Marcus Arlt

A Hybrid Ring‐Opening Metathesis Polymerization Catalyst Based on an Engineered Variant of the β‐Barrel Protein FhuA

A β-barrel protein hybrid catalyst was prepared by covalently anchoring a Grubbs-Hoveyda type olefin metathesis catalyst at a single accessible cysteine amino acid in the barrel interior of a variant of β-barrel transmembrane protein ferric hydroxamate uptake protein component A (FhuA). Activity of this hybrid catalyst type was demonstrated by ring-opening metathesis polymerization of a 7-oxanorbornene derivative in aqueous solution.

Hybrid Ruthenium ROMP Catalysts Based on an Engineered Variant of β‐Barrel Protein FhuA ΔCVFtev: Effect of Spacer Length

A biohybrid ring-opening olefin metathesis polymerization catalyst based on the reengineered β-barrel protein FhuA ΔCVF(tev) was chemically modified with respect to the covalently anchored Grubbs-Hoveyda type catalyst. Shortening of the spacer (1,3-propanediyl to methylene) between the N-heterocyclic carbene ligand and the cysteine site 545 increased the ROMP activity toward a water-soluble 7-oxanorbornene derivative. The cis/trans ratio of the double bond in the polymer was influenced by the hybrid catalyst.

Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries

During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error‐prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance. In this report, bsla (543 bp, small lipase gene from Bacillus subtilis) was divided into three fragments (147, 192, 204 bp). Each fragment was subjected to an epPCR with a high mutation load (22, 31, and 33 mutations per kb) in order to increase the number of identified beneficial positions while maintaining a fraction of active population which can efficiently be screened in agar plate or microtiter plate format. The use of this “casting epPCR” process termed as (cepPCR), doubles the number of identified beneficial positions (from 14% to 29%), when compared to standard epPCR for the BSLA enzyme model. A further increase to 39% of beneficial positions is obtainable through combination of cepPCR with the transversion biased sequence saturation mutagenesis (SeSaM) method. Furthermore, sequencing of up to 600 mutations per fragment provided valuable insights into the correlation of total throughput and number of identified beneficial positions as well as how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns. Biotechnol. Bioeng. 2017;114: 1921–1927.

2-Methyltetrahydrofuran and cyclopentylmethylether: two green solvents for efficient purification of membrane proteins like FhuA.

β-Barrel shaped membrane proteins are attractive hosts for hybrid catalysts in which reactions are controlled through space. Production and extraction of β-barrel shaped membrane proteins in gram scale is challenging due to their hydrophobicity. Solvent mixtures such as chloroform/methanol (CM) are widely used for membrane protein extraction but toxicity and mutagenicity were reported in several cases. 2-Methyltetrahydrofuran (2-MeTHF) and cyclopentylmethylether (CPME) are two green (reduction of solvent-related environmental damage in chemical production) and potentially efficient solvents for membrane protein purification. On the example of the ferric hydroxamate uptake protein component A (FhuA) a 4-Step method was developed to provide gram amounts of highly purified FhuA: cell disruption (Step 1), removal of membrane protein impurities with n-octyl-poly-oxyethylene (oPOE) (Step 2), dissolution of membranes and FhuA precipitation (Step 3), and refolding using urea and dialysis with polyethylene-polyethyleneglycol (PE-PEG; Step 4) resulted in high FhuA purity (95% 2-MeTHF, 80% CPME; 70mg FhuA per liter fermenter broth). Structural integrity of FhuA protein was confirmed by circular dichroism (CD) and a translocation functionality assay.

Artificial Diels–Alderase based on the transmembrane protein FhuA

Summary Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVFtev). 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.

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.

CHAPTER 3:Channel Protein FhuA as a Promising Biomolecular Scaffold for Bioconjugates

The ferric hydroxamate uptake protein component A, FhuA, is a large monomeric transmembrane protein. FhuA functions as a receptor for ferrichrome and the structurally closely related antibiotic albomycin. In addition to its biological importance, FhuA is a robust protein scaffold that can be genetically modified and is stable under a broad range of conditions. By removing the globular cork domain (deletion of amino acids 1–160), FhuA became a large passive diffusion channel (FhuA Δ1–160) with an inner diameter of about 2.0 nm. FhuA was reconstituted in liposomes and polymersomes for controlled compound release responding to reducing agents and UV light. FhuA was also re-engineered to increase its length, enlarge its diameter and harbour single functional groups (–SH and –NH2). FhuA Δ1–159 Ext with an increased hydrophobic region was generated and inserted more efficiently into polymer membranes. FhuA Δ1–159 Exp has an enlarged diameter and shows increased diffusion kinetics. The remarkable resistance of FhuA variants to organic solvents and high temperatures makes it suitable as a scaffold for accommodating hybrid catalysts to perform chemical reactions. By substituting the amino acid residues surrounding the coupling site in the interior of the FhuA channel, one can also optimize the accessibility of the coupling site and the enantioselectivity.