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Methylobacterium species, the representative bacteria distributed in phyllosphere region of plants, often synthesize carotenoids to resist harmful UV radiations. Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C30 backbone. However, its exact structure remains unknown. In the present study, the carotenoid produced by M. extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopenedioc acid (1), a glycosylated C30 carotenoid. Furthermore, the genes related to the C30 carotenoid synthesis were investigated. Squalene, the precursor of the C30 carotenoid, is synthesized by the co-occurrence of META1p1815, META1p1816 and META1p1817. Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1. By using gene deletion and gene complementation experiments, the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4′-diapolycopene-4,4′-dioic acid to carotenoid 1. In conclusion, the structure and biosynthetic genes of carotenoid 1 produced by M. extorquens AM1 were firstly characterized in this work, which shed lights on engineering M. extorquens AM1 for producing carotenoid 1 in high yield.

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals, owing to its high degree of reduction, abundance, and low price. Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals. Alternatively, synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms, such as Escherichia coli. Owing to the complex metabolic pathways, limited availability of genetic tools, and methanol/formaldehyde toxicity, the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility. This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms. It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol.

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals, owing to its high degree of reduction, abundance, and low price. Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals. Alternatively, synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms, such as Escherichia coli. Owing to the complex metabolic pathways, limited availability of genetic tools, and methanol/formaldehyde toxicity, the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility. This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms. It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol. [ABSTRACT FROM AUTHOR]

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

Lanthanide elements have been recently recognized as "new life metals" yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.

Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo β-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

The Radical SAM (RS) enzyme PqqE catalyzes the first step in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, forming a new carbon-carbon bond between two side chains within the ribosomally synthesized peptide substrate PqqA. In addition to the active site RS 4Fe-4S cluster, PqqE is predicted to have two auxiliary Fe-S clusters, like the other members of the SPASM domain family. Here we identify these sites and examine their structure using a combination of X-ray crystallography and Mössbauer and electron paramagnetic resonance (EPR) spectroscopies. X-ray crystallography allows us to identify the ligands to each of the two auxiliary clusters at the C-terminal region of the protein. The auxiliary cluster nearest the RS site (AuxI) is in the form of a 2Fe-2S cluster ligated by four cysteines, an Fe-S center not seen previously in other SPASM domain proteins; this assignment is further supported by Mössbauer and EPR spectroscopies. The second, more remote cluster (AuxII) is a 4Fe-4S center that is ligated by three cysteine residues and one aspartate residue. In addition, we examined the roles these ligands play in catalysis by the RS and AuxII clusters using site-directed mutagenesis coupled with EPR spectroscopy. Lastly, we discuss the possible functional consequences that these unique AuxI and AuxII clusters may have in catalysis for PqqE and how these may extend to additional RS enzymes catalyzing the post-translational modification of ribosomally encoded peptides.

Herein, a novel laccase gene, Melac13220, was amplified from Methylobacterium extorquens and successfully expressed in Escherichia coli with a molecular weight of approximately 50 kDa. The purified Melac13220 had no absorption peak at 610 nm and remained silent within electron paramagnetic resonance spectra, suggesting that Melac13220 belongs to the non-blue laccase group. Both inductively coupled plasma spectroscopy/optical emission spectrometry (ICP-OES) and isothermal titration calorimetry (ITC) indicated that one molecule of Melac13220 can interact with two iron ions. Furthermore, the optimal temperature of Melac13220 was 65 °C. It also showed a high thermolability, and its half-life at 65 °C was 80 min. Melac13220 showed a very good acid environment tolerance; its optimal pH was 1.5. Cu2+ and Co2+ can slightly increase enzyme activity, whereas Fe2+ could increase Melac13220′s activity five-fold. Differential scanning calorimetry (DSC) indicated that Fe2+ could also stabilize Melac13220. Unlike most laccases, Melac13220 can efficiently decolorize Congo Red and Indigo Carmine dyes even in the absence of a redox mediator. Thus, the non-blue laccase from Methylobacterium extorquens shows potential application value and may be valuable for environmental protection, especially in the degradation of dyes at low pH. [ABSTRACT FROM AUTHOR]

Biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is initiated when the precursor peptide, PqqA, is recognized and bound by the RiPP precursor peptide recognition element (RRE), PqqD, for presentation to the first enzyme in the pathway, PqqE. Unlike other RiPP-producing, postribosomal peptide synthesis (PRPS) pathways in which the RRE is a component domain of the first enzyme, PqqD is predominantly a separate scaffolding protein that forms a ternary complex with the precursor peptide and first tailoring enzyme. As PqqD is a stable, independent RRE, this makes the PQQ pathway an ideal PRPS model system for probing RRE interactions using nuclear magnetic resonance (NMR). Herein, we present both the solution NMR structure of Methylobacterium extorquens PqqD and results of 1H-15N HSQC binding experiments that identify the PqqD residues involved in binding the precursor peptide, PqqA, and the enzyme, PqqE. The reported structural model for an independent RRE, along with the mapped binding surfaces, will inform future efforts both to understand and to manipulate PRPS pathways.

The ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is a dehydrogenase cofactor synthesized by, but not exclusively used by, certain prokaryotes. RiPPs represent a rapidly expanding and diverse class of natural products-many of which have therapeutic potential-and the biosynthetic pathways for these are gaining attention. Five gene products from the pqq operon (PqqA, PqqB, PqqC, PqqD, and PqqE) are essential for PQQ biosynthesis. The substrate is the peptide PqqA, which is presented to the radical SAM enzyme PqqE by the small protein PqqD. PqqA is unstructured in solution, and only binds to PqqE when in complex with PqqD. PqqD is a member of a growing family of RiPP chaperone proteins (or domains in most cases) that present their associated peptide substrates to the initial RiPP biosynthesis enzymes. An X-ray crystal structure exists for dimeric Xanthomonas campestris PqqD (PDB ID: 3G2B), but PqqD is now known to act as a monomer under physiological conditions. In this study, the PqqD truncation from naturally fused Methylobacterium extorquens (Mex) PqqCD was overexpressed in Escherichia coli and MexPqqA was chemically synthesized. Solution NMR (1)H-,(15)N-HSQC chemical shift studies have identified the PqqD residues involved in binding PqqA, and (1)H, (13)C, and (15)N peak assignments for PqqD alone and for PqqD bound to PqqA are reported herein.

Pyrroloquinoline quinone (PQQ) is a product of a ribosomally synthesized and post-translationally modified pathway consisting of five conserved genes, pqqA-E. PqqE is a radical S-adenosylmethionine (RS) protein with a C-terminal SPASM domain, and is proposed to catalyze the formation of a carbon-carbon bond between the glutamate and tyrosine side chains of the peptide substrate PqqA. PqqD is a 10-kDa protein with an unknown function, but is essential for PQQ production. Recently, in Klebsiella pneumoniae (Kp), PqqD and PqqE were shown to interact; however, the stoichiometry and KD were not obtained. Here, we show that the PqqE and PqqD interaction transcends species, also occurring in Methylobacterium extorquens AM1 (Me). The stoichiometry of the MePqqD and MePqqE interaction is 1:1 and the KD, determined by surface plasmon resonance spectroscopy (SPR), was found to be ∼12 μm. Moreover, using SPR and isothermal calorimetry techniques, we establish for the first time that MePqqD binds MePqqA tightly (KD ∼200 nm). The formation of a ternary MePqqA-D-E complex was captured by native mass spectrometry and the KD for the MePqqAD-MePqqE interaction was found to be ∼5 μm. Finally, using a bioinformatic analysis, we found that PqqD orthologues are associated with the RS-SPASM family of proteins (subtilosin, pyrroloquinoline quinone, anaerobic sulfatase maturating enzyme, and mycofactocin), all of which modify either peptides or proteins. In conclusion, we propose that PqqD is a novel peptide chaperone and that PqqD orthologues may play a similar role in peptide modification pathways that use an RS-SPASM protein.

Abstract Background Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. Results In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The K m and k cat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s−1, 1.28 mM and 153.14 s−1, and 2.34 mM and 1.15 s−1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. Conclusions These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3′hydroxyl of (p)ppGpp in a Mn2+ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP+ reduction, which facilitate estimation of kinetic constants. Furthermore, by using this assay technique we confirmed TLC observations and also revealed that purified small alarmone hydrolase (SAHMex) from Methylobacterium extorquens displays a strong hydrolase activity toward (p)ppApp but only negligible activity toward (p)ppGpp. In contrast, the substrate specificity of the hydrolase present in catalytically active N-terminal domain of the RSH protein from Streptococcus equisimilis (RelSeq) includes (p)ppGpp but not (p)ppApp. It is noteworthy that the RSH protein from M. extorquens (RSHMex) has been recently shown to synthesize both (p)ppApp and (p)ppGpp. [ABSTRACT FROM AUTHOR]

