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.

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.

Advances in ultrahigh-throughput screening for directed enzyme evolution.

Enzymes are versatile catalysts and their synthetic potential has been recognized for a long time. In order to exploit their full potential, enzymes often need to be re-engineered or optimized for a given application. (Semi-) rational design has emerged as a powerful means to engineer proteins, but requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies, which consist of iterative rounds of diversity generation and screening, can improve an enzyme's properties with virtually no structural knowledge. Current diversity generation methods grant us access to a vast sequence space (libraries of >1012 enzyme variants) that may hide yet unexplored catalytic activities and selectivity. However, the time investment for conventional agar plate or microtiter plate-based screening assays represents a major bottleneck in directed evolution and limits the improvements that are obtainable in reasonable time. Ultrahigh-throughput screening (uHTS) methods dramatically increase the number of screening events per time, which is crucial to speed up biocatalyst design, and to widen our knowledge about sequence function relationships. In this review, we summarize recent advances in uHTS for directed enzyme evolution. We shed light on the importance of compartmentalization to preserve the essential link between genotype and phenotype and discuss how cells and biomimetic compartments can be applied to serve this function. Finally, we discuss how uHTS can inspire novel functional metagenomics approaches to identify natural biocatalysts for novel chemical transformations.

Combinatorial assembly of ferredoxin-linked modules in Escherichia coli yields a testing platform for Rnf-complexes.

The Rnf complex is a membrane-bound ferredoxin(Fd):NAD(P)+ oxidoreductase (Fno) that couples Fd oxidation to vectorial H+ /Na+ transport across the cytoplasmic membrane. Here, we produced two putative Rnf-complexes from Clostridioides difficile (Cd-Rnf) and Clostridium ljungdahlii (Cl-Rnf) for the first time in Escherichia coli. A redox-responsive low-expression system enabled Rnf assembly in the membranes of E. coli as confirmed by in vitro activity measurements. To study the physiological effects of Rnf on the metabolism of E. coli, we assembled additional Fd-dependent enzymes by plasmid-based multigene expression: (a) an Fd-linked butyrate pathway (But) from C. difficile, (b) an [FeFe]-hydrogenase (Hyd) to modulate the redox state of Fd, and (c) heterologous ferredoxins as electron carriers. The hydrogenase efficiently modulated butyrate formation by H2 -mediated Fd reoxidation under nitrogen. In its functionally assembled state, Rnf severely impaired cell growth. Including Hyd in the But/Rnf background, in turn, restored normal growth. Our findings suggest that Rnf mediates reverse electron flow from NADH to Fd, which requires E. coli's F-type ATPase to function in its reverse, ATP hydrolyzing direction. The reduced Fd is then reoxidized by endogenous Fd:NAD(P)H oxidoreductase (Fpr), which regenerates NADH and, thereby, initiates a futile cycle fueled by ATP hydrolysis. The introduction of hydrogenase interrupts this futile cycle under N2 by providing an efficient NAD(P)+ -independent Fd reoxidation route, whereas under H2 , Hyd outcompetes Rnf for Fd reduction. This is the first report of an Rnf complex being functionally produced and physiologically investigated in E. coli.

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.

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.

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.

Development of a methodological approach for the characterization of bioaerosols in exhaust air from pig fattening farms with MALDI-TOF mass spectrometry.

In this paper, we evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a cultivation-independent, routinely applicable approach to identify microbial fractions in bioaerosol emission samples. We developed a streamlined protocol in line with the German state-of-the-art impingement sampling guideline. Following isokinetic sampling, a fast and reliable pre-treatment methodology involving a series of cascade filtration steps was implemented, which produced fractions for spectrometric measurement devoid of interfering substances. We sampled the exhaust air from eight pig fattening farms around western Germany, which yielded two sets of samples for both method development and validation. For method development, in total 65 bacterial isolates were produced directly from the exhaust air samples, taxonomically classified by 16S rRNA-Gene sequencing, and subjected to MALDI-TOF analysis. In this way, we could assign fingerprint biomarkers to classified bacterial genera or even species to build up a preliminary reference database. For verification of the novel methodology and application of the reference database, we subjected the second set of exhaust air samples to the developed protocol. Here, 18 out of 21 bacterial species deposited in the database were successfully retrieved, including organisms classified in risk group 2, which might be used to evaluate the pathogenic potential of sampled exhaust air. Overall, this study pursues an entirely new approach to rapidly analyze airborne microbial fractions.

A flexible toolbox to study protein-assisted metalloenzyme assembly in vitro.

