A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigCCopyright: BioVI
Stefanie Brands, Hannah U. C. Brass, Andreas S. Klein, Jörg Pietruszka, Anna Joëlle Ruff, and Ulrich Schwaneberg, “A colourimetric high-throughput screening system for directed evolution of prodigiosin ligase PigC,” Chem. Commun., 2020, doi: 10.1039/D0CC02181D.
A two-step screening assay enables directed evolution of prodigiosin ligase PigC to perform biosynthesis of bioactive prodiginine compounds.
Prodigiosin synthetase PigC is a membrane bound ATP-ligase that catalyses the final condensation step in the biosynthesis pathway of the natural product prodigiosin, a tripyrrolic red pigment, which occurs naturally in several bacterial species. The compound class of prodiginines is famous for their prominent colour, which makes colonies of prodiginine-producing bacteria appear like little droplets of blood. Several prodiginine bioactivities have so far been discovered, which moved the compounds into the focus of industry: One artificial prodiginine derivative, Obatoclax Mesylate GX15-070, is currently on phase II clinical trials as candidate to treat different forms of cancer.
As chemical synthesis of prodiginines unfortunately is not trivial, biocatalysis remains a promising approach to get the compounds into hand. In our PigC screening assay, we combined chemical synthesis of prodiginine precursors (colourless in solution) with PigC biocatalysis (during which the red prodiginines are formed). In an agar prescreening, red and active colonies are selected for expression in 96-well format, where pyrrolic precursors are supplemented and prodiginines finally extracted and photometrically quantified by their red colour.
This concept makes the exchange of a pyrrolic precursor by a more promising pyrrole derivative easy, in our case, we screened with 2,3-dimethyl pyrrole instead of the natural PigC substrate 2-methyl-3-amyl pyrrole. Short-chain prodiginines have shown an increased bioactivity in anticancer autophagy tests. As 2,3-dimethyl pyrrole is not readily accepted by PigC, enzyme engineering campaigns on PigC are required to extend the synthetase’s substrate scope. This is where a suitable high-throughput screening method for detection of beneficial PigC variants comes into action: a PigC random mutagenesis library was generated in Pseudomonas putida KT2440 and screened for PigC variants with enhanced acceptance of the prodiginine precursor. After screening of approximately 3000 clones, a variant with one amino acid exchange in the substrate-binding domain stood out with a nearly 3-fold increased prodiginine yield. The variant’s catalytic efficiency with 2,3-dimethyl pyrrole (K M/k cat) proofed to be 2.9-fold higher over the wild type, and also its stability at 30°C was slightly increased, which enabled the production of higher amount of prodiginines in the screening assay.
This paper was written in the division Molecular Bioeconomy. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Culture and Science within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-00213).
Loop engineering of aryl sulfotransferase B for improving catalytic performance in regioselective sulfationCopyright: BioVI
Yu Ji, Shohana Islam, Haiyang Cui, Gaurao V. Dhoke, Mehdi D. Davari, Alan M. Mertens, Ulrich Schwaneberg, Catalysis Science & Technology, 2020, 10, 2369-2377 DOI: 10.1039/D0CY00063A
Copyright: Catalysis Science & Technology
Reengineering of loop12 and loop 13 of aryl sulfotransferase B yielded variant with high sulfate transfer efficiency up to 94%.
Catechol sulfates act in our body as important antioxidants and often have anti-inflammatory properties. This study focuses on reengineering of loop 12 and loop 13 of aryl sulfotransferase B from Desulfitobacterium hafniense in order to improve the sulfate transfer efficiency of six catechols. The obtained aryl sulfotransferase B variants were generated in a KnowVolution campaign using the random mutagenesis method SeSaM and the multi-site saturation method OmniChange. The catalytic activity and catalytic efficiency of the final variant were improved for all six investigated catechols when compared to the wild type (e.g., 13.6-fold improvement of catalytic activity for 3-bromocatechol). HPLC-MS analysis confirmed the improved sulfate stoichiometry of aryl sulfotransferase B variant with a transfer efficiency up to 94% for 3-methylcatechol in comparison to 24% for the wild type. A molecular understanding of the improved sulfation activity of the variant was achieved through molecular docking studies and electron effects of catechol substituents were analyzed by Hammett equation. Enzymatic sulfation of catechol as well as substituted catechols by aryl sulfotransferases opens up an environmentally friendly route for chemo- and / or regioselective sulfation of valuable compounds, such as mycamine, sodium picosulfate, and minoxidil sulfate.
