FhuA: From Iron-Transporting Transmembrane Protein to Versatile Scaffolds through Protein Engineering.

Protein engineering has emerged as a powerful methodology to tailor the properties of proteins. It empowers the design of biohybrid catalysts and materials, thereby enabling the convergence of materials science, chemistry, and medicine. The choice of a protein scaffold is an important factor for performance and potential applications. In the past two decades, we utilized the ferric hydroxamate uptake protein FhuA. FhuA is, from our point of view, a versatile scaffold due to its comparably large cavity and robustness toward temperature as well as organic cosolvents. FhuA is a natural iron transporter located in the outer membrane of Escherichia coli (E. coli). Wild-type FhuA consists of 714 amino acids and has a ß-barrel structure composed of 22 antiparallel ß-sheets, closed by an internal globular "cork" domain (amino acids 1-160). FhuA is robust in a broad pH range and toward organic cosolvents; therefore, we envisioned FhuA to be a suitable platform for various applications in (i) biocatalysis, (ii) materials science, and (iii) the construction of artificial metalloenzymes.(i) Applications in biocatalysis were achieved by removing the globular cork domain (FhuA_Δ1-160), thereby creating a large pore for the passive transport of otherwise difficult-to-import molecules through diffusion. Introducing this FhuA variant into the outer membrane of E. coli facilitates the uptake of substrates for downstream biocatalytic conversion. Furthermore, removing the globular "cork" domain without structural collapse of the ß-barrel protein allowed the use of FhuA as a membrane filter, exhibiting a preference for d-arginine over l-arginine.(ii) FhuA is a transmembrane protein, which makes it attractive to be used for applications in non-natural polymeric membranes. Inserting FhuA into polymer vesicles yielded so-called synthosomes (i.e., catalytic synthetic vesicles in which the transmembrane protein acted as a switchable gate or filter). Our work in this direction enables polymersomes to be used in biocatalysis, DNA recovery, and the controlled (triggered) release of molecules. Furthermore, FhuA can be used as a building block to create protein-polymer conjugates to generate membranes.(iii) Artificial metalloenzymes (ArMs) are formed by incorporating a non-native metal ion or metal complex into a protein. This combines the best of two worlds: the vast reaction and substrate scope of chemocatalysis and the selectivity and evolvability of enzymes. With its large inner diameter, FhuA can harbor (bulky) metal catalysts. Among others, we covalently attached a Grubbs-Hoveyda-type catalyst for olefin metathesis to FhuA. This artificial metathease was then used in various chemical transformations, ranging from polymerizations (ring-opening metathesis polymerization) to enzymatic cascades involving cross-metathesis. Ultimately, we generated a catalytically active membrane by copolymerizing FhuA and pyrrole. The resulting biohybrid material was then equipped with the Grubbs-Hoveyda-type catalyst and used in ring-closing metathesis.The number of reports on FhuA and its various applications indicates that it is a versatile building block to generate hybrid catalysts and materials. We hope that our research will inspire future research efforts at the interface of biotechnology, catalysis, and material science in order to create biohybrid systems that offer smart solutions for current challenges in catalysis, material science, and medicine.

Biofuel blending reduces particle emissions from aircraft engines at cruise conditions.

Aviation-related aerosol emissions contribute to the formation of contrail cirrus clouds that can alter upper tropospheric radiation and water budgets, and therefore climate. The magnitude of air-traffic-related aerosol-cloud interactions and the ways in which these interactions might change in the future remain uncertain. Modelling studies of the present and future effects of aviation on climate require detailed information about the number of aerosol particles emitted per kilogram of fuel burned and the microphysical properties of those aerosols that are relevant for cloud formation. However, previous observational data at cruise altitudes are sparse for engines burning conventional fuels, and no data have previously been reported for biofuel use in-flight. Here we report observations from research aircraft that sampled the exhaust of engines onboard a NASA DC-8 aircraft as they burned conventional Jet A fuel and a 50:50 (by volume) blend of Jet A fuel and a biofuel derived from Camelina oil. We show that, compared to using conventional fuels, biofuel blending reduces particle number and mass emissions immediately behind the aircraft by 50 to 70 per cent. Our observations quantify the impact of biofuel blending on aerosol emissions at cruise conditions and provide key microphysical parameters, which will be useful to assess the potential of biofuel use in aviation as a viable strategy to mitigate climate change.