[Display omitted] • Genome-scale FBA simulations guiding the construction of photo-methylotrophic metabolism in a methanol utilizer of Methylobacterium extorquens. • Light-driven ATP generation maintaining the metabolic activity of non-growth cells during a two-stage fermentation process. • Photo-methylotrophic cell factory increasing the titer, yield, and productivity of 3-HP. • Transcriptomics revealing light-driven methanol consumption and oxidation as key contributing factor for the increase of 3-HP production. • Photo-methylotrophy opening up novel opportunities for increasing the production of energy- and reducing equivalent-consuming chemicals. This study explored the potential of methanol as a sustainable feedstock for biomanufacturing, focusing on Methylobacterium extorquens , a well-established representative of methylotrophic cell factories. Despite this bacterium's long history, its untapped photosynthetic capabilities for production enhancement have remained unreported. Using genome-scale flux balance analysis, it was hypothesized that introducing photon fluxes could boost the yield of 3-hydroxypropionic acid (3-HP), an energy- and reducing equivalent-consuming chemicals. To realize this, M. extorquens was genetically modified by eliminating the negative regulator of photosynthesis, leading to improved ATP levels and metabolic activity in non-growth cells during a two-stage fermentation process. This modification resulted in a remarkable 3.0-fold increase in 3-HP titer and a 2.1-fold increase in its yield during stage (II). Transcriptomics revealed that enhanced light-driven methanol oxidation, NADH transhydrogenation, ATP generation, and fatty acid degradation were key factors. This development of photo-methylotrophy as a platform technology introduced novel opportunities for future production enhancements. [ABSTRACT FROM AUTHOR]

Abstract Background 3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol. Results Here we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1_4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase. Conclusions We demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol.

The objective of this study was to investigate the possibility of using natural and bacteria-modified Erzurum clayey soil with Methylobacterium extorquens as an alternative to high cost commercial adsorbent materials for the removal of copper from aqueous solution. The copper concentrations in the samples of the polluted river water and CuCl2 solutions treated by the natural and bacteria-modified Erzurum clayey soil (ECS) have been determined by spectrophotometric method. Firstly, the surface of ECS was modified with M. extorquens and surface functionality was increased. Then, the adsorption of Cu (II) from solution phases was studied with respect to varying metal concentration, pH, and temperature and agitation time. The maximum adsorption of Cu (II) for natural and bacteria-modified Erzurum clayey soil was observed at pH: 5.0. At different copper concentrations, copper adsorption analysis was performed on 1 g using clay soil or modified clay soil. Maximum adsorption of Cu (II) was obtained as 45.7 and 48.1 mg g-1 at initial concentration (50 mg/50 mL) and optimal conditions by natural and bacteria-modified clay soil, respectively. The copper concentration was decreased in the substantial amount of the leachates solutions of natural and bacteria-modified clay soil. Langmuir and Freundlich isotherms were used to describe the adsorption behavior of Cu (II) ions. The results showed that modified clay soil had a high level of adsorption capacity for copper ion. The various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were analyzed to observe the nature of adsorption. The structural properties of the natural and bacteria-modified-ECS have been characterized by SEM, FTIR and XRD techniques. Consequently, it was concluded that the bacteria-modified clay soil could be successfully used for the removal of the copper ions from the aqueous solutions.

In bacteria, the so-called stringent response is responsible for adaptation to changing environmental conditions. This response is mediated by guanosine derivatives [(p)ppGpp], synthesized by either large mono-functional RelA or bi-functional SpoT (synthesis and hydrolysis) enzymes in β- and γ-proteobacteria, such as Escherichia coli. In Firmicutes and α-, δ-, and 𝜀-proteobacteria, large bifunctional Rel-SpoT-homologs (RSH), often accompanied by small (p)ppGpp synthetases and/or hydrolases devoid of regulatory domains, are responsible for (p)ppGpp turnover. Here, we report on surprising in vitro and in vivo properties of an RSH enzyme from Methylobacterium extorquens (RSHMex). We find that this enzyme possesses some unique features, e.g., it requires cobalt cations for the most efficient (p)ppGpp synthesis, in contrast to all other known specific (p)ppGpp synthetases that require Mg2+. In addition, it can synthesize pppApp, which has not been demonstrated in vitro for any Rel/SpoT/RSH enzyme so far. In vivo, our studies also show that RSHMex is active in Escherichia coli cells, as it can complement E. coli ppGpp0 growth defects and affects rrnB P1-lacZ fusion activity in a way expected for an RSH enzyme. These studies also led us to discover pppApp synthesis in wild type E. coli cells (not carrying the RSHMex enzyme), which to our knowledge has not been demonstrated ever before. In the light of our recent discovery that pppApp directly regulates E. coli RNAP transcription in vitro in a manner opposite to (p)ppGpp, this leads to a possibility that pppApp is a new member of the nucleotide second-messenger family that is widely present in bacterial species.