In situ monitoring of membrane protein insertion into block copolymer vesicle membranes and their spreading via potential-assisted approachCopyright: BioVI
T. Mirzaei Garakani, Z. Liu, U. Glebe, J. Gehrmann, J. Lazar, M. A. S. Mertens, M. Möller, N. Hamzelui, L. Zhu, U. Schnakenberg, A. Böker, U. Schwaneberg, ACS Applied Materials & Interfaces, 2019, DOI: 10.1021/acsami.9b09302
Copyright: ACS Applied Materials & Interfaces
A new approach to produce solid-supported biomimetic membranes via potential-assisted vesicle spreading was developed.
Transmembrane proteins incorporated in solid-supported hybrid membranes can act as selective transporters or used for catalysis. The hydrophobic region of transmembrane proteins allows their incorporation into polymer membranes. The triblock copolymer poly(2-methyl oxazoline)-blockpoly(dimethylsiloxane)-block-poly(2-methyl oxazoline) and an engineered variant of the outer membrane protein Ferric hydroxamate uptake protein component A (FhuA) were used for the potential-assisted spreading of synthosomes on gold electrodes. FhuA lacking the cork domain (FhuA ∆1-160) was a useful candidate to achieve large passive diffusion channels in synthosomes. Isothermal titration calorimetry was used to analyze the insertion of FhuA into the polymer vesicles. The charge of the polymer was used to spread FhuA-containing vesicles on electrodes by electrostatic interactions. The potential-assisted vesicle spreading system was shown to produce homogenous synthosomes more efficiently than common spin- and dip-coating techniques. Functionality of the spread synthosomes was analyzed by passive ion transport response and through electrochemical impedance spectroscopy analysis. Solid-supported biomimetic membranes formed via potential-assisted spreading could be an interesting system for the development of new biosensors and as useful pores for drug delivery and water purification.
Engineering of laccase CueO for improved electron transfer in bioelectrocatalysis by semi-rational designCopyright: BioVI
Lingling Zhang, Haiyang Cui, Gaurao V. Dhoke, Zhi Zou, Daniel F. Sauer, Mehdi D. Davari, and Ulrich Schwaneberg, Chem. Eur. J., 2020. DOI: 10.1002/chem.201905598
Copyright: Chem. EUR. J. J.
The fifth copper binding site of CueO is very important to electron transfer kinetics of bioelectrocatalytic oxygen reduction.
Different from other laccases, CueO is found to possess an extra copper binding site (i.e., the fifth copper binding site). To understand the role of the fifth copper in bioelectrocatalysis, semi-rational design was applied to generate a site-saturation mutagenesis library at the four coordination positions (M355, D360, D439, and M441). Thanks to the electrochemical screening platform established before ( Angew. Chem. 2019, 131, 4610 ), 11 improved variants were identified with over 2.5-fold increased currents. Molecular dynamics (MD) simulation suggested two reasons for the improvement: an increase in localized structural stability and a decrease of distance between the fifth copper and the first copper. It may guide a novel way to tailor laccases and perhaps other oxidoreductases for bioelectrocatalytic applications.
The simulation section was supported by division Computational Biology. This work was financed by Alexander von Humboldt Foundation and was immediately highlighted by Nature Reviews Chemistry once it was published.
Advances in ultrahigh-throughput screening for directed enzyme evolutionCopyright: BioVI
Ulrich Markel, Khalil D. Essani, Volkan Besirlioglu, Johannes Schiffels, Wolfgang R. Streit and Ulrich Schwaneberg*, Chem. Soc. Rev., 2020, 49, 233-262. DOI: 10.1039/C8CS00981C
Here, we review state-of-the-art and up-and-coming ultrahigh-throughput methods for the screening of large libraries in directed enzyme evolution.