Full-endoscopic bilateral over-the-top decompression in lumbar central stenosis: surgical technique and outcomes.

Decompression of spinal stenosis represents one of the most commonly performed procedures in spine surgery. With constantly increasing patient age and changing demographics, reducing the invasiveness of surgical procedures has become increasingly important. Over the past decades, microsurgical decompression has been established as a gold standard technique for the surgical treatment of spinal stenosis. In comparison with open techniques or surgeries that were performed with loop lenses, which required larger skin incisions, and which consecutively raised the access-related collateral damage, the microscope served to significantly reduce the invasiveness of the decompression interventions. Advantages included smaller skin incisions, reduced collateral tissue damage, less blood loss, lower infection rates and wound healing problems, shorter hospital stay, and multiple others, as widely known across various MIS techniques. For the same reasons as outlined above, the introduction of full-endoscopic surgical techniques aims to further reduce the invasiveness of surgical interventions. The present manuscript provides a delineation of the surgical technique of LE-ULBD (Lumbar Endoscopic Unilateral Laminotomy for Bilateral Decompression), gives an overview on the current state of literature, and aims to put this surgery into context with other currently available decompression techniques.

Protein Nanopore Membranes Prepared by a Simple Langmuir-Schaefer Approach.

Filtration through membranes with nanopores is typically associated with high transmembrane pressures and high energy consumption. This problem can be addressed by reducing the respective membrane thickness. Here, a simple procedure is described to prepare ultrathin membranes based on protein nanopores, which exhibit excellent water permeance, two orders of magnitude superior to comparable, industrially applied membranes. Furthermore, incorporation of either closed or open protein nanopores allows tailoring the membrane's ion permeability. To form such membranes, the transmembrane protein ferric hydroxamate uptake protein component A (FhuA) or its open-pore variant are assembled at the air-water interface of a Langmuir trough, compressed to a dense film, crosslinked by glutaraldehyde, and transferred to various support materials. This approach allows to prepare monolayer or multilayer membranes with a very high density of protein nanopores. Freestanding membranes covering holes up to 5 µm in diameter are visualized by atomic force microscopy (AFM), helium ion microscopy, and transmission electron microscopy. AFM PeakForce quantitative nanomechanical property mapping (PeakForce QNM)  demonstrates remarkable mechanical stability and elastic properties of freestanding monolayer membranes with a thickness of only 5 nm. The new protein membrane can pave the way to energy-efficient nanofiltration.

A Photoclick-Based High-Throughput Screening for the Directed Evolution of Decarboxylase OleT.

Enzymatic oxidative decarboxylation is an up-and-coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof-of-principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co-substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed-up the directed evolution of OleT and other decarboxylases.

An artificial ruthenium-containing ß-barrel protein for alkene-alkyne coupling reaction.

A modified Cp*Ru complex, equipped with a maleimide group, was covalently attached to a cysteine of an engineered variant of Ferric hydroxamate uptake protein component: A (FhuA). This synthetic metalloprotein catalyzed the intermolecular alkene-alkyne coupling of 3-butenol with 5-hexynenitrile. When compared with the protein-free Cp*Ru catalyst, the biohybrid catalyst produced the linear product with higher regioselectivity.

OF-Pelvis classification of osteoporotic sacral and pelvic ring fractures.