The wilting of tomato (Fusarium oxysporum) promotes the excessive use of synthetic fungicides, which are not economically or environmentally viable. This work proposes the use of Methylobacterium extorquens to determine its effect on plant development and the signaling pathway, for the defense of tomato plants, against phytopathogens. From tomato seeds var. SUN 7705 embedded and plants sprinkled with the bacteria at a concentration of 109 CFU mL-1, were challenged to the plant with the fungus in two tests. Variables of root length in seedlings, plant height, leaf amplitude, yield, total dry weight of the plant and dry root weight were measured. A completely randomized design with six treatments and four repetitions was used, two plants formed the experimental unit. Likewise, an essay was carried out on the expression of defense genes, in tomato seedlings of the same variety. Significant statistical differences (p≤ 0.05) were found between treatments embedded and sprinkled with M. extorquens and the treatments inoculated with F. oxysporum and the controls, reflecting in the seedling root length, plant height, leaf amplitude, dry weight of the plant and the root, as well as in the yield. However, the genes encoding the phenylalanine ammonium lyase (PAL), protein (PR-6), superoxide dismutase (SOD) and lipoxygenase (LOX) enzyme group, were not significantly expressed in seedlings treated with M. extorquens. [ABSTRACT FROM AUTHOR]

Summary: Until recently, rare‐earth elements (REEs) had been thought to be biologically inactive. This view changed with the discovery of the methanol dehydrogenase XoxF that strictly relies on REEs for its activity. Some methylotrophs only contain xoxF, while others, including the model phyllosphere colonizer Methylobacterium extorquens PA1, harbor this gene in addition to mxaFI encoding a Ca2+‐dependent enzyme. Here we found that REEs induce the expression of xoxF in M. extorquens PA1, while repressing mxaFI, suggesting that XoxF is the preferred methanol dehydrogenase in the presence of sufficient amounts of REE. Using reporter assays and a suppressor screen, we found that lanthanum (La3+) is sensed both in a XoxF‐dependent and independent manner. Furthermore, we investigated the role of REEs during Arabidopsisthaliana colonization. Element analysis of the phyllosphere revealed the presence of several REEs at concentrations up to 10 μg per g dry weight. Complementary proteome analyses of M. extorquens PA1 identified XoxF as a top induced protein in planta and a core set of La3+‐regulated proteins under defined artificial media conditions. Among these was a REE‐binding protein that is encoded next to a gene for a TonB‐dependent transporter. The latter was essential for REE‐dependent growth on methanol indicating chelator‐assisted uptake of REEs. [ABSTRACT FROM AUTHOR]

In bacteria, the so-called stringent response is responsible for adaptation to changing environmental conditions. This response is mediated by guanosine derivatives [(p)ppGpp], synthesized by either large mono-functional RelA or bi-functional SpoT (synthesis and hydrolysis) enzymes in β - and γ - proteobacteria, such as Escherichia coli. In Firmicutes and α - , δ - , and 𝜀 - proteobacteria, large bifunctional Rel-SpoT-homologs (RSH), often accompanied by small (p)ppGpp synthetases and/or hydrolases devoid of regulatory domains, are responsible for (p)ppGpp turnover. Here, we report on surprising in vitro and in vivo properties of an RSH enzyme from Methylobacterium extorquens (RSHMex). We find that this enzyme possesses some unique features, e.g., it requires cobalt cations for the most efficient (p)ppGpp synthesis, in contrast to all other known specific (p)ppGpp synthetases that require Mg2+. In addition, it can synthesize pppApp, which has not been demonstrated in vitro for any Rel/SpoT/RSH enzyme so far. In vivo , our studies also show that RSHMex is active in Escherichia coli cells, as it can complement E. coli ppGpp0 growth defects and affects rrnB P1- lacZ fusion activity in a way expected for an RSH enzyme. These studies also led us to discover pppApp synthesis in wild type E. coli cells (not carrying the RSHMex enzyme), which to our knowledge has not been demonstrated ever before. In the light of our recent discovery that pppApp directly regulates E. coli RNAP transcription in vitro in a manner opposite to (p)ppGpp, this leads to a possibility that pppApp is a new member of the nucleotide second-messenger family that is widely present in bacterial species. [ABSTRACT FROM AUTHOR]