Enzymes often need to be re-engineered or optimized in order to exploit their full potential. (Semi-) rational design requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies can improve an enzyme’s properties without structural knowledge by iterative rounds of diversity generation and screening. Current diversity generation methods allow us to generate huge libraries but conventional screening on agar plates or in microtiter plates fails to interrogate the full generated diversity in reasonable time. Ultrahigh-throughput screening methods drastically increase the number of enzyme variants we can screen and speed up biocatalyst design ultimately widening our knowledge about sequence function relationships. In the present review, we summarize recent advances in ultrahigh-throughput screening for the directed evolution of enzymes. We discuss the importance of compartmentalization to link genotype and phenotype and illustrate how cells and biomimetic compartments can serve this function. Finally, we discuss how new functional metagenomics approaches can profit from ultrahigh-throughput screening to identify natural biocatalysts for novel chemical transformations.
CompassR: A new rule for recombination of beneficial substitutions in directed evolutionCopyright: BioVI
Haiyang Cui, Hao Cao, Haiying Cai, Karl Erich Jaeger, Mehdi D. Davari, Ulrich Schwaneberg, Chemistry A European Journal, 2019, https://doi.org/10.1002/chem.201903994
The Computer-assisted Recombination – CompassR – strategy is a selection filter for experimentalists to recombine beneficial substitutions in order to gradually improve enzyme performance and maximize improvements through recombination.
Directed evolution has matured into a powerful method for improving enzymes in catalysis and medical applications as documented by the Nobel Prize in chemistry in 2018. A main remaining challenge is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after recombination of 3 to 4 beneficial substitutions. The latter limits researchers to harness the benefits from beneficial substitutions that have been identified in directed evolution campaigns and to exploit nature’s potential in generating better enzymes. The Computer-assisted Recombination – CompassR – strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding. The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of the Bacillus subtilis lipase A. Analysis of their relative free energy of folding was used to deduct the CompassR rule that substitutions with a relative free energy of folding below +0.36 kcal/mol can be efficiently recombined, variants with a free energy of folding between +0.36 and +7.52 kcal/mol were active and inactive indicating unpredictable behavior, folding energies over +7.52 kcal/mol mostly yielded inactive variants. The finally obtained recombinant with four amino acid substitutions had a 2.7-fold improved specific activity in 18.3 % ionic liquid [BMIM][Cl]. Notably, the BSLA variants with 3 and 4 substitutions were better performing than the variants with 2 substitutions. In essence, the deducted CompassR allows to recombine beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time-efficient manner.
This work was realized in the division Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187). Haiyang Cui is financially supported by the China Scholarship Council (CSC) scholarship.
Chemoenzymatic cascade for stilbene production from cinnamic acid catalyzed by ferulic acid decarboxylase and an artificial metatheaseCopyright: BIO VI
M. A. Stephanie Mertens,‡ Daniel F. Sauer,‡ Ulrich Markel, Johannes Schiffels, Jun Okuda,* Ulrich Schwaneberg,* Catalysis Science & Technology, 2019, DOI: 10.1039/C9CY01412H
Copyright: Catalysis Science & Technology
The combination of a decarboxylase and an artificial metathease in a chemoenzymatic cascade reaction for stilbene production with efficient removal of metal contamination is reported.
A chemoenzymatic cascade reaction involving a biocatalyst and a biohybrid catalyst for the production of stilbene derivatives was designed. Stepwise conversion of cinnamic acid as a renewable resource to valuable compounds was achieved in one pot in aqueous solution and under mild reaction conditions. The ferulic acid decarboxylase FDC1 from Saccharomyces cerevisiae was used for the conversion of cinnamic acid. In a following reaction, cross-metathesis of the styrene intermediate was performed with an artificial metathease, FhuA-GH, based on a Grubbs-Hoveyda catalyst incorporated into an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A, FhuA. Intermediate workup steps and isolation of the styrene intermediates was not required, as both reaction steps proceeded in aqueous solution. In comparison to the protein-free catalyst, cascade reactions with the artificial metathease revealed a significant lower metal content after a simple extraction step. The cascade reaction is the first example of the combination of biocatalysts and biohybrid catalysts for efficient removal of metal impurities in the product fraction.
Molecular understanding of enzyme and ionic liquid interactions provide a general protein engineering strategy for their sustainable biocatalytic applicationsCopyright: BIO VI
Pramanik S.†, Dhoke G. V.†, Jaeger KE., Schwaneberg U., Davari MD. ACS Sustainable Chem. Eng., 2019, https://doi.org/10.1021/acssuschemeng.9b00752 †shared co-authorship
Copyright: ACS Sustainable Chem. Eng.
