Genetic Tools for the Industrially Promising Methanotroph Methylomicrobium buryatense

Abstract

c 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. Methane, the principal component of natural gas, is a promising feedstock for industrial biotechnology. Domestic natural gas supplies are increasing, and natural gas prices have remained lower than oil prices over the last decade (1, 2). Methane has also been identified as a prime mitigation target for reducing greenhouse gas emissions (3). Aerobic methanotrophs oxidize methane under ambient temperatures and pressures and are genetically tractable, making these bacteria potentially useful biocatalysts for converting natural gas into chemicals and liquid fuels via metabolic engineering (1, 4).Previous industrial interest in aerobic methanotrophs has included attempts to produce biomass for use as single-cell protein or carotenoids in animal feed (5, 6). However, production of biomass from natural gas is complicated by the fact that nonmethane components of natural gas can be toxic to methanotrophs and can also provide substrates for growth of contaminating nonmethanotrophic bacterial species (5). Methylomicrobium buryatense is a gammaproteobacterial (type I) methanotroph that was isolated directly on natural gas from a soda lake in the Transbaikal region of Russia, a harsh environment with fluctuating temperatures, salinity, and pH levels (7). M. buryatense is naturally adapted to this environment and moderately haloalkaliphilic and consequently grows robustly in pure culture on natural gas, with increased resistance to contamination. The genome of this species was also recently sequenced and annotated by the Joint Genome Institute (8), enabling genomic manipulation for metabolic engineering. These characteristics make M. buryatense a strain of interest for industrial biotechnology.Genetic manipulation of methanotrophic bacteria is often a slow process with marker exchange mutagenesis typically taking weeks per mutation, making it difficult to construct multiple mutations in a timely manner (9, 10). Counterselection prot...

Quorum Sensing in a Methane-Oxidizing Bacterium

“…We saw maximum accumulation (approximately 330 nM) of the 3-OH-C 10 -HSL signal during stationary phase. Next, to determine the sensitivity of MbaR to 3-OH-C 10 -HSL in its native host, we constructed an M. tundripaludum reporter strain wherein the signal- responsive open reading frame (ORF) mbaI was replaced with the catechol dioxygenase reporter gene xylE (25). This mbaI::xylE reporter strain was responsive to as little as 3 nM 3-OH-C 10 -HSL (Fig.…”

“…For both electroporation and mating, successful integrants (single crossovers) were selected on NMS plates containing kanamycin (50 g/ml). Subsequent sucrose counterselection was performed as previously described (25).…”

XoxF Acts as the Predominant Methanol Dehydrogenase in the Type I Methanotroph Methylomicrobium buryatense

“…M. buryatense 5GB1C and its derivatives were grown in modified nitrate mineral salts medium (NMS2) as previously described (3). For growth without copper, the 500ϫ high-purity trace elements solution was modified to contain 1.0 g of Na 2 sucrose, 100 g of hygromycin/ml, 50 g of kanamycin/ml, 30 g of zeocin/ml, 30 M lanthanum chloride (unless a different concentration is specified) (Sigma-Aldrich), and 30 M cerium chloride (Sigma-Aldrich).…”

“…Methanotrophs, or methane-oxidizing bacteria, utilize methane as their carbon and energy source and are prime candidates for the industrial bioconversion of methane (1). Renewed interest in the industrial use of methanotrophs has come about partially because of the discovery of rapidly growing strains and new tools for genetic manipulation, allowing for fast-paced metabolic engineering (3,4). The success of metabolic engineering strategies in these methanotrophs depends upon a strong foundation of knowledge concerning the metabolic pathways that methanotrophs employ, both in the laboratory and in their natural environments, and an understanding of how various branches of metabolic pathways are regulated.…”

Lanthanide-Dependent Regulation of Methanol Oxidation Systems in Methylobacterium extorquens AM1 and Their Contribution to Methanol Growth

“…ethylotrophs have gained worldwide interest as platforms for the production of value-added chemicals from singlecarbon compounds, turning atmospheric pollutants like methane and methanol into green chemicals, including biofuels and biodegradable plastics (1)(2)(3)(4)(5). A key step in this process is the oxidation of methanol to formaldehyde, which is carried out by different enzymes, including methanol dehydrogenase (MeDH) and alcohol oxidase, depending on the specific methylotroph (6,7).…”