OBJECTIVES: Osteoporotic fractures of the pelvis (OFP) are an increasing issue in orthopedics. Current classification systems (CS) are mostly CT-based and complex and offer only moderate to substantial inter-rater reliability (interRR) and intra-rater reliability (intraRR). MRI is thus gaining importance as a complement. This study aimed to develop a simple and reliable CT- and MRI-based CS for OFP. METHODS: A structured iterative procedure was conducted to reach a consensus among German-speaking spinal and pelvic trauma experts over 5 years. As a result, the proposed OF-Pelvis CS was developed. To assess its reliability, 28 experienced trauma and orthopedic surgeons categorized 25 anonymized cases using X-ray, CT, and MRI scans twice via online surveys. A period of 4 weeks separated the completion of the first from the second survey, and the cases were presented in an altered order. While 13 of the raters were also involved in developing the CS (developing raters (DR)), 15 user raters (UR) were not deeply involved in the development process. To assess the interRR of the OF-Pelvis categories, Fleiss' kappa (κF) was calculated for each survey. The intraRR for both surveys was calculated for each rater using Kendall's tau (τK). The presence of a modifier was calculated with κF for interRR and Cohen's kappa (κC) for intraRR. RESULTS: The OF-Pelvis consists of five subgroups and three modifiers. Instability increases from subgroups 1 (OF1) to 5 (OF5) and by a given modifier. The three modifiers can be assigned alone or in combination. In both surveys, the interRR for subgroups was substantial: κF = 0.764 (Survey 1) and κF = 0.790 (Survey 2). The interRR of the DR and UR was nearly on par (κF Survey 1/Survey 2: DR 0.776/0.813; UR 0.748/0.766). The agreement for each of the five subgroups was also strong (κF min.-max. Survey 1/Survey 2: 0.708-0.827/0.747-0.852). The existence of at least one modifier was rated with substantial agreement (κF Survey 1/Survey 2: 0.646/0.629). The intraRR for subgroups showed almost perfect agreement (τK = 0.894, DR: τK = 0.901, UR: τK = 0.889). The modifier had an intraRR of κC = 0.684 (DR: κC = 0.723, UR: κC = 0.651), which is also considered substantial. CONCLUSION: The OF-Pelvis is a reliable tool to categorize OFP with substantial interRR and almost perfect intraRR. The similar reliabilities between experienced DRs and URs demonstrate that the training status of the user is not important. However, it may be a reliable basis for an indication of the treatment score.

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design.

Invited for the cover of this issue is the group of Ulrich Schwaneberg at the Institute of Biotechnology, RWTH-Aachen University and DWI Lebniz Institute of Interactive Materials. The picture calls for special attention to be paid to the extra Cu binding site of Copper efflux Oxidase (CueO), due to its predominant function in tuning the electrocatalytic kinetics towards oxygen reduction. Read the full text of the article at 10.1002/chem.201905598.

Engineering of Laccase CueO for Improved Electron Transfer in Bioelectrocatalysis by Semi-Rational Design.

Copper efflux oxidase (CueO) from Escherichia coli is a special bacterial laccase due to its fifth copper binding site. Herein, it is discovered that the fifth Cu occupancy plays a crucial and favorable role of electron relay in bioelectrocatalytic oxygen reduction. By substituting the residues at the four coordinated positions of the fifth Cu, 11 beneficial variants are identified with ≥2.5-fold increased currents at -250 mV (up to 6.13 mA cm-2 ). Detailed electrocatalytic characterization suggests the microenvironment of the fifth Cu binding site governs the electrocatalytic current of CueO. Additionally, further electron transfer analysis assisted by molecular dynamics (MD) simulation demonstrates that an increase in localized structural stability and a decrease of distance between the fifth Cu and the T1 Cu are two main factors contributing to the improved kinetics of CueO variants. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.

Anchor Peptide-Mediated Surface Immobilization of a Grubbs-Hoveyda-Type Catalyst for Ring-Opening Metathesis Polymerization.

