title: Genetic tools for the industrially promising methanotroph Methylomicrobium buryatense.
abstract: Aerobic 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 for Methylomicrobium buryatense, a haloalkaliphilic gammaproteobacterial (type I) methanotroph. M. buryatense was 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 in M. buryatense by 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 make M. buryatense a promising model system for studying aerobic methanotroph physiology and enable metabolic engineering in this bacterium for industrial biocatalysis of methane.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA awpuri@uw.edu.
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Puri
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Owen
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
authors.forename: Frances
authors.initials: F
authors.lastname: Chu
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
authors.forename: Ted
authors.initials: T
authors.lastname: Chavkin
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA eScience Institute, University of Washington, Seattle, Washington, USA.
authors.forename: David A C
authors.initials: DA
authors.lastname: Beck
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA.
authors.forename: Marina G
authors.initials: MG
authors.lastname: Kalyuzhnaya
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington, USA Department of Microbiology, University of Washington, Seattle, Washington, USA.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Lidstrom
mesh_terms: D003227:Conjugation, Genetic; D017353:Gene Deletion; D022761:Gene Transfer, Horizontal; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008697:Methane / Q000378:metabolism; D008772:Methylococcaceae / Q000235:genetics* / Q000254:growth & development; D008967:Molecular Biology / Q000379:methods*; D010084:Oxidation-Reduction; D010957:Plasmids
chemical_list: D008697:Methane