic growth. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify novel genetic determinants of methanotrophy. These CRISPRi screening methodologies can also be applied to high-throughput engineering approaches for isolation of improved methanotroph biocatalysts. 

ABSTRACT Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH₄as a carbon and energy source. Evidence suggests that some methanotrophs, includingMethylococcus capsulatus, can also use CO₂as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH₄and CO₂metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show thatM. capsulatusexpresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO₂-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO₂limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles inM. capsulatusmetabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO₂evolution from CH₄-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH₄to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH₄biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH₄into CO₂and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, includingMethylococcus capsulatus, can also use CO₂as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show thatM. capsulatuscarbonic anhydrases are critical for this bacterium to optimally utilize CO₂. We developed an engineered strain with improved CO₂utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH₄conversion bioprocesses.