Adhesion promoting peptides have been reported to enable efficient enzyme immobilization on various material surfaces. Here we report the first immobilization of a synthetic Grubbs-Hoveyda (GH) type catalyst on two different materials (silica and polypropylene). To this end, the GH catalyst was coupled to an engineered (F16C) variant of the adhesion promoting peptide LCI through thiol-maleimide "click" reaction. Immobilization was performed in an oriented manner through the adhesion promoting peptide by simple incubation with the materials in water and subsequent washing with water and tetrahydrofuran. The immobilized GH catalyst was probed in ring-opening metathesis polymerization of a norbornene derivative to alter the surface properties in a layer-by-layer fashion.

Directed aryl sulfotransferase evolution toward improved sulfation stoichiometry on the example of catechols.

Sulfation is an important way for detoxifying xenobiotics and endobiotics including catechols. Enzymatic sulfation occurs usually with high chemo- and/or regioselectivity under mild reaction conditions. In this study, a two-step p-NPS-4-AAP screening system for laboratory evolution of aryl sulfotransferase B (ASTB) was developed in 96-well microtiter plates to improve the sulfate transfer efficiency toward catechols. Increased transfer efficiency and improved sulfation stoichiometry are achieved through the two-step screening procedure in a one-pot reaction. In the first step, the p-NPS assay is used (detection of the colorimetric by-product, p-nitrophenol) to determine the apparent ASTB activity. The sulfated product, 3-chlorocatechol-1-monosulfate, is quantified by the 4-aminoantipyrine (4-AAP) assay in the second step. Comparison of product formation to p-NPS consumption ensures successful directed evolution campaigns of ASTB. Optimization yielded a coefficient of variation below 15% for the two-step screening system (p-NPS-4-AAP). In total, 1760 clones from an ASTB-SeSaM library were screened toward the improved sulfation activity of 3-chlorocatechol. The turnover number (kcat = 41 ± 2 s-1) and catalytic efficiency (kcat/KM = 0.41 µM-1 s-1) of the final variant ASTB-M5 were improved 2.4- and 2.3-fold compared with ASTB-WT. HPLC analysis confirmed the improved sulfate stoichiometry of ASTB-M5 with a conversion of 58% (ASTB-WT 29%; two-fold improvement). Mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) confirmed the chemo- and regioselectivity, which yielded exclusively 3-chlorocatechol-1-monosulfate. For all five additionally investigated catechols, the variant ASTB-M5 achieved an improved kcat value of up to 4.5-fold and sulfate transfer efficiency was also increased (up to 2.3-fold).

Towards the Evolution of Artificial Metalloenzymes-A Protein Engineer's Perspective.

Incorporating artificial metal-cofactors into protein scaffolds results in a new class of catalysts, termed biohybrid catalysts or artificial metalloenzymes. Biohybrid catalysts can be modified chemically at the first coordination sphere of the metal complex, as well as at the second coordination sphere provided by the protein scaffold. Protein-scaffold reengineering by directed evolution exploits the full power of nature's diversity, but requires validated screening and sophisticated metal cofactor conjugation to evolve biohybrid catalysts. In this Minireview, we summarize the recent efforts in this field to establish high-throughput screening methods for biohybrid catalysts and we show how non-chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we shed light on the potential of this field and challenges that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.

Improved riboflavin production with Ashbya gossypii from vegetable oil based on 13C metabolic network analysis with combined labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR.

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B2. In order to meet the constantly increasing demands for improved production processes, it appears essential to better understand the underlying metabolic pathways of the vitamin. Here, we used a highly sophisticated set-up of parallel 13C tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes in the overproducing strain A. gossypii B2 during growth and subsequent riboflavin production from vegetable oil as carbon source, yeast extract, and supplemented glycine. The studies provided a detailed picture of the underlying metabolism. Glycine was exclusively used as carbon-two donor of the vitamin's pyrimidine ring, which is part of its isoalloxazine ring structure, but did not contribute to the carbon-one metabolism due to the proven absence of a functional glycine cleavage system. The pools of serine and glycine were closely connected due to a highly reversible serine hydroxymethyltransferase. Transmembrane formate flux simulations revealed that the one-carbon metabolism displayed a severe bottleneck during initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting carbon-one pool was successfully replenished by time-resolved feeding of small amounts of formate and serine, respectively. This increased the intracellular availability of glycine, serine, and formate and resulted in a final riboflavin titer increase of 45%.

