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.

FIG 1

Analysis of M. buryatense 5GB1 glycogen synthase (glgA1, glgA2) mutants. Growth curves and doubling times (A) and intracellular glycogen contents (B) for wild-type and glycogen synthase mutants. Data represent the means from 3 replicates ± standard deviations.

FIG 2

Genetic tools for M. buryatense. (A) Sucrose counterselection vector pCM433kanT for unmarked allelic exchange. (B) Minimized IncP-based broad-host-range vector pAWP78 for plasmid-based gene expression. In both cases, the vector backbone was amplified using the annotated primers to add inserts of interest (AP186 and AP187 for pCM433kanT; AP259 and AP254 for pAWP78; see Table S1 in the supplemental material).

FIG 3

Loss of M. buryatense 5GB1 native plasmid results in more genetically tractable variants capable of being conjugated with small IncP-based plasmids. (A) Selection scheme used to obtain the more genetically tractable variant strain 5GB1S. This selection was repeated twice. (B) Number of transconjugants containing the small IncP-based plasmid pCM66 for M. buryatense 5GB1, selected strain 5GB1S, and a strain intentionally cured of the native plasmid, 5GB1C. N.D., not detected. (C) Genome sequence reads mapped to chromosome versus native plasmid for strains 5GB1 and 5GB1S. RPKM, reads per kilobase of gene per millions of reads in the sample.

FIG 4

dTomato fluorescence increases over time after cell breakage. Cell extract was incubated at 37°C, and fluorescence (excitation, 535 nm; emission, 590 nm) was measured with a fixed gain at the time points indicated. Data represent the means from 3 replicates ± standard deviations.

FIG 5

Relative promoter strengths in M. buryatense. Fluorescence (excitation, 535 nm; emission, 590 nm) was measured in cell extracts of M. buryatense 5GB1S containing probe vectors with promoter-dTomato fusions and normalized to protein content. Data represent the mean results for 3 distinct transconjugants ± standard deviations.