Methylobacterium Extorquens AM1 Research Articles

Cerium Oxide and Neodymium Oxide Phytoextraction by Ryegrass in Bioenhanced Hydroponic Environments

Sustainable technologies for the recovery of rare earth elements (REE) from waste need to be developed to decrease the volume of ore mining extractions and its negative environmental consequences, while simultaneously restoring previously impacted lands. This is critical due to the extensive application of REE in everyday life from electronic devices to energy and medical technologies, and the dispersed distribution of REE resources in the world. REE recovery by plants has been previously studied but the feasibility of REE phytoextraction from a poorly soluble solid phase (i.e., nanoparticles) by different plant species has been rarely investigated. In this study, the effect of biostimulation and bioaugmentation on phytorecovery of REE nanoparticles (REE-NP) was investigated by exposing ryegrass seeds to REE-NP in hydroponic environments. This was studied in two sets of experiments: bioaugmentation (using CeO2 nanoparticles and Methylobacterium extorquens AM1 pure culture), and biostimulation (using CeO2 or Nd2O3 nanoparticles and endogenous microorganisms). Addition of M. extorquens AM1 in bioaugmentation experiment including 500 mg/L CeO2 nanoparticles could not promote the nanoparticles accumulation in both natural and surface-sterilized treatments. However, it enhanced the translocation of Ce from roots to shoots in sterile samples. Moreover, another REE-utilizing bacterium, Bacillus subtilis, was enriched more than M. extorquens in control samples (no M. extorquens AM1), and associated with 52% and 14% higher Ce extraction in both natural (165 μg/gdried-plant) and surface-sterilized samples (136 μg/gdried-plant), respectively; showing the superior effect of endogenous microorganisms’ enrichment over bioaugmentation in this experiment. In the biostimulation experiments, up to 705 μg/gdried-plant Ce and 19,641 μg/gdried-plant Nd could be extracted when 500 mg/L REE-NP were added. Furthermore, SEM-EDS analysis of the surface and longitudinal cross-sections of roots in Nd2O3 treatments confirmed surface and intracellular accumulation of Nd2O3-NP. These results demonstrate stimulation of endogenous microbial community can lead to an enhanced REE phytoaccumulation.

Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1)

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Scalable and Consolidated Microbial Platform for Rare Earth Element Leaching and Recovery from Waste Sources.

Chemical methods for the extraction and refinement of technologically critical rare earth elements (REEs) are energy-intensive, hazardous, and environmentally destructive. Current biobased extraction systems rely on extremophilic organisms and generate many of the same detrimental effects as chemical methodologies. The mesophilic methylotrophic bacterium Methylobacterium extorquens AM1 was previously shown to grow using electronic waste by naturally acquiring REEs to power methanol metabolism. Here we show that growth using electronic waste as a sole REE source is scalable up to 10 L with consistent metal yields without the use of harsh acids or high temperatures. The addition of organic acids increases REE leaching in a nonspecific manner. REE-specific bioleaching can be engineered through the overproduction of REE-binding ligands (called lanthanophores) and pyrroloquinoline quinone. REE bioaccumulation increases with the leachate concentration and is highly specific. REEs are stored intracellularly in polyphosphate granules, and genetic engineering to eliminate exopolyphosphatase activity increases metal accumulation, confirming the link between phosphate metabolism and biological REE use. Finally, we report the innate ability of M. extorquens to grow using other complex REE sources, including pulverized smartphones, demonstrating the flexibility and potential for use as a recovery platform for these critical metals.

Real flue gas CO2 hydrogenation to formate by an enzymatic reactor using O2- and CO-tolerant hydrogenase and formate dehydrogenase.

It is challenging to capture carbon dioxide (CO2), a major greenhouse gas in the atmosphere, due to its high chemical stability. One potential practical solution to eliminate CO2 is to convert CO2 into formate using hydrogen (H2) (CO2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O2), potential inhibitors. Our proposed CO2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O2, which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H2 and CO2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction. We anticipated that this enzyme system would enable the utilization of diverse H2 and CO2 sources, including waste gases, biomass, and gasified plastics.

Characterization of C30 carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1

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.

