Overexpression of native carbonic anhydrases increases carbon conversion efficiency in the methanotrophic biocatalyst Bath.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH as a carbon and energy source. Evidence suggests that some methanotrophs, including , can also use CO as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH and CO metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that expresses 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 CO-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO limitation. 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 in metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO evolution from CH-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 CH to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH into CO and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including , can also use CO as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that carbonic anhydrases are critical for this bacterium to optimally utilize CO. We developed an engineered strain with improved CO utilization 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 CH conversion bioprocesses.