Abstract The fractionation of carbon and chlorine stable isotopes of dichloromethane (CH 2 Cl 2 , DCM) upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed. Isotope effects for individual steps of the multi-stage DCM degradation process, including transfer across the cell wall from the aqueous medium to the cell cytoplasm, dehalogenase binding, and catalytic reaction, were considered. The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of C Cl bonds, and not by inflow of DCM into cells. Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures, presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess. Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes (Δδ13C/Δδ37Cl), increasing corresponding ratio from 7.2–8.6 to 9.6–10.5 under conditions of glutathione deficiency. This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM, unlike the uncatalyzed reaction, which is a one-stage process, as shown by quantum-chemical modeling. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Multiple genes encoding putative PHB synthases (up to 5 in single strain) were found in Methylobacterium genomes. As a result of phylogenetic analysis proteins PhaC1, PhaC2, PhaC3 were identified as class I PHB synthases, PhaC4 proteins were identified as class III PHB synthases, while PhaC5 apparently belongs to uncharacterized class of PHB synthases. Firstly, the recombinant class I PBH synthase (EC 2.3.1.B2) encoded by phaC1 gene from Methylobacterium extorquens AM1 was purified and characterized. Molecular mass of enzyme monomer was 78 kDa. Michaelis constant (Km) for PhaC1 was 1,3 mM and maximal reaction rate (Vmax) was 0,1 μmol∙min–1∙mg–1. The deletion mutant of Methylobacterium extorquens in the phaC gene was obtained which is promising for further study of peculiarities of methylobacteria’s PHB biosynthesis

N-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. N-alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics. Chemical synthesis of N-methylated amino acids often is limited by incomplete stereoselectivity, over-alkylation or the use of hazardous chemicals. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of N-methylglutamate from simple carbon sources and monomethylamine. P. putida KT2440, which is generally recognized as safe and grows with glucose and the alternative feedstock glycerol as sole carbon and energy source, was engineered for the production of N-methylglutamate using heterologous enzymes from Methylobacterium extorquens. About 3.9 g L−1N-methylglutamate accumulated within 48 h in shake flask cultures with minimal medium containing monomethylamine and glycerol. A fed-batch cultivation process yielded a N-methylglutamate titer of 17.9 g L−1.

Rare earth elements (REE) are valuable raw materials in our modern life. Extensive REE application from electronic devices to medical instruments and wind turbines, and non-uniform distribution of these resources around the world, make them strategically and economically important for countries. Current REE physical and chemical mining and recycling methods could have negative environmental consequences, and biologically-mediated techniques could be applied to overcome this issue. In this study, the bioextraction of cerium oxide and neodymium oxide nanoparticles (REE-NP) by a pure culture Methylobacterium extorquens AM1 (ATCC®14718™) was investigated in batch experiments. Results show that adding up to 1000 ppm CeO 2 or Nd 2 O 3 nanoparticles (REE-NP) did not seem to affect the bacterial growth over 14-days contact time. Effect of methylamine hydrochloride as an essential electron donor and carbon source for microbial oxidation and growth was also observed inasmuch as there was approximately no growth when it does not exist in the medium. Although very low concentrations of cerium and neodymium in the liquid phase were measured, concentrations of 45 μg/g cell Ce and 154 μg/g cell Nd could be extracted by M. extorquens AM1. Furthermore, SEM-EDS and STEM-EDS confirmed surface and intracellular accumulation of nanoparticles. These results confirmed the ability of M. extorquens to accumulate REE nanoparticles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Three high‐resolution X‐ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP‐ribose occupying the pyridine nucleotide‐binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate‐binding site is accessible in the enzyme resting state or NAD+‐bound forms, the substrate‐bound form exhibits a closed conformation. This conformational change involves the transition of an α‐helix to a 310‐helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)‐malate. [ABSTRACT FROM AUTHOR]