A general protein engineering strategy was suggested through molecular understanding of enzyme and ionic liquid interactions based on computational study.
In this publication, interactions between Bacillus subtilis lipase A (BSLA) and four imidazolium-based ionic liquids were studied using molecular dynamic simulations. This study provides first insight that ionic liquids co-solvents do not alter the overall and local BSLA conformation. The effect on the reduction of activity was attributed to surface interactions of the ionic liquid cations, which removed essential water molecules from the BSLA surface.Furthermore, the comparison of simulation results with experimental full site-saturation mutagenesis BSLA libraries confirmed that most of beneficial positions for resistance improvement are located at the ionic liquid cations binding regions. Taken together, these findings suggest that surface charge engineering might be a general protein engineering strategy to improve BSLA resistance in ionic liquids and is most likely applicable to other lipases and α/β-hydrolases.
This work was realized in the division of Computational Biology and was supported by computing resources granted by JARA-HPC from RWTH Aachen University (JARA0187).
Science praises our recent research on plant pest managementCopyright: Green Chemistry
The BiFuProt surface coating platform was highly appreciated in a recent news article published by Science as a new plant protection technology. The Science article quoted leading plant scientists. "With the current scale of the soybean rust problem, and the rapid evolution of resistance against multiple fungicides, any addition to the toolbox would be welcome," says Nichola Hawkins at Rothamsted Research in Harpenden, U.K. and Ralph Hückelhoven at the Technical University of Munich in Germany is quoted that the BiFuProt-technology is promising and "It opens a treasure box of solutions".
The protection of crops by pesticides is often challenging due to a very low persistence time and low rain fastness on the plant surface. A reduction of pesticide usage while preserving crop productivity demands new strategies for pest and disease control. In our current research highlight published in Green Chemistry we developed a platform technology that enables the functionalization of the aerial crop surface for sustainable disease management. We demonstrated an alternative way to protect soybean leaves from its most severe disease, the Asian soybean rust (Phakopsora pachyrhizi), by functionalizing the leaves with bifunctional fusion proteins or in short BiFuProts.
"With the current scale of the soybean rust problem, and the rapid evolution of resistance against multiple fungicides, any addition to the toolbox would be welcome."
- Nichola Hawkins (Rothamsted Research, Harpenden, Großbritannien)
The first domain binds to the wax layers of plant leaves, the second domain prevents the germination of Phakopsora pachyrhizi spores. In detail, the amphiphilic anchor peptides LCI, Thanatin, Tachystatin A2, and Lactoferricin B were genetically fused to the reporter protein eGFP and investigated for plant leaf binding. eGFP-LCI and eGFP-Thanatin strongly bound in a rainfast manner to the surface of soybean, barley, and corn leaves. Especially, eGFP-Thanatin bound to the soybean leaves and withstood high temperature, sunlight, and biotic degradation for at least 17 days. The weak binding of eGFP-Thanatin and eGFP-LCI to the wax-depleted mutant of barley or corn leaves indicated that the peptides mainly bind to the surface waxes of leaves. As a fusion partner for Thanatin, the antimicrobial peptide Dermaseptin 01 was selected. The bifunctional peptide Dermaseptin 01-Thanatin inhibited the germination of Phakopsora pachyrhizi spores in vitro and reduces Asian soybean rust disease by in a rainfast manner. It is very likely that state-of-the-art protein engineering strategies such as PePevo combined with a KnowVolution campaign will enable the design of peptides with tuned binding strength and persistence to match application demands. We expect that bifunctional peptides or proteins consisting of plant-attaching anchor peptides and pesticidal peptides or proteins have the potential to fight essentially any plant pest and disease in a rainfast manner.
This innovative research was performed in the framework of Bioeconomy Science Center (BioSC), which was funded by the Ministry of Culture and Science of the German State of North Rhine-Westphalia under the NRW Strategy Project BioSC. The BOOST FUND Project BiFuProts (Bifunctional fusion proteins for plant protection) team comprised Professor Ulrich Schwaneberg and Dr. Felix Jakob (initiator/coordinator; RWTH Aachen), Professor Uwe Conrath and Dr. Caspar Langenbach (RWTH Aachen University), Professor Lutz Schmitt (Heinrich-Heine-University Düsseldorf) and Professor Georg Noga, Dr. Mauricio Husche, and Dr. Shyam Pariyar (University of Bonn)
"It opens a treasure box of solutions".
