Metabolic engineering in methanotrophic bacteria

Kalyuzhnaya, Marina G.; Puri, Aaron W.; Lidstrom, Mary E.

doi:10.1016/j.ymben.2015.03.010

Cited by 275 publications

(175 citation statements)

References 111 publications

Supporting

Mentioning

162

Contrasting

Unclassified

Order By: Relevance

mentioning

confidence: 99%

“…The industrial use of microorganisms to convert methane into useful chemicals or biofuels represents one way to mitigate atmospheric methane (1,2). 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).…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

2016

Self Cite

View full text Add to dashboard Cite

Many methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense, XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive. IMPORTANCEAerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph.A n increasing surplus in the global methane budget exists due to human activity. The industrial use of microorganisms to convert methane into useful chemicals or biofuels represents one way to mitigate atmospheric methane (1, 2). 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.The majority of methanotrophs have two systems for oxidizing methane to methanol: the parti...

show abstract

mentioning

confidence: 99%

mentioning

confidence: 99%

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

2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…Aerobic methanotrophs are found in two major groups, the gammaproteobacterial methanotrophs (type I) and the alphaproteobacterial methanotrophs (type II) (2). Most studies and biotechnological efforts have focused on well-characterized species, such as Methylococcus capsulatus Bath, Methylosinus trichosporium OB3b, and Methylocystis parvus OBBP (6). In recent years, a variety of new strains have been isolated, including thermophilic, psychrophilic, acidophilic, alkaliphilic, and halophilic methanotrophs, which have expanded the physiological range of aerobic methanotrophs (7,8).…”

mentioning

confidence: 99%

“…Moreover, some isolates show robust growth and promise for use in biotechnological applications. Type I methanotrophs are particularly well suited for use in industrial processes, as they utilize the highly efficient ribulose monophosphate pathway for formaldehyde assimilation into biomass (2,6). A particularly promising type I methanotroph for industrial use is Methylomicrobium buryatense 5G, a haloalkaliphilic strain that was isolated directly on natural gas from a soda lake in the Transbaikal region of Russia, a harsh environment with fluctuating temperatures, salinities, and pH levels (7).…”

mentioning

confidence: 99%

“…At present, conjugation is a method commonly used for the transformation of methanotrophs (6). Recently, to metabolically engineer M. buryatense 5GB1, Puri et al developed a number of genetic tools for use with the strain, including a sucrose counterselection system and a small replicable vector, and these were found to be sufficient for genetic manipulation (9).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Yan

Chu

Puri

et al. 2016

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains. Methane, the principal component of natural gas and biogas, is a cheap, abundant, and renewable energy and carbon source. However, methane is also the second most prevalent greenhouse gas (1). Therefore, technologies to efficiently convert methane to chemicals or fuels can bring new sustainable solutions to a number of industries with large environmental footprints. Methane-oxidizing bacteria (methanotrophs) are able to use methane as their sole source of carbon and energy and thus are promising systems for methane-based bioconversion (2, 3). The bioconversion of methane to industrial products (single-cell proteins, biopolymers, lipids, etc.) using aerobic methanotrophs has been studied for approximately 50 years but has had little enduring success (4). Current biological engineering and systems biology approaches provide new opportunities for metabolic engineering of methanotrophs. However, to be an industrial workhorse like Escherichia coli, an industrial methanotroph needs to have a high growth rate, and efficient genetic tools for its manipulation and a fundamental knowledge base are needed.Many methanotrophs have been isolated and characterized since the classic study of Whittenbury et al. (5). Aerobic methanotrophs are found in two major groups, the...

show abstract