Biointeraction of cerium oxide and neodymium oxide nanoparticles with pure culture Methylobacterium extorquens AM1

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 CeO2 or Nd2O3 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/gcell Ce and 154 μg/gcell 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.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life**

AbstractCertain f‐block elements—the lanthanides—have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide‐dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide‐dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one‐carbon metabolism if they possess the correct size and a +III oxidation state.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Over the last decade, the biological relevance of certain f-elements - the lanthanides - was established in the context of methylotrophic bacteria. The respective strains incorporate these 4f-elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. Here, we investigate if actinides, the radioactive 5f-elements, can replace the essential 4f-elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF-mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides, when presented to a mixture of equal amounts of lanthanides together with americium and curium. Bacterial uptake is confirmed by time-resolved laser-induced fluorescence spectroscopy, liquid scintillation counting, and inductively-coupled plasma mass-spectrometry. Additionally, we show catalytic activity of the lanthanide-dependent enzyme upon reconstitution with actinides. Our combined invivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism; if they possess the correct size and a +III oxidation state. Furthermore, bacteria capable of utilizing actinides will be able to mobilize these elements in the environment. This may lead to applications for bioremediation and recycling/separation of lanthanides and actinides.

Co-inoculant Response of Microbial Consortia on Physiology of Blackgram [Vigna mungo (L.) Hepper] Seed Germination

Background: Blackgram (Vigna mungo (L.) Hepper) belonging to the Leguminoceae family is rich in protein. In this crop, biological seed treatment is an environmentally sound approach for improving the vigour of seeds. Besides legume-Rhizobium symbiosis, several other beneficial microbes play a crucial role in vigour enhancement in blackgram. Methods: The surface sterilised seeds were first coated with 20% gum acacia followed by the coating with bioinoculants viz., T0 - Control (Dry seed), T1 - Rhizobium sp. BMBS + Arbuscular Mycorrhizal Fungi (AMF) + Methylobacterium extorquens AM1 and T2 - Rhizobium sp. BMBS + AMF + Bacillus velezensis. Result: Inoculation of blackgram seeds with the Rhizobium sp. BMBS and AMF with Methylobacterium extorquens AM1 resulted in a significant increase in germination (97%), dry matter production (0.237 g 10 seedlings-1), vigour index (22.99) and seed mobilization efficiency (1.11) as compared to control seeds. The biochemical parameters such as á-amylase and proteases were found to be significantly higher in the Rhizobium sp. BMBS + AMF + Methylobacterium extorquens AM1 inoculated seeds. Changes in root exudates composition due to co-inoculation assessed through GC-MS, indicated compounds with antioxidant and antimicrobial activities. Our results confirmed that the positive interaction of rhizobial strain BMBS and AMF with Methylobacterium extorquens AM1 may emerge as a novel bio-inoculant for sustainable pulse productivity.

Sodium formate redirects carbon flux and enhances heterologous mevalonate production in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 (AM1), a model strain of methylotrophic cell factories (MeCFs) could be used to produce fine chemicals from methanol. Synthesis of heterologous products usually needs reducing cofactors, but AM1 growing on methanol lack reducing power. Formate could be used as a reducing agent. In this study, mevalonic acid (MEV) yield of 0.067 gMEV/g methanol was reached by adding 10mmol L-1 sodium formate in MEV accumulating stage (at 72h). The yield was improved by 64.57%, and represented the highest yield reported to date. 13 C-labeling experiments revealed global effects of sodium formate on metabolic pathways in engineered Methylobacterium extorquens AM1. Sodium formate significantly increased the ratios of reducing equivalents, enhanced the metabolic rate of pathways demanding reducing cofactors and redirected the carbon flux to MEV synthesis. As a result, coupling formate to methanol-based production provide a promising way for converting C1 substances to useful chemical products.

Structural insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.

Construction of a Bioelectrochemical Dihydrogen/Formate Interconversion System and a Bio-Solar Cell