- Ralph Hückelhoven (Technical University of Munich, Germany)
Plant health has already been an emerging research field within the Schwaneberg group in collaboration with plant scientist and chemists in last five year. The plant nutrient release technology (GreenGel) was successfully demonstrated on the example of cucumber iron deficiency in the BioSC funded project GreenGel. Dr. Felix Jakob and Professor Schwaneberg (coordinator; RWTH Aachen) were teamed up in GreenGel project with Professor Pich (RWTH Aachen University) and Professor Goldbach (University of Bonn). The GreenRelease technology is based on the compound loaded microgels (containers) that are decorated with plant adhesion promoting peptides and was published in Angewandte Chemie as hot paper in 2017. Based on the success of GreenGel and BiFuProts, the FocusLab greenRelease for Plant Health FocusLab has been funded (2.3 Mio€; for three years from January 2018 onwards) for translational research. The FocusLab is a part of BioSC as well and we teamed up with the groups of Professor Pich (RWTH Aachen University), Professor Conrath (RWTH Aachen University), Professor Noga (University of Bonn), Professor. Bröring (University of Bonn), Professor Knief (University of Bonn), Professor Groth (HHU Düsseldorf), Professor Gohlke (HHU Düsseldorf), and Professor Schurr (Forschungszentrum Jülich) to perform groundbreaking research in the field of plant health and protection in the upcoming years.
Tailor-made membrane channel enables chiral separation of racematesCopyright: BioVI
Deepak Anand, Gaurao V. Dhoke, Julia Gehrmann, Tayebeh M. Garakani, Mehdi D. Davari, Marco Bocola, Leilei Zhu, and Ulrich Schwaneberg, Chemical Communications, 2019. DOI: 10.1039/c9cc00154a.
Copyright: Chem. Commun.
Chiral resolution of arginine enantiomers was achieved through an engineered Escherichia coli outer membrane protein FhuA.
Chiral molecules are of large economic value in chemical, pharmaceutical, and food industries. Separation of enantiomers can be achieved by various methods but it still remains a challenging task. Chiral protein-polymer membranes would be an attractive, cost-effective, and scalable alternative for chiral separation. The main challenges lie in the design of filter regions within the channel proteins and the development of screening systems to identify chiral channel protein variants. In the present study, we report for the first time a chiral β-barrel channel based on ferric hydroxamate uptake component A – FhuA, an outer membrane protein of E. coli. Two filter regions were identified and redesigned through screening of multi-site saturation mutagenesis libraries, in order to achieve the chiral separation of a D-/L-arginine racemate. Screening resulted in identification of FhuAF4 variant showing an improved enantiomeric excess of 24% at 52% conversion compared to the parent FhuA variant. Interestingly, even a subtle change of just two amino acids considerably influenced the selectivity of the FhuA channel. Steered molecular dynamic simulations indicated that the separation is based on diffusivity differences of two enantiomers through FhuAF4. It is likely that with the identified filter region and multi-site saturation libraries further improvements are achievable for separation of other amino acids and a broader range of enantiomers. The chiral FhuA channel proteins would be an excellent scaffold for generation of chiral membranes based on protein-polymer conjugates with a high potential for novel and scalable downstream processes.
We kindly acknowledge the German Federal Ministry of Education and Research (BMBF) for providing financial support to the Chiral Membranes I project. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under the project RWTH0116.
Biohybrid catalysts for sequential one-pot reactions based on an engineered transmembrane proteinCopyright: BioVI
D. F. Sauer, Y. Qu, M. A. S. Mertens, J. Schiffels, T. Polen, U. Schwaneberg*, J. Okuda*, Catal. Sci. Tech. 2019, 9, 942-946. DOI: 10.1039/C8CY02236D
Compartmentalization of organometallic catalysts was achieved by generation of their corresponding biohybrid catalysts based on the transmembrane protein FhuA to enable cascade reactions.
