Nature-Inspired Strategies for Sustainable Degradation of Synthetic Plastics.

Synthetic plastics have become integral to our daily lives, yet their escalating production, limited biodegradability, and inadequate waste management contribute to environmental contamination. Biological plastic degradation is one promising strategy to address this pollution. The inherent chemical and physical properties of synthetic plastics, however, pose challenges for microbial enzymes, hindering the effective degradation and the development of a sustainable biological recycling process.

Heme o Isolation and Analysis Using Heme o Synthase Heterologously Expressed in Escherichia coli.

Heme o is an Fe-porphyrin involved in the majority of aerobic respiration pathways found in all three domains of life. In eukaryotes and most aerobic prokaryotes, heme o functions solely as the precursor for the synthesis of heme a, a necessary cofactor for most heme-copper terminal oxidases. In some prokaryotes, such as Escherichia coli (E.

Recent mechanistic developments for cytochrome c nitrite reductase, the key enzyme in the dissimilatory nitrate reduction to ammonium pathway.

Cytochrome c nitrite reductase, NrfA, is a soluble, periplasmic pentaheme cytochrome responsible for the reduction of nitrite to ammonium in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway, a vital reaction in the global nitrogen cycle. NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we summarize the latest progress in elucidating the reaction mechanism of ammonia production, including new findings about the active site architecture of NrfA, as well as recent results that elucidate electron transfer and storage in the pentaheme scaffold of this enzyme.

Evidence that the catalytic mechanism of heme a synthase involves the formation of a carbocation stabilized by a conserved glutamate.

In eukaryotes and many aerobic prokaryotes, the final step of aerobic respiration is catalyzed by an aa-type cytochrome c oxidase, which requires a modified heme cofactor, heme a. The conversion of heme b, the prototypical cellular heme, to heme o and ultimately to heme a requires two modifications, the latter of which is conversion of a methyl group to an aldehyde, catalyzed by heme a synthase (HAS). The N- and C-terminal halves of HAS share homology, and each half contains a heme-binding site.

Technoeconomic evaluation of recent process improvements in production of sugar and high-value lignin co-products via two-stage Cu-catalyzed alkaline-oxidative pretreatment.

Background: A lignocellulose-to-biofuel biorefinery process that enables multiple product streams is recognized as a promising strategy to improve the economics of this biorefinery and to accelerate technology commercialization. We recently identified an innovative pretreatment technology that enables of the production of sugars at high yields while simultaneously generating a high-quality lignin stream that has been demonstrated as both a promising renewable polyol replacement for polyurethane applications and is highly susceptible to depolymerization into monomers. This technology comprises a two-stage pretreatment approach that includes an alkaline pre-extraction followed by a metal-catalyzed alkaline-oxidative pretreatment.

Skeletal Ni electrode-catalyzed C-O cleavage of diaryl ethers entails direct elimination via benzyne intermediates.

Diaryl ethers undergo electrocatalytic hydrogenolysis (ECH) over skeletal Ni cathodes in a mild, aqueous process that achieves direct C-O cleavage without initial benzene ring saturation. Mechanistic studies find that aryl phenyl ethers with a single para or meta functional group (methyl, methoxy, or hydroxy) are selectively cleaved to the substituted benzene and phenol, in contrast to recently reported homogeneous catalytic cleavage processes. Ortho positioning of substituents reverses this C-O bond selectivity, except for the 2-phenoxyphenol case.

From Lignin to Valuable Aromatic Chemicals: Lignin Depolymerization and Monomer Separation via Centrifugal Partition Chromatography.

Lignin has long been recognized as a potential feedstock for aromatic molecules; however, most lignin depolymerization methods create a complex mixture of products. The present study describes an alkaline aerobic oxidation method that converts lignin extracted from poplar into a collection of oxygenated aromatics, including valuable commercial compounds such as vanillin and -hydroxybenzoic acid. Centrifugal partition chromatography (CPC) is shown to be an effective method to isolate the individual compounds from the complex product mixture.

Biosynthesis and trafficking of heme and heme : new structural insights and their implications for reaction mechanisms and prenylated heme transfer.

Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases contain a prenylated heme (typically heme or occasionally heme ) in the active site.

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome Nitrite Reductase (NrfA).

Cytochrome nitrite reductases (CNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time.

Nucleophilic Thiols Reductively Cleave Ether Linkages in Lignin Model Polymers and Lignin.

Lignin may serve as a renewable feedstock for the production of chemicals and fuels if mild, scalable processes for its depolymerization can be devised. The use of small organic thiols represents a bioinspired strategy to cleave the β-O-4 bond, the most common linkage in lignin. In the present study, synthetic β-O-4 linked polymers were treated with organic thiols, yielding up to 90 % cleaved monomer products.

Cytochrome nitrite reductase from the bacterium represents a new NrfA subclass.

Cytochrome nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized NrfA.

Multiple Mechanisms Mapped in Aryl Alkyl Ether Cleavage via Aqueous Electrocatalytic Hydrogenation over Skeletal Nickel.