Modern societies have energy and environmental issues such as fossil fuel depletion and excessive carbon dioxide (CO2) emissions. Therefore, an effective use of renewable energy resources instead of fossil fuels is essential for the development of a sustainable energy economy. Particularly, dihydrogen (H2), formic acid (HCOOH) and sunlight are expected to be new energy sources. These advantages are as follows; H2 emits no harmful substances in use; HCOOH has a high energy density and is a liquid fuel that can be easily stored and transported; sunlight is universally available on the ground. However, the electrochemical conversion of the above energy has the following problems; a catalyst for H2 oxidation, platinum, is expensive; byproducts such as carbon monoxide are generated in CO2 reduction at usual electrodes; solar cell materials are sometimes expensive and environmentally harmful. In order to overcome these problems, we focused on biocatalysts, which are ecofriendly, and have high catalytic performance and selectivity under mild conditions. Furthermore, we constructed two electro-enzymatic energy conversion devices: an H2/HCOO- interconversion system and a bio-solar cell.In an H2/HCOO- interconversion system, we applied two enzymes which catalyze bidirectional redox reactions: [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F (H2ase; EC 1.12.2.1) and tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1 (FoDH1; EC 1.2.1.2). H2ase and FoDH1 catalyze the H+/H2 and CO2/HCOO- interconversion, respectively, and we attempted to generate HCOO- without external power supply by the coupling of two bioelectrocatalytic reactions: direct electron transfer (DET)-type H2 oxidation catalyzed by H2ase and mediated electron transfer (MET)-type CO2 reduction catalyzed by FoDH1 using benzyl viologen (BV) as a mediator (H2 + CO2 → HCOO- + H+). H2ase was adsorbed on a bioanode functionalized with p-phenylenediamine, which leads to orientations suitable for DET-type bioelectrocatalysis of H2ase, while FoDH1 and BV were adsorbed on a biocathode. In addition, gas-diffusion-type electrodes were employed for high-speed supply of gaseous substrates. The two electrodes were short-circuited in a cell, and CO2 and H2 gasses were spontaneously supplied from the outside of the biocathode and the bioanode, respectively. The amount of formate in the electrolyte solution increased almost linearly with the reaction time. On the other hand, when the feed gasses were replaced with Ar gas to remove CO2 and H2 from the system, the amount of formate decreased with the time. This is due to the reversed reaction: (HCOO- + H+ → H2 + CO2). These results mean that the difference in equilibrium potentials of the H+/H2 and CO2/HCOO- couples determines the driving force of the reaction, which is reversed depending on conditions, and the interconversion between H2 and HCOO- was experimentally demonstrated.A bio-solar cell is an electric energy device mimicking natural photosynthesis and extracts excited electrons outside the photosynthetic system. In this study, we constructed a bio-solar cell using the thylakoid membrane from spinach as an anodic photo-bioelectrocatalyst which oxidizes water (H2O) to dioxygen (O2) and the recombinant bilirubin oxidase (BOD; EC 1.3.3.5) produced by expressing the gene of BOD from Myrothecium verucarria in Pichia pastoris as a cathodic bioelectrocatalyst which reduces O2 to H2O. In addition, we investigated the characteristics of a thylakoid membrane-functionalized bioanode with a mediator of hexaammineruthenium(III/II) ion ([Ru(NH3)6]3+/2+), which has the following characteristics: fast electrode kinetics, high stability and solubility, and somewhat low redox potential. The photo-driven bioanode prepared by applying the mixture of thylakoid membranes and water-dispersed multi-walled carbon nanotubes on a gold-spattered indium tin oxide (ITO) electrode showed the photocurrent caused by MET-type photo-bioelectrocatalytic H2O oxidation in a buffer (pH 7.0) containing 5 mM Ru(NH3)6Cl3 under the light condition. The performance of the bioanode (0.2 mA cm- 2 at 0 V vs. Ag|AgCl|sat. KCl) is better than that in the previous report using 1,2-naphthoquinone as a mediator. Then, a bio-solar cell was constructed by combining the bioanode with the BOD-modified gas-diffusion-type biocathode, and O2 was spontaneously supplied from the outside of the biocathode. The cell exhibited an open-circuit voltage of 0.61 V and a maximum power density of 50 μW cm- 2 at a cell voltage of 0.42 V under quiescent conditions. To the best of our knowledge, the power density of our cell unit is the highest among those reported for the bio-solar cells to date.These works lead to the artificial photosynthetic system, which generates H2 or HCOO- from sunlight and H2O (and CO2), and they might be a breakthrough for the sustainable energy economy.