In the present publication we showed that incorporation of organometallic catalysts into protein scaffolds enable cascade reactions in one pot, which were not possible by simply mixing the protein free catalysts. Bibenzyl derivatives (1,2-diphenylethane derivatives), which are valuable compounds in drug design or are used as building blocks in natural compound synthesis, were synthesized starting from styrene derivatives. In the first step, olefin cross-metathesis of the styrene derivatives was performed with a Ru-based Grubbs-Hoveyda type catalyst. In the subsequent step, hydrogenation of the stilbene intermediate was achieved with a Rh-type catalyst without isolation of the stilbene. This route was not possible by simply mixing the two organometallic catalysts in one pot. The strategy to compartmentalize the organometallic catalysts with proteins is promising approach and offers the possibility to keep reactions in a homogeneous fashion. The latter removes diffusion barriers through mass transfer between phases. This work was conducted in the division Next Generation Biocatalysis in cooperation with Professor Jun Okuda (Institute of Inorganic Chemistry, RWTH Aachen University). This research was financially supported by the DFG and the BMBF. A generous gift of metal precursors by Umicore, Frankfurt, is acknowledged.
In this study, the bacterial laccase CueO from Escherichia coli was chosen to perform directed evolution towards lowering the overpotential of cathodic oxygen reduction. A robust and efficient 8-channel electrochemical screening platform was developed for the first time to evaluate CueO variants generated by random mutagenesis and subsequent site-saturation mutagenesis (SSM). The enzyme immobilization could be accomplished in 20 seconds and directly from crude cell lysates. Two positions adjacent to the coordinated ligands of the T1 copper site, was identified as a main region that contribute to improvements in the onset potential. A remarkable increased onset potential of 0.54 V was obtained with two amino acid substitutions. Finally, the cathode generated open circuit potential of 0.56 V as well as 1.72-fold enhanced power output for the enzymatic biofuel cell comprising the generated CueO variant coupled with a glucose dehydrogenase. The developed directed evolution protocol offers a promising methodology beyond chemistry and materials science in improving laccase properties such as substrate specificity, turnover efficiency, and tolerance to harsh-environment conditions.
This work was supported by the Alexander von Humboldt Foundation.
Targeting microplastic particles in the void of diluted suspensionsCopyright: BioVI
In the present publication, the application of the anchor peptide Tachystatin A2 as adhesion promoter is described, which enhances the degradation of polyester-polyurethane nanoparticles by the cutinase Tcur1278.
Directed evolution of bacterial laccase CueO for enzymatic biofuel cellsCopyright: BioVI
Lingling Zhang, Haiyang Cui, Zhi Zou, Tayebeh Mirzaei Garakani, Catalina Novoa-Henriquez, Bahareh Jooyeh, and Ulrich Schwaneberg, Angew. Chem. Int. Ed, 2019. DOI: 1002/ange.201814069
Copyright: Environ. Int.
Directed evolution of CueO via electrochemical screening found substitutions that led to the dramatic overpotential decrease of 0.12 V, making CueO competitive to fungal laccases and applicable in enzymatic biofuel cells.
The accumulation of microplastics in the environment as well as in the food chain will be a grand upcoming challenge for our society. Polyurethanes belong to the accumulating plastics as they are widely used in medical, e.g. catheters, and industrial products, especially as foams. Polyurethane is a man-made polymer and therefore not abundant in nature. Hence, only a few fungal and microbial strains as well as enzymes, such as polyurethaneases and cutinases, have been reported to efficiently degrade polyurethane. An estimated degradation of plastic in nature takes at least 50 to 100 years. A minimization of existing environmental pollution requires sustainable management strategies for microplastics. Material binding peptides like anchor peptides strongly bind to synthetic polymers such as polypropylene, polyethylene terephthalate, and polyurethane. In this publication, we report the fusion of the anchor peptide Tachystatin A2 to the bacterial cutinase Tcur1278. The fusion enzyme Tcur1278-Tachystatin A2 accelerated the degradation of polyester-polyurethane nanoparticles by a factor of 6.6 in comparison to wild-type Tcur1278 without any anchor peptide. Additionally, the degradation half-life of polyester-polyurethane nanoparticles was reduced from 41.8 h to 6.2 h indicating a 6.7-fold improvement in a highly diluted polyester-polyurethane suspension by the fusion enzyme. Taken together, anchor peptides provide a versatile tool for a targeted degradation of micro- and nanoplastics at ambient temperature in highly diluted particle suspensions.