A method to stably immobilize microbes on electrodes was developed. Resting cells of Methylobacterium extorquens AM1( Me AM1) were caged within multiwalled carbon nanotubes (MWNTs)by adding the cells to a water dispersion of MWNTs then allowing the resulting mixture to dry on electrodes. The Me AM1-MWCNTs electrode thus obtained displayed excellent activities in the bidirectional bioelectrocatalysis due to formate dehydrogenase(s) in the resting cells; formate oxidation and carbon dioxide reduction proceeded at steady-state catalytic current densities of 0.6 ± 0.1 and −0.8 ± 0.1 mA cm −2 , respectively, using methyl viologen as mediator under very mild conditions (pH 7.0, atmospheric pressure, and 37 °C). In addition, the catalytic signal was stable for more than one week under continuous operation. [ABSTRACT FROM AUTHOR]

Methylobacterium strains can use one-carbon compounds, such as methanol, for methylotrophic growth. In addition to methanol, a few strains also utilize dichloromethane, a major industrial chlorinated solvent pollutant. With a fully assembled and annotated genome, M. extorquens DM4 is the reference bacterium for aerobic dichloromethane degradation. The doublet N-terminal oriented proteomics (dN-TOP) strategy was applied to further improve its genome annotation and a differential proteomics approach was performed to compare M. extorquens DM4 grown either with methanol or dichloromethane as the sole source of carbon and energy. These approaches led to experimental confirmation of 259 hypothetical proteins, correction of 78 erroneous predicted start codons, discovery of 39 new proteins and annotation of 66 signal peptides, including essential enzymes involved in methylotrophic growth. Significance Dichloromethane (methylene chloride, CH 2 Cl 2 , DCM) is one of the most widely used industrial halogenated solvents and a potential carcinogen. Microbial rehabilitation of worldwide-contaminated sites involves DCM breakdown by bacteria that are able to grow using this pollutant as their sole carbon and energy source. The most-studied methylotrophic DCM degrader is Methylobacterium extorquens strain DM4. Proteomic studies of the Methylobacterium genus have been performed previously, but genome-wide investigation of N-termini of expressed proteins has not yet been performed. Differential quantitative proteomic analysis also opens new research perspectives to better monitor and understand bacterial growth with DCM. [ABSTRACT FROM AUTHOR]

Organisms are either heterotrophic or autotrophic, meaning that they cover their carbon requirements by assimilating organic compounds or by fixing inorganic carbon dioxide (CO 2 ). The conversion of a heterotrophic organism into an autotrophic one by metabolic engineering is a long-standing goal in synthetic biology and biotechnology, because it ultimately allows for the production of value-added compounds from CO 2 . The heterotrophic Alphaproteobacterium Methylobacterium extorquens AM1 is a platform organism for a future C1-based bioeconomy. Here we show that M. extorquens AM1 provides unique advantages for establishing synthetic autotrophy, because energy metabolism and biomass formation can be effectively separated from each other in the organism. We designed and realized an engineered strain of M. extorquens AM1 that can use the C1 compound methanol for energy acquisition and forms biomass from CO 2 by implementation of a heterologous Calvin-Benson-Bassham (CBB) cycle. We demonstrate that the heterologous CBB cycle is active, confers a distinct phenotype, and strongly increases viability of the engineered strain. Metabolic 13 C-tracer analysis demonstrates the functional operation of the heterologous CBB cycle in M. extorquens AM1 and comparative proteomics of the engineered strain show that the host cell reacts to the implementation of the CBB cycle in a plastic way. While the heterologous CBB cycle is not able to support full autotrophic growth of M. extorquens AM1, our study represents a further advancement in the design and realization of synthetic autotrophic organisms. [ABSTRACT FROM AUTHOR]

Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low μM (0.1 to 10 μM) range. We designed this assay to follow LaIII and EuIII depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as PrIII, or to monitor the depletion of LaIII from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS). IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria. [ABSTRACT FROM AUTHOR]