Molecular characterization of native pink pigmented facultative methylotrophs of chilli and their induced systemic resistance mechanism in management of anthracnose

Aim: This study aimed to assess the plant growth promotional ability and biocontrol efficiency of native pink pigmented facultative methylotrophs (PPFMs) of chilli for management of chilli anthracnose disease through induced systemic resistance mechanism under pot culture experiments. Methodology: The selected native PPFMs were identified by analysis of 16S rRNA gene sequence. These isolates along with reference strain (Methylobacterium extorquens AM1) were inoculated to healthy chilli plants. The pathogen (Colletotrichum capsici) was later inoculated to these chilli plants, and induced systemic resistance (ISR) molecules were estimated at different intervals from one day after challenge inoculation of pathogen (DAI) upto 7 DAI. Influence of PPFM on plant growth parameters, yield, capsaicin content and PPFMs population load on phyllosphere and rhizosphere was studied. Results: The results of 16S rRNA gene sequence analysis revealed selected isolates as Methylobacterium populi (PPFM6) and Methylobacterium radiotolerans (PPFM170). The ISR molecules tested were significantly influenced by PPFM isolates. The peroxidase and phenyl alanine ammonia lyase (PALase) activity increased from 1 DAI to 7 DAI whereas chitinase, polyphenol oxidase (PPO) and phenol contents increased upto 5 DAI, after which gradual decrease was noticed. Inoculation of PPFMs to chilli crop significantly improved plant height, dry matter and chlorophyll content. Significantly lesser disease incidence, more yield and more capsaicin content was observed with application of M. populi as compared to control. Interpretation: The present study highlights native M. populi (PPFM6) of chilli as an effective plant growth promoter, exhibiting significant biocontrol efficiency against C. capsici.

Coupled Electrochemical and Microbial Catalysis for the Production of Polymer Bricks.

Power‐to‐X technologies have the potential to pave the way towards a future resource‐secure bioeconomy as they enable the exploitation of renewable resources and CO2. Herein, the coupled electrocatalytic and microbial catalysis of the C5‐polymer precursors mesaconate and 2S‐methylsuccinate from CO2 and electric energy by in situ coupling electrochemical and microbial catalysis at 1 L‐scale was developed. In the first phase, 6.1±2.5 mm formate was produced by electrochemical CO2 reduction. In the second phase, formate served as the substrate for microbial catalysis by an engineered strain of Methylobacterium extorquens AM‐1 producing 7±2 μm and 10±5 μm of mesaconate and 2S‐methylsuccinate, respectively. The proof of concept showed an overall conversion efficiency of 0.2 % being 0.4 % of the theoretical maximum.

Expression of Formate-Tetrahydrofolate Ligase Did Not Improve Growth but Interferes With Nitrogen and Carbon Metabolism of Synechocystis sp. PCC 6803.

The introduction of alternative CO2-fixing pathways in photoautotrophic organism may improve the efficiency of biological carbon fixation such as minimizing the carbon loss due to photorespiration. Here, we analyzed the effects of creating a formate entry point into the primary metabolism of the cyanobacterium Synechocystis sp. PCC 6803. The formate-tetrahydrofolate ligase (FTL) from Methylobacterium extorquens AM1 was expressed in Synechocystis to enable formate assimilation and reducing the loss of fixed carbon in the photorespiratory pathway. Transgenic strains accumulated serine and 3-phosphoglycerate, and consumed more 2-phosphoglycolate and glycine, which seemed to reflect an efficient utilization of formate. However, labeling experiments showed that the serine accumulation was not due to the expected incorporation of formate. Subsequent DNA-microarray analysis revealed profound changes in transcript abundance due to ftl expression. Transcriptome changes were observed in relation to serine and glycine metabolism, C1-metabolism and particularly nitrogen assimilation. The data implied that ftl expression interfered with the signaling the carbon/nitrogen ratio in Synechocystis. Our results indicate that the expression of new enzymes could have a severe impact on the cellular regulatory network, which potentially hinders the establishment of newly designed pathways.

Adaptively evolved Escherichia coli for improved ability of formate utilization as a carbon source in sugar-free conditions