This work was financed by the German Federal Ministry of Education and Research and the German Federation of Industrial Research Associations. We acknowledge Prof. Wolfgang Zimmermann and Dr. Ren Wei for the sequence of the cutinase Tcur1278. We thank Thorsten Palmer for dynamic light scattering measurements and finally the Center for Chemical Polymer Technology especially Sabrina Mallmann for field emission scanning electron microscopy.
Towards evolution of artificial metalloenzymes – A protein engineer’s perspectiveCopyright: BioVI
In the present publication, an overview of the early approaches of directed evolution of biohybrid catalyst is given.
The incorporation of artificial metal-cofactors into protein scaffolds yields a new class of catalysts termed biohybrid catalysts or artificial metalloenzymes. In addition to modification of the artificial cofactor, these biohybrid catalysts can be modified at the second coordination sphere provided by the protein scaffold. Protein engineering provides tremendous potential to tailor such biohybrid catalysts but requires a robust screening system and sophisticated metal cofactor conjugation. In this minireview, we give an overview of the recent efforts in this field. We describe high-throughput screening methods applied for the directed evolution of biohybrid catalysts and we illustrate how non-chiral catalysts catalyze reactions enantioselectively by highlighting the first successes in this emerging field. Furthermore, we summarize the potential and limitations that need to be overcome to advance from biohybrid catalysts to true artificial metalloenzymes.
We gratefully acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) through the International Research Training Group ‘Selectivity in Chemo- and Biocatalysis’ (SeleCa) and the Bundesministerium für Bildung und Forschung (BMBF) (FKZ: 031B0297). #: These authors contributed equally
Enzyme-polyelectrolyte complexes boost the catalytic performance of enzymesCopyright: Bio VI
Martin J. Thiele, Mehdi D. Davari, Melanie König, Isabell Hofmann, Niklas O. Junker, Tayebeh Mirzaei Garakani, Ljubica Vojcic, Jörg Fitter, Ulrich Schwaneberg, ACS Catalysis, 2018, DOI: 10.1021/acscatal.8b02935
In the present publication, a molecular understanding was reported on polyelectrolyte-enzyme complexes which boost enzymatic activity in multi-component systems.
In this publication, a molecular understanding of the boosting effect of polyacrylic acid -PAA-) and poly-L-γ-glutamic acid -γ-PGA- for a nonspecific subtilisin protease was generated through biophysical characterization, e.g. fluorescence correlation and circular dichroism spectroscopies, isothermal titration calorimetry, molecular dynamics simulations and protease reengineering, e.g. site-saturation mutagenesis. This study revealed that enthalpically driven interactions via key amino acid residues close to the protease Ca2+ binding sites cause the boosting effect in protease activity. On the molecular level electrostatic interactions result in the formation of protease-polyelectrolyte complexes. Site-saturation mutagenesis on 6 positions yielded an increased proteolytic performance against a complex protein mixture -trademark CO3; up to ~300% and ~70%- in the presence of PAA and γ-PGA. Being able to fine-tune interactions between proteins and negatively charged polymers through integrative use of computational design, protein reengineering and biophysical characterization proved to be an efficient workflow to improve protease performance.
This research was partially funded by Henkel AG & Co. KGaA, Düsseldorf, Germany, as part of the Henkel Innovation Campus for Advanced Sustainable Technologies -HICAST- project. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169.
Directed evolution of sortase A for efficient site-specific bioconjugations in organic co-solventsCopyright: Bio VI
Evolved sortase A variants enable efficient site-specific conjugation of peptide/amine in organic co-solvents
Directed evolution of sortase A in 45% (v/v) DMSO, as co-solvent, yielded variants M1 and M3 with 2.2-fold increased resistance and 6.3-fold increased catalytic efficiency compared to sortase A wild-type, respectively. The generated variants were used to expand sortase-mediated ligation in organic co-solvent with hydrophobic substrates. Variant M3 showed up to 4.7-fold increased activity for peptide-peptide/peptide-amines in DMSO/DMF co-solvents compared to the wild-type. Structure-function relationship of sortase A in DMSO co-solvent were investigated by computational studies, which revealed that conformational mobility is important for the resistance gained by sortase A in organic co-solvent.