PqqE is a radical S-adenosyl- l-methionine ( SAM) enzyme that catalyzes the initial reaction of pyrroloquinoline quinone ( PQQ) biosynthesis. PqqE belongs to the SPASM (subtilosin/ PQQ/anaerobic sulfatase/mycofactocin maturating enzymes) subfamily of the radical SAM superfamily and contains multiple Fe -S clusters. To characterize the Fe -S clusters in PqqE from Methylobacterium extorquens AM1, Cys residues conserved in the N-terminal signature motif ( CX3 CX2C) and the C-terminal seven-cysteine motif ( CX9-15 GX4 CX n CX2 CX5 CX3 CX nC; n = an unspecified number) were individually or simultaneously mutated into Ser. Biochemical and Mössbauer spectral analyses of as-purified and reconstituted mutant enzymes confirmed the presence of three Fe -S clusters in PqqE: one [4Fe -4S]2+ cluster at the N-terminal region that is essential for the reductive homolytic cleavage of SAM into methionine and 5′-deoxyadenosyl radical, and one each [4Fe -4S]2+ and [2Fe -2S]2+ auxiliary clusters in the C-terminal SPASM domain, which are assumed to serve for electron transfer between the buried active site and the protein surface. The presence of [2Fe -2S]2+ cluster is a novel finding for radical SAM enzyme belonging to the SPASM subfamily. Moreover, we found uncommon ligation of the auxiliary [4Fe -4S]2+ cluster with sulfur atoms of three Cys residues and a carboxyl oxygen atom of a conserved Asp residue. [ABSTRACT FROM AUTHOR]

Hopanoids are sterol-like membrane lipids widely used as geochemical proxies for bacteria. Currently, the physiological role of hopanoids is not well understood, and this represents one of the major limitations in interpreting the significance of their presence in ancient or contemporary sediments. Previous analyses of mutants lacking hopanoids in a range of bacteria have revealed a range of phenotypes under normal growth conditions, but with most having at least an increased sensitivity to toxins and osmotic stress. We employed hopanoid-free strains of Methylobacterium extorquens DM4, uncovering severe growth defects relative to the wild-type under many tested conditions, including normal growth conditions without additional stressors. Mutants overproduce carotenoids–the other major isoprenoid product of this strain–and show an altered fatty acid profile, pronounced flocculation in liquid media, and lower growth yields than for the wild-type strain. The flocculation phenotype can be mitigated by addition of cellulase to the medium, suggesting a link between the function of hopanoids and the secretion of cellulose in M. extorquens DM4. On solid media, colonies of the hopanoid-free mutant strain were smaller than wild-type, and were more sensitive to osmotic or pH stress, as well as to a variety of toxins. The results for M. extorquens DM4 are consistent with the hypothesis that hopanoids are important for membrane fluidity and lipid packing, but also indicate that the specific physiological processes that require hopanoids vary across bacterial lineages. Our work provides further support to emerging observations that the role of hopanoids in membrane robustness and barrier function may be important across lineages, possibly mediated through an interaction with lipid A in the outer membrane. [ABSTRACT FROM AUTHOR]

Methylobacterium extorquens AM1 (AM1) has shown promise for use in industry due to its ability to use methanol as a sole carbon and energy source. Mevalonic acid (MEV) is an important platform chemical for the biosynthesis of isoprenoids, and a building block in the synthesis of polymers. In our previous study, the growth and MEV production of AM1-dcel-mvt strain were found to be unstable in the culture media tested (CHOI, MP, MC and HP), which contained ammonium salts, magnesium, calcium, buffers, methanol and trace elements. In this study, we developed a novel MC medium based on MP and CHOI, which improved the growth rate and the reproducibility and replicability of culture process considerably. AM1-dcel-mvt cells cultivated in MC displayed a more homogenous doubling time than cells in other media, which could explain their more stable growth. The optimized MO medium containing a higher phosphate buffer concentration and lower trace elements was developed, which enhanced MEV production by 0.61-fold to 340 mg/L. In fed-batch fermentation in a 5 L fermenter, a MEV titre of 2.59 g/Land a dry cell weight (DCW) of 9.03 g/L were obtained, representing the highest MEV production reported to date for the AM1-dcel-mvt strain. The MO medium facilitate the stable growth of AM1, and improve the industrial-scale production of MEV. [ABSTRACT FROM AUTHOR]

Malyl-CoA lyase (MCL) is an Mg2+-dependent enzyme that catalyzes the reversible cleavage of (2 S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56 Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg2+ is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. This domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism. [ABSTRACT FROM AUTHOR]

