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Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences., (© 2022. The Author(s).)

Methanococcus maripaludis is a rapidly growing, hydrogenotrophic, and genetically tractable methanogen with unique capabilities to convert formate and CO 2 to CH 4 . The existence of genome-scale metabolic models and an established, robust system for both large-scale and continuous cultivation make it amenable for industrial applications. However, the lack of molecular tools for differential gene expression has hindered its application as a microbial cell factory to produce biocatalysts and biochemicals. In this study, a library of differentially regulated promoters was designed and characterized based on the pst promoter, which responds to the inorganic phosphate concentration in the growth medium. Gene expression increases by 4- to 6-fold when the medium phosphate drops to growth-limiting concentrations. Hence, this regulated system decouples growth from heterologous gene expression without the need for adding an inducer. The minimal pst promoter is identified and contains a conserved AT-rich region, a factor B recognition element, and a TATA box for phosphate-dependent regulation. Rational changes to the factor B recognition element and start codon had no significant impact on expression; however, changes to the transcription start site and the 5' untranslated region resulted in the differential protein production with regulation remaining intact. Compared to a previous expression system based upon the histone promoter, this regulated expression system resulted in significant improvements in the expression of a key methanogenic enzyme complex, methyl-coenzyme M reductase, and the potentially toxic arginine methyltransferase MmpX.

Iron-sulfur (Fe-S) clusters are among the oldest and most versatile cofactors present in all domains of life. Many bacterial and eukaryotic Fe-S proteins have been well-characterized, whereas the archaeal ones are less studied. Fe-S proteins are particularly abundant and play essential roles in methanogenic archaea. Methanococcus maripaludis is a model methanogen with available genetic tools. Here, we describe the techniques for anaerobic cultivation of M. maripaludis with formate, liposome-mediated transformation, expression and anoxic affinity purification of Fe-S proteins, Fe-S cluster reconstitution, and analysis of Fe-S proteins by UV-visible absorption spectroscopy., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Efficient encapsulation of tetraethylenepentamine (TEPA), as an example aliphatic amine, was achieved by an emulsion-templated, in situ polymerization. Hydrophobically modified clay nanoplatelets were employed as emulsifiers to obtain water-in-oil (W/O) dispersions followed by interfacial polymerization between a portion of the TEPA cargo and polymethylene polyphenylene isocyanate (PMPPI). The resultant capsules exhibit spherical shape, desirable thermal stability, modest barrier properties, and shear-induced release in an epoxide monomer mixture. Most importantly, a significant gain in capsule barrier properties was realized by introducing poly(allyl amine) (pAAm) as an interface-selective reactive additive in the Pickering emulsions. In addition to the fundamental interest of pAAm localization and interface-selective reactivity, this microencapsulation system for aliphatic amines has technological potential in coating, self-healing, and drug-delivery applications.

Aluminum coordination in the framework of USY and ZSM-5 zeolites containing charge-compensating cations (NH4+, H+, or Cu+) was investigated by Al K-edge EXAFS and XANES. This work was performed using a newly developed in-situ cell designed especially for acquiring soft X-ray absorption data. Both tetrahedrally and octahedrally coordinated Al were observed for hydrated H-USY and H-ZSM-5, in good agreement with 27Al NMR analyses. Upon dehydration, water desorbed from the zeolite, and octahedrally coordinated Al was converted progressively to tetrahedrally coordinated Al. These observations confirmed the hypothesis that the interaction of water with Brønsted acid protons can lead to octahedral coordination of Al without loss of Al from the zeolite lattice. When H+ is replaced with NH4+ or Cu+, charge compensating species that absorb less water, less octahedrally coordinated Al was observed. Analysis of Al K-edge EXAFS data indicates that the Al-O bond distance for tetrahedrally coordinated Al in dehydrated USY and ZSM-5 is 1.67 angstroms. Simulation of k3chi(k) for Cu+ exchanged ZSM-5 leads to an estimated distance between Cu+ and framework Al atoms of 2.79 angstroms.

Cu-exchanged Y zeolite was investigated in order to determine the location of the copper cations relative to the zeolite framework and to determine which Cu cations are active for the oxidative carbonylation of methanol to dimethyl carbonate (DMC). Cu-Y zeolite was prepared by vapor-phase exchange of H-Y with CuCl. The oxidation state, local coordination, and bond distances of Al and Cu were determined using Al K-edge and Cu K-edge X-ray absorption spectroscopy (XAS). Complimentary information was obtained by H2 temperature-programmed reduction and by in-situ infrared spectroscopy. Cu-Y has a Cu/Al ratio of unity and very little occluded CuCl. The average Al-O and Al-Cu bond distances are 1.67 angstroms and 2.79 angstroms, respectively, and the average Cu-O and Cu-Si(Al) bond distances are 1.99 angstroms and 3.13 angstroms, respectively. All of the Cu exchanged is present as Cu+ in sites I', II, and III'. Cu-Y is active for the oxidative carbonylation of methanol, and at low reactant contact time produces DMC as the primary product. With increasing reactant contact time, DMC formation decreases in preference to the formation of dimethoxy methane (DMM) and methylformate (MF). The formation of DMM and MF is attributed to the hydrogenation of DMC and the hydrogenolysis of DMM, respectively. Observation of the catalyst under reaction conditions reveals that most of the copper cations remain as Cu+, but some oxidation of Cu+ to Cu2+ does occur. It is also concluded that only those copper cations present in site II and III' positions are accessible to the reactants, and hence are catalytically active. The dominant adsorbed species on the surface are methoxy groups, and adsorbed CO is present as a minority species. The relationship of these observations to the kinetics of DMC synthesis is discussed.

A new pseudo-tetrahedral Co(II) complex has been grafted onto the surface of SBA-15 and successfully utilized for the catalytic oxidization of alkylaromatic substrates with tert-butyl hydroperoxide (TBHP)via an H-atom transfer mechanism.

A nonaqueous molecular precursor grafting approach was employed for the generation of well-defined surface structures featuring Cu on a mesoporous silica support. X-ray absorption measurements (XANES and EXAFS) were used to determine that [CuOSi(OtBu)3]4 provided 100% isolated Cu(I) sites upon grafting (without thermal treatment), whereas [CuOtBu]4 gave isolated species with most of the original Cu-O-Cu linkages intact, but in a more relaxed straight chain form. Upon heating under inert conditions, the vast majority of Cu in the materials from [CuOSi(OtBu)3]4 remained as isolated Cu(I) sites (up to 88% isolation), with significant stabilization provided from the -OSi(OtBu)3 ligands. In stark contrast, approximately 100% of the Cu in the materials generated from [CuOtBu]4 was readily reduced upon heating, forming isolated Cu metal particles with an average diameter of 0.7 nm.