BackgroundFormate converted from CO2 reduction has great potential as a sustainable feedstock for biological production of biofuels and biochemicals. Nevertheless, utilization of formate for growth and chemical production by microbial species is limited due to its toxicity or the lack of a metabolic pathway. Here, we constructed a formate assimilation pathway in Escherichia coli and applied adaptive laboratory evolution to improve formate utilization as a carbon source in sugar-free conditions.ResultsThe genes related to the tetrahydrofolate and serine cycles from Methylobacterium extorquens AM1 were overexpressed for formate assimilation, which was proved by the 13C-labeling experiments. The amino acids detected by GC/MS showed significant carbon labeling due to biomass production from formate. Then, 150 serial subcultures were performed to screen for evolved strains with improved ability to utilize formate. The genomes of evolved mutants were sequenced and the mutations were associated with formate dehydrogenation, folate metabolism, and biofilm formation. Last, 90 mg/L of ethanol production from formate was achieved using fed-batch cultivation without addition of sugars.ConclusionThis work demonstrates the effectiveness of the introduction of a formate assimilation pathway, combined with adaptive laboratory evolution, to achieve the utilization of formate as a carbon source. This study suggests that the constructed E. coli could serve as a strain to exploit formate and captured CO2.

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide-Dependent Methanol Dehydrogenase Activity.

Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with LaIII , CeIII , and NdIII using their physiological electron acceptor, XoxG, display Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG-XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.

Continuous Culture Adaptation of Methylobacterium extorquens AM1 and TK 0001 to Very High Methanol Concentrations.

The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O-acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O-acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently.

A Periplasmic Binding Protein for Pyrroloquinoline Quinone.

Pyrroloquinoline quinone (PQQ) is an essential redox cofactor in bacterial calcium- and lanthanide-dependent alcohol dehydrogenases. Although certain bacteria are known to synthesize and secrete PQQ, little is known about trafficking of this cofactor within and between cells. Here, we show that a previously uncharacterized periplasmic (solute) binding protein from Methylobacterium extorquens AM1, here renamed PqqT, binds 1 equiv of PQQ with high affinity ( Kd = 50 nM). UV-visible and spectrofluorometric titrations establish that PqqT binds an unhydrated form of PQQ with distinct spectral features from the cofactor in free solution. To our knowledge, PqqT is the first solute-binding protein identified for PQQ and the first protein implicated in cellular trafficking of the cofactor. We propose that PqqT, which is encoded adjacent to a putative ATP-binding cassette transporter in the M. extorquens genome, is involved in uptake of exogenous PQQ to supplement endogenous cofactor biosynthesis. These results support the emerging importance of PQQ transfer within microbial and microbe-host communities.

Designing and Engineering Methylorubrum extorquens AM1 for Itaconic Acid Production

Methylorubrum extorquens (formerly Methylobacterium extorquens) AM1 is a methylotrophic bacterium with a versatile lifestyle. Various carbon sources including acetate, succinate and methanol are utilized by M. extorquens AM1 with the latter being a promising inexpensive substrate for use in the biotechnology industry. Itaconic acid (ITA) is a high-value building block widely used in various industries. Given that no wildtype methylotrophic bacteria are able to utilize methanol to produce ITA, we tested the potential of M. extorquens AM1 as an engineered host for this purpose. In this study, we successfully engineered M. extorquens AM1 to express a heterologous codon-optimized gene encoding cis-aconitic acid decarboxylase. The engineered strain produced ITA using acetate, succinate and methanol as the carbon feedstock. The highest ITA titer in batch culture with methanol as the carbon source was 31.6 ± 5.5 mg/L, while the titer and productivity were 5.4 ± 0.2 mg/L and 0.056 ± 0.002 mg/L/h, respectively, in a scaled-up fed-batch bioreactor under 60% dissolved oxygen saturation. We attempted to enhance the carbon flux toward ITA production by impeding poly-β-hydroxybutyrate accumulation, which is used as carbon and energy storage, via mutation of the regulator gene phaR. Unexpectedly, ITA production by the phaR mutant strain was not higher even though poly-β-hydroxybutyrate concentration was lower. Genome-wide transcriptomic analysis revealed that phaR mutation in the ITA-producing strain led to complex rewiring of gene transcription, which might result in a reduced carbon flux toward ITA production. Besides poly-β-hydroxybutyrate metabolism, we found evidence that PhaR might regulate the transcription of many other genes including those encoding other regulatory proteins, methanol dehydrogenases, formate dehydrogenases, malate:quinone oxidoreductase, and those synthesizing pyrroloquinoline quinone and thiamine co-factors. Overall, M. extorquens AM1 was successfully engineered to produce ITA using acetate, succinate and methanol as feedstock, further supporting this bacterium as a feasible host for use in the biotechnology industry. This study showed that PhaR could have a broader regulatory role than previously anticipated, and increased our knowledge of this regulator and its influence on the physiology of M. extorquens AM1.