The biotechnological production of the methyl methacrylate precursor 2-hydroxyisobutyric acid (2-HIBA) via bacterial poly-3-hydroxybutyrate (PHB) overflow metabolism requires suitable (R)-3-hydroxybutyryl coenzyme A (CoA)-specific coenzyme B12-dependent mutases (RCM). Here, we characterized a predicted mutase from Bacillus massiliosenegalensis JC6 as a mesophilic RCM closely related to the thermophilic enzyme previously identified in Kyrpidia tusciae DSM 2912 (M.-T. Weichler et al., Appl Environ Microbiol 81:4564-4572, 2015, https://doi.org/10.1128/AEM.00716-15). Using both RCM variants, 2-HIBA production from methanol was studied in fed-batch bioreactor experiments with recombinant Methylobacterium extorquens AM1. After complete nitrogen consumption, the concomitant formation of PHB and 2-HIBA was achieved, indicating that both sets of RCM genes were successfully expressed. However, although identical vector systems and incubation conditions were chosen, the metabolic activity of the variant bearing the RCM genes from strain DSM 2912 was severely inhibited, likely due to the negative effects caused by heterologous expression. In contrast, the biomass yield of the variant expressing the JC6 genes was close to the wild-type performance, and 2-HIBA titers of 2.1 g liter-1 could be demonstrated. In this case, up to 24% of the substrate channeled into overflow metabolism was converted to the mutase product, and maximal combined 2-HIBA plus PHB yields from methanol of 0.11 g g-1 were achieved. Reverse transcription-quantitative PCR analysis revealed that metabolic genes, such as methanol dehydrogenase and acetoacetyl-CoA reductase genes, are strongly downregulated after exponential growth, which currently prevents a prolonged overflow phase, thus preventing higher product yields with strain AM1. [ABSTRACT FROM AUTHOR]

Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA–derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. 13 C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67 g/L, which was equivalent to an overall yield of 0.055 mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation. [ABSTRACT FROM AUTHOR]

The performance of an exogenous bacterium, Methylobacterium extorquens, in inducing bioxidation of methane from landfill gas (LFG) was assessed in a laboratory scale bioreactor. The study show that enhanced oxidation of methane is attained when the bacteria are introduced into the landfill soil. The maximum percentage reduction of methane fraction from LFG when the bioreactor was inoculated with the methanotrophic bacteria was 94.24 % in aerobic treatment process and 99.97 % in anaerobic process. In the experiments with only the indigenous microorganisms present in the landfill soil, the maximum percentage reduction of methane for the same flow rate of LFG was 59.67 % in aerobic treatment and 45 % in anaerobic treatment. The methane oxidation efficiency of this exogenous methanotrophic bacterium can be considered to be the optimum in anaerobic condition and at a flow rate of 0.6 L/m/min when the removal percentage is 99.95 %. The results substantiate the use of exogenous microorganisms as potential remediation agents of methane in LFG. [ABSTRACT FROM AUTHOR]

Methylobacterium species, the representative bacteria distributed in phyllosphere region of plants, often synthesize carotenoids to resist harmful UV radiations. Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C 30 backbone. However, its exact structure remains unknown. In the present study, the carotenoid produced by M. extorquens AM1 was isolated and its structure was determined as 4-[2- O -11 Z -octadecenoyl-β-glucopyranosyl]-4,4'-diapolycopenedioc acid ( 1 ), a glycosylated C 30 carotenoid. Furthermore, the genes related to the C 30 carotenoid synthesis were investigated. Squalene, the precursor of the C 30 carotenoid, is synthesized by the co-occurrence of META1p1815, META1p1816 and META1p1817. Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1 . By using gene deletion and gene complementation experiments, the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4'-diapolycopene-4,4'-dioic acid to carotenoid 1 . In conclusion, the structure and biosynthetic genes of carotenoid 1 produced by M. extorquens AM1 were firstly characterized in this work, which shed lights on engineering M. extorquens AM1 for producing carotenoid 1 in high yield., Competing Interests: The paper entitled “Characterization of C30 carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1” was submitted to Synthetic and Systems Biotechnology. All authors declare that they do not have any financial or commercial conflict of interest in connection with the work submitted., (© 2023 The Authors.)

Chloromethane is an ozone-depleting gas, produced predominantly from natural sources, that provides an important carbon source for microbes capable of consuming it. Chloromethane catabolism has been difficult to study owing to the challenging genetics of its native microbial hosts. Since the pathways for chloromethane catabolism show evidence of horizontal gene transfer, we reproduced this transfer process in the laboratory to generate new chloromethane-catabolizing strains in tractable hosts. We demonstrate that six putative accessory genes improve chloromethane catabolism, though heterologous expression of only one of the six is strictly necessary for growth on chloromethane. In contrast to growth of Methylobacterium strains with the closely-related compound dichloromethane, we find that chloride export does not limit growth on chloromethane and, in general, that the ability of a strain to grow on dichloromethane is uncorrelated with its ability to grow on chloromethane. This heterologous expression system allows us to investigate the components required for effective chloromethane catabolism and the factors that limit effective catabolism after horizontal transfer.