We present here detailed mechanistic studies of electrocatalytic hydrogenation (ECH) in aqueous solution over skeletal nickel cathodes to probe the various paths of reductive catalytic C-O bond cleavage among functionalized aryl ethers relevant to energy science. Heterogeneous catalytic hydrogenolysis of aryl ethers is important both in hydrodeoxygenation of fossil fuels and in upgrading of lignin from biomass. The presence or absence of simple functionalities such as carbonyl, hydroxyl, methyl, or methoxyl groups is known to cause dramatic shifts in reactivity and cleavage selectivity between sp C-O and sp C-O bonds.

PreDSLpmo: A neural network-based prediction tool for functional annotation of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs), a family of copper-dependent oxidative enzymes, boost the degradation of polysaccharides such as cellulose, chitin, and others. While experimental methods are used to validate LPMO function, a computational method that can aid experimental methods and provide fast and accurate classification of sequences into LPMOs and its families would be an important step towards understanding the breadth of contributions these enzymes make in deconstruction of recalcitrant polysaccharides. In this study, we developed a machine learning-based tool called PreDSLpmo that employs two different approaches to functionally classify protein sequences into the major LPMO families (AA9 and AA10).

Performance of three delignifying pretreatments on hardwoods: hydrolysis yields, comprehensive mass balances, and lignin properties.

Background: In this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading.

Biomimetic Reductive Cleavage of Keto Aryl Ether Bonds by Small-Molecule Thiols.

The nucleophilic and reductive properties of thiolates and thiols make them ideal candidates as redox mediators via the thiol/disulfide couple. One mechanism for biological lignin depolymerization entails reduction of keto aryl ether bonds by an S 2 mechanism with the thiol redox mediator glutathione. In this study, mimicking this chemistry in a simple protein- and metal-free process, several small organic thiols are surveyed for their ability to cleave aryl keto ethers that model the β-O-4 linkages found in partially oxidized lignin.

Cox15 interacts with the cytochrome dimer within respiratory supercomplexes as well as in the absence of cytochrome oxidase.

The heme molecule is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome oxidase. Heme is synthesized from heme by a multi-spanning inner membrane protein, heme synthase (Cox15 in the yeast ). The insertion of heme is critical for cytochrome oxidase function and assembly, but this process has not been fully elucidated.

Enhancement of sugar recovery and ethanol production from wheat straw through alkaline pre-extraction followed by steam pretreatment.

To improve sugar recovery and ethanol production from wheat straw, a sequential two-stage pretreatment process combining alkaline pre-extraction and acid catalyzed steam treatment was investigated. The results showed that alkaline pre-extraction using 8% (w/w) sodium hydroxide at 80 °C for 90 min followed by steam pretreatment with 3% (w/w) sulfur dioxide at 151 °C for 16 min was sufficient to prepare a substrate that could be efficiently hydrolyzed at high solid loadings. Moreover, alkaline pre-extraction reduced the process severity of steam pretreatment and decreased the generation of inhibitory compounds.

Integrated experimental and technoeconomic evaluation of two-stage Cu-catalyzed alkaline-oxidative pretreatment of hybrid poplar.

Background: When applied to recalcitrant lignocellulosic feedstocks, multi-stage pretreatments can provide more processing flexibility to optimize or balance process outcomes such as increasing delignification, preserving hemicellulose, and maximizing enzymatic hydrolysis yields. We previously reported that adding an alkaline pre-extraction step to a copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment process resulted in improved sugar yields, but the process still utilized relatively high chemical inputs (catalyst and HO) and enzyme loadings. We hypothesized that by increasing the temperature of the alkaline pre-extraction step in water or ethanol, we could reduce the inputs required during Cu-AHP pretreatment and enzymatic hydrolysis without significant loss in sugar yield.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.

Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase.

The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery.

Isotopic fractionation associated with [NiFe]- and [FeFe]-hydrogenases.

Rationale: Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling.

Methods: We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases.

Effective alkaline metal-catalyzed oxidative delignification of hybrid poplar.

Background: Strategies to improve copper-catalyzed alkaline hydrogen peroxide (Cu-AHP) pretreatment of hybrid poplar were investigated. These improvements included a combination of increasing hydrolysis yields, while simultaneously decreasing process inputs through (i) more efficient utilization of H2O2 and (ii) the addition of an alkaline extraction step prior to the metal-catalyzed AHP pretreatment. We hypothesized that utilizing this improved process could substantially lower the chemical inputs needed during pretreatment.

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster.

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell.

Chemical and structural changes associated with Cu-catalyzed alkaline-oxidative delignification of hybrid poplar.

Background: Alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2'-bipyridine complexes has previously been determined to substantially improve the enzymatic hydrolysis of woody plants including hybrid poplar as a consequence of moderate delignification. In the present work, cell wall morphological and lignin structural changes were characterized for this pretreatment approach to gain insights into pretreatment outcomes and, specifically, to identify the extent and nature of lignin modification.

Results: Through TEM imaging, this catalytic oxidation process was shown to disrupt cell wall layers in hybrid poplar.