A number of metalloenzymes harbor unique cofactors, which are incorporated into the apo-enzymes via protein-assisted maturation. In the case of [NiFe]-hydrogenases, minimally seven maturation factors (HypABCDEF and a specific endopeptidase) are involved, making these enzymes an excellent example for studying metallocenter assembly in general. Here, we describe an innovative toolbox to study maturation involving multiple putative gene products. The two core elements of the system are a modular, combinatorial cloning system and a cell-free maturation system, which is based on recombinant Escherichia coli extracts and/or purified maturases. Taking maturation of the soluble, oxygen-tolerant [NiFe]-hydrogenase (SH) from Cupriavidus necator as an example, the capacities of the toolbox are illustrated. In total 18 genes from C. necator were analyzed, including four SH-structural genes, the SH-dedicated hyp-genes and a second set of hyp-genes putatively involved in maturation of the Actinobacterium-like hydrogenase (AH). The two hyp-sets were either expressed in their entirety from single vectors or split into functional modules, which enabled flexible approaches to investigate limitations, specificities and the capabilities of individual constituents to functionally substitute each other. Affinity-tagged Hyp-Proteins were used in pull-down experiments to demonstrate direct interactions between dedicated or non-related constituents. The dedicated Hyp-set from C. necator exhibited the highest maturation efficiency in vitro. Constituents of non-related maturation machineries were found to interact with and to accomplish partial activation of SH. In contrast to homologues of the Hyp-family, omission of the SH-specific endopeptidase HoxW completely abolished in vitro maturation. We detected stoichiometric imbalances inside the recombinant production system, which point to limitations by the cyanylation complex HypEF and the premature subunit HoxH. Purification of HoxW revealed strong indications for the presence of a putative [4Fe-4S]-cluster, which is unique among this class of maturases. Results are discussed in the context of [NiFe]-hydrogenase maturation, and in light of the capacity of the novel toolbox.

An innovative cloning platform enables large-scale production and maturation of an oxygen-tolerant [NiFe]-hydrogenase from Cupriavidus necator in Escherichia coli.

Expression of multiple heterologous genes in a dedicated host is a prerequisite for approaches in synthetic biology, spanning from the production of recombinant multiprotein complexes to the transfer of tailor-made metabolic pathways. Such attempts are often exacerbated, due in most cases to a lack of proper directional, robust and readily accessible genetic tools. Here, we introduce an innovative system for cloning and expression of multiple genes in Escherichia coli BL21 (DE3). Using the novel methodology, genes are equipped with individual promoters and terminators and subsequently assembled. The resulting multiple gene cassettes may either be placed in one vector or alternatively distributed among a set of compatible plasmids. We demonstrate the effectiveness of the developed tool by production and maturation of the NAD(+)reducing soluble [NiFe]-hydrogenase (SH) from Cupriavidus necator H16 (formerly Ralstonia eutropha H16) in E. coli BL21Star™ (DE3). The SH (encoded in hoxFUYHI) was successfully matured by co-expression of a dedicated set of auxiliary genes, comprising seven hyp genes (hypC1D1E1A2B2F2X) along with hoxW, which encodes a specific endopeptidase. Deletion of genes involved in SH maturation reduced maturation efficiency substantially. Further addition of hoxN1, encoding a high-affinity nickel permease from C. necator, considerably increased maturation efficiency in E. coli. Carefully balanced growth conditions enabled hydrogenase production at high cell-densities, scoring mg·(Liter culture)(-1) yields of purified functional SH. Specific activities of up to 7.2±1.15 U·mg(-1) were obtained in cell-free extracts, which is in the range of the highest activities ever determined in C. necator extracts. The recombinant enzyme was isolated in equal purity and stability as previously achieved with the native form, yielding ultrapure preparations with anaerobic specific activities of up to 230 U·mg(-1). Owing to the combinatorial power exhibited by the presented cloning platform, the system might represent an important step towards new routes in synthetic biology.

Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.

The butyrogenic genes from Clostridium difficile DSM 1296(T) have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD(+)-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.

Facile analysis of short-chain fatty acids as 4-nitrophenyl esters in complex anaerobic fermentation samples by high performance liquid chromatography.

Short-chain fatty acids are crucial intermediates in the conversion of biomass to methane. Due to the complexity of raw biomass, volatile fatty acids (including n- and branched-chain compounds) as well as arylacetic and arylpropionic acids arise from digestion of carbohydrates, proteins and lipids. The development of a simple extraction procedure in combination with internal standardization and facile 4-nitrophenyl-labelling via oxalylchloride-generated acylchlorides enabled robust separation and quantification of the target compounds in crude biological samples like raw cattle manure and biogas fermenter contents. Detection limits of <100 µM and error rates of less than 4% for the quantification of individual compounds in a concentration range up to 50 mM for non-diluted samples suggest that the novel method might be of general advantage for the routine quantification of short-chain fatty acids in complex biological samples including complex fermentation media.