Complex waste stream valorization through combined enzymatic hydrolysis and catabolic assimilation by Pseudomonas putida.

Biogenic waste-derived feedstocks for production of fuels, chemicals, and materials offer great potential supporting the transition to net-zero and greater circularity. However, such feedstocks are heterogeneous and subject to geographical and seasonal variability. Here, we show that, through careful strain selection and metabolic engineering, Pseudomonas putida can be employed to permit efficient co-utilization of highly heterogeneous substrate compositions derived from hydrolyzed mixed municipal-like waste fractions (food, plastic, organic, paper, cardboard, and textiles) for growth and synthesis of exemplar bioproducts.

Kilo-Scale GMP Synthesis of Renewable Semisynthetic Vaccine-Grade Squalene.

Emulsions of the triterpene squalene ((6,10,14,18)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, CAS 111-02-4) have been used as adjuvants in influenza vaccines since the 1990s. Traditionally sourced from shark liver oil, the overfishing of sharks and concomitant reduction in the oceanic shark population raises sustainability issues for vaccine adjuvant grade squalene. We report a semisynthetic route to squalene meeting current pharmacopeial specifications for use in vaccines that leverages the ready availability of -β-farnesene ((6)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, CAS 18794-84-8), manufactured from sustainable sugarcane via a yeast fermentation process.

The prFMNH-binding chaperone LpdD assists UbiD decarboxylase activation.

The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution. However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical Escherichia coli UbiD.

Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study.

Semi-synthetic terpenoids with differential adjuvant properties as sustainable replacements for shark squalene in vaccine emulsions.

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and β-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene.

Heterologous production and biophysical characterization of catabolic Nitratireductor pacificus pht-3B reductive dehalogenase.

Reductive dehalogenases provide a possible route to the biotechnological remediation of widespread anthropogenic environmental organohalide contamination. These bacterial enzymes employ cobalamin and an internal electron transfer chain of two [4Fe-4S] clusters to remove halide ions from organohalides, leaving an organic molecule more amenable to further transformations. Detailed protocols for the cloning, heterologous expression, purification, crystallization and characterization of the catabolic dehalogenase from Nitratireductor pacificus pht-3B (NpRdhA) are presented, together with insight into enzyme turnover, substrate selectivity and the use of electron paramagnetic resonance (EPR) spectroscopy as an active site probe.

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase.

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied.

Structural basis of terephthalate recognition by solute binding protein TphC.

Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC.

UbiD domain dynamics underpins aromatic decarboxylation.

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge.

A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code.

Structure and Mechanism of PA0254/HudA, a prFMN-Dependent Pyrrole-2-carboxylic Acid Decarboxylase Linked to Virulence.

The UbiD family of reversible (de)carboxylases depends on the recently discovered prenylated-FMN (prFMN) cofactor for activity. The model enzyme ferulic acid decarboxylase (Fdc1) decarboxylates unsaturated aliphatic acids via a reversible 1,3-cycloaddition process. Protein engineering has extended the Fdc1 substrate range to include (hetero)aromatic acids, although catalytic rates remain poor.

The In Vitro Production of prFMN for Reconstitution of UbiD Enzymes.

Prenylated flavin (prFMN) is a modified FMN cofactor, the isoalloxazine is extended by an additional six membered nonaromatic ring. The modification confers azomethine ylide characteristics on the oxidised prFMN, allowing it to support the reversible nonoxidative decarboxylation of unsaturated acids by the UbiD family of decarboxylases. In absence of a chemical synthesis route for prFMN, enzymatic production by the flavin prenyltransferase, UbiX, is required for in vitro reconstitution of prFMN-dependent enzymes.

Estimation of elastic properties of an additively manufactured lattice using resonant ultrasound spectroscopy.

Development of improved approaches in the characterization of additively manufactured structures continues to be a topic of interest for the advanced manufacturing community. This article will investigate an approach using resonant ultrasound spectroscopy (RUS) to determine the effective elastic constants of an orthotropic lattice structure. The evaluation is performed on a cube shaped 316 L stainless steel test specimen, constructed using selective laser melting techniques.

Catabolic Reductive Dehalogenase Substrate Complex Structures Underpin Rational Repurposing of Substrate Scope.