Directed OmniChange Evolution Converts P450 BM3 into an Alkyltrimethylammonium Hydroxylase.

Cetyl-trimethylammonium bromide (CTAB) is a widely used cationic surfactant that is biodegradable in nature. CTAB biodegradation requires hydroxylation in the first step, which is rate-limiting and crucial for solubility in water. In this study, the OmniChange multi-site mutagenesis method was applied to reengineer the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four previously reported positions (R47, Y51, F87, and L188). 1740 clones from the P450 BM3 OmniChange library were screened with the NADPH depletion assay. A total of 696 clones were rescreened with the NADPH depletion and an Ampliflu™ Red/ horseradish peroxidase based H2 O2 detection assay. Several improved P450 BM3 variants were identified and finally four were kinetically characterized with respect to CTAB hydroxylation, based on both performance and coupling efficiency. Based on NADPH consumption, the P450 BM3 variant P3A8 (R47E/Y51M/F87V/L188E) displayed an initial activity (64.9±4.8 s-1 , 13.5-fold increased activity compared with wild-type P450 BM3), which nearly matches the specific activity for its natural fatty acid substrate (palmitic acid (32-122 s-1 )). Variant P3A8 showed high coupling efficiency (92.5 %), whereas wild-type P450 BM3 displayed a low coupling efficiency (0.5 %). HPLC-MS/MS detection confirmed that P3A8 and P2E7 (R47D/Y51L/F87V/L188A) form 13 and 35 times more 2-hydroxylated CTAB than P450 BM3. In addition, di-hydroxylated CTAB products were detected for all four investigated P450 BM3 variants (up to a yield of 77 %; P3A8). Di-hydroxylated quaternary amines are highly interesting bolaform surfactants with a high hydrophilicity (surface contact angle: θ=16.7°).

A hydroquinone-specific screening system for directed P450 evolution.

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 µM) and a broad linear detection range (5 to 250 µM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M).

Olefin metathesis catalysts embedded in ß-barrel proteins: creating artificial metalloproteins for olefin metathesis.

This review summarizes the recent progress of Grubbs-Hoveyda (GH) type olefin metathesis catalysts incorporated into the robust fold of ß-barrel proteins. Anchoring strategies are discussed and challenges and opportunities in this emerging field are shown from simple small-molecule transformations over ring-opening metathesis polymerizations to in vivo olefin metathesis.

2-Methyl-2,4-pentanediol (MPD) boosts as detergent-substitute the performance of ß-barrel hybrid catalyst for phenylacetylene polymerization.

Covering hydrophobic regions with stabilization agents to solubilize purified transmembrane proteins is crucial for their application in aqueous media. The small molecule 2-methyl-2,4-pentanediol (MPD) was used to stabilize the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA) utilized as host for the construction of a rhodium-based biohybrid catalyst. Unlike commonly used detergents such as sodium dodecyl sulfate or polyethylene polyethyleneglycol, MPD does not form micelles in solution. Molecular dynamics simulations revealed the effect and position of stabilizing MPD molecules. The advantage of the amphiphilic MPD over micelle-forming detergents is demonstrated in the polymerization of phenylacetylene, showing a ten-fold increase in yield and increased molecular weights.

Selective Metal-Free Hydrosilylation of CO2 Catalyzed by Triphenylborane in Highly Polar, Aprotic Solvents.

Triphenylborane (BPh3 ) in highly polar, aprotic solvents catalyzes hydrosilylation of CO2 effectively under mild conditions to provide silyl formates with high chemoselectivity (>95 %) and without over-reduction. This system also promotes reductive hydrosilylation of tertiary amides as well as dehydrogenative coupling of silane with alcohols.

Artificial Diels-Alderase based on the transmembrane protein FhuA.

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVF(tev)). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels-Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.