Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

Abstract

ABSTRACTAerobic methanotrophs oxidize methane at ambient temperatures and pressures and are therefore attractive systems for methane-based bioconversions. In this work, we developed and validated genetic tools forMethylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph.M. buryatensewas isolated directly on natural gas and grows robustly in pure culture with a 3-h doubling time, enabling rapid genetic manipulation compared to many other methanotrophic species. As a proof of concept, we used a sucrose counterselection system to eliminate glycogen production inM. buryatenseby constructing unmarked deletions in two redundant glycogen synthase genes. We also selected for a more genetically tractable variant strain that can be conjugated with small incompatibility group P (IncP)-based broad-host-range vectors and determined that this capability is due to loss of the native plasmid. These tools makeM. buryatensea promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.