Construction of a broad-host-range Anderson promoter series and particulate methane monooxygenase promoter variants expand the methanotroph genetic toolbox.

Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products. These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases. Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs. However, the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria, which hinders genetic and metabolic engineering of these biocatalysts. As such, expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products. Here, we determined the copy number of three broad-host-range plasmids in Bath and OB3b, representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs, respectively. Further, we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in and OB3b, but the synthetic series had limited range. Thus, we mutagenized the native particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic, constitutive promoters functional not only in , but also in . Collectively, the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.