Reductive dehalogenases are responsible for the reductive cleavage of carbon-halogen bonds during organohalide respiration. A variety of mechanisms have been proposed for these cobalamin and [4Fe-4S] containing enzymes, including organocobalt, radical, or cobalt-halide adduct based catalysis. The latter was proposed for the oxygen-tolerant cataboli reductive dehalogenase (NpRdhA).

Heterologous expression of cobalamin dependent class-III enzymes.

The family of cobalamin class-III dependent enzymes is composed of the reductive dehalogenases (RDases) and related epoxyqueuosine reductases. RDases are crucial for the energy conserving process of organohalide respiration. These enzymes have the ability to reductively cleave carbon-halogen bonds, present in a number of environmentally hazardous pollutants, making them of significant interest for bioremediation applications.

Ferulic Acid Decarboxylase Controls Oxidative Maturation of the Prenylated Flavin Mononucleotide Cofactor.

Prenylated flavin mononucleotide (prFMN) is a recently discovered modified flavin cofactor containing an additional nonaromatic ring, connected to the N5 and C6 atoms. This cofactor underpins reversible decarboxylation catalyzed by members of the widespread UbiD enzyme family and is produced by the flavin prenyltransferase UbiX. Oxidative maturation of the UbiX product prFMNH to the corresponding oxidized prFMN is required for ferulic acid decarboxylase (Fdc1; a UbiD-type enzyme) activity.

Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code.

Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function.

Enzymatic control of cycloadduct conformation ensures reversible 1,3-dipolar cycloaddition in a prFMN-dependent decarboxylase.

The UbiD enzyme plays an important role in bacterial ubiquinone (coenzyme Q) biosynthesis. It belongs to a family of reversible decarboxylases that interconvert propenoic or aromatic acids with the corresponding alkenes or aromatic compounds using a prenylated flavin mononucleotide cofactor. This cofactor is suggested to support (de)carboxylation through a reversible 1,3-dipolar cycloaddition process.

Arginine to Lysine Mutations Increase the Aggregation Stability of a Single-Chain Variable Fragment through Unfolded-State Interactions.

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here, we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behavior of scFv mutants harboring single or multiple site-specific arginine to lysine mutations in denaturing buffers.

Inspection of plate weldments through an interposed layer of viscoelastic material using an elastic-electromagnetic scanning method.

In this article, an elastic-microwave based non-destructive evaluation method is presented to inspect for cracks in weldments and thinning of coated steel plates. The approach uses a microwave interferometer operating at 94 GHz to record the total surface displacement of a coated steel plated as it is driven by an incident elastic field. These spatiotemporal data coupled with wavefield processing algorithms provide powerful detection and localization capabilities.

The UbiX flavin prenyltransferase reaction mechanism resembles class I terpene cyclase chemistry.

The UbiX-UbiD enzymes are widespread in microbes, acting in concert to decarboxylate alpha-beta unsaturated carboxylic acids using a highly modified flavin cofactor, prenylated FMN (prFMN). UbiX serves as the flavin prenyltransferase, extending the isoalloxazine ring system with a fourth non-aromatic ring, derived from sequential linkage between a dimethylallyl moiety and the FMN N5 and C6. Using structure determination and solution studies of both dimethylallyl monophosphate (DMAP) and dimethyallyl pyrophosphate (DMAPP) dependent UbiX enzymes, we reveal the first step, N5-C1' bond formation, is contingent on the presence of a dimethylallyl substrate moiety.

Heterologous production, reconstitution and EPR spectroscopic analysis of prFMN dependent enzymes.

The recent discovery of the prenylated FMN (prFMN) cofactor has led to a renewed interest in the prFMN-dependent UbiD family of enzymes. The latter catalyses the reversible decarboxylation of alpha-beta unsaturated carboxylic acids and features widely in microbial metabolism. The flavin prenyltransferase UbiX synthesizes prFMN from reduced FMN and phosphorylated dimethylallyl precursors.

Enzymatic Carboxylation of 2-Furoic Acid Yields 2,5-Furandicarboxylic Acid (FDCA).

The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO levels in vitro.

NADPH-Driven Organohalide Reduction by a Nonrespiratory Reductive Dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-dependent enzymes that mostly act as the terminal oxidoreductases in the bacterial organohalide respiration process. This process often leads to detoxification of recalcitrant organohalide pollutants. While low cell yields and oxygen sensitivity hamper the study of many reductive dehalogenases, this is not the case for the nonrespiratory reductive dehalogenase NpRdhA from Nitratireductor pacificus.