This work was financed by the Chinese Scholarship Council-CSC. Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169.
KnowVolution campaign of an aryl sulfotransferase increases activity toward cellobiose sulfatation
Islam, S., Laaf, D., Infanzón, B., Pelantová, H., Davari, M. D., Jakob, F., Křen, V., Elling, L., and Schwaneberg, U. Chemistry - A European Journal , 2018 . doi:10.1002/chem.201803729
Glycosaminoglycans are mostly sulfated polysaccharides playing significant roles in signal transduction, anti-coagulation, detoxification, and many more. Full chemical synthesis of glycosaminoglycans is still a very hard task due to many synthesis steps that affect the final yields. A suitable and sustainable alternative is represented by the in vitro synthesis of sulfated polysaccharides such as cellulose or chitin that mimic functionalities of glycosaminoglycans. Enzymatic sulfation of polysaccharide building blocks by sulfotransferases is synthetically attractive due to their ability to perform highly chemoselective sulfation in aqueous solution and at ambient temperature. The bacterial aryl sulfotransferase B - ASTB was reengineered to improve the sulfation activity toward the cellulose building block, cellobiose. A full KnowVolution campaign was performed. After screening 3,067 ASTB variants, Leu446 and Val579 were identified as beneficial positions to show positive effects on the ASTB activity.
Advancement of an aryl sulfotransferase toward a synthetically attractive sulfation agent for saccharides
Computational studies suggested that Leu446Pro conveys more flexibility to the substrate-binding site and Val579Lys is a distal substitution. Finally, the recombination of Leu446Pro and Val579Lys yielded a variant ASTB-M5 with up to 7.6-fold increased specific activity of ASTB toward cellobiose. A monosulfation of cellobiose was confirmed via mass spectrometry indicating a very selective sulfation. Furthermore, conversion was increased from 33.8% to 87.1% by the ASTB variant in the synthesis of the monosulfated glycosaminoglycan-building block, N-acetylglucosamine. Structure elucidation confirmed a partially regioselective monosulfation at C-3 and C-4 position, opposed to the chemically preferred C-6.
This work was a successful collaboration between Professor Schwaneberg and Professor Elling from RWTH Aachen and Professor Křen from Czech Academy of Sciences. Our work was financed by the projects FuPol, BioSulfa and the German Federal Ministry of Education and Research. For more detail, access this publication in our Research Highlights and Chemistry – A European Journal website.
Directed OmniChange evolution converts P450 BM3 into an alkyltrimethylammonium hydroxylaseCopyright: Bio VI
Yu Ji, Alan Maurice Mertens, Christoph Gertler, Sallama Fekiri, Merve Keser, Daniel F. Sauer, Kilian E. C. Smith, and Ulrich Schwaneberg*, Chemistry - A European Journal, 2018, doi:10.1002/chem. 201803806
Reengineering of the P450 BM3 substrate specificity towards the hydroxylation of CTAB by OmniChange multi-site mutagenesis method
Bolaform surfactants are a novel class of compounds with a wide range of industrial and technical applications. Bolaform surfactants are capable of forming of very small micelles and therefore are more effective than contemporary surfactants.
However, their production is expensive and involves the use of strong acids and large amounts of solvents. An alternative green synthesis route is the direct hydroxylation through monooxygenases as performed in nature.
In this study, the OmniChange multi-site mutagenesis method was applied for reengineering of the P450 BM3 substrate specificity towards the hydroxylation of CTAB by simultaneous mutagenesis of four relevant positions. Improved variants were identified in a two-step screening system. Then 10 promising P450 BM3 variants were analyzed by HPLC-MS/MS. Four P450 BM3 variants had significantly improved productivities and were kinetically characterized after purification. Interestingly all four variants were capable to di-hydroxylate CTAB and coupling efficiency up to 92.5% were obtained.
Notably, di-hydroxylation products of CTAB with bolaform surfactant properties have for the first time been produced. Bolaform surfactants are biodegradable and sustainable surfactants that offer excellent solubility properties and novel possibilities for drug delivery and/or compound formulations. Additionally, the two-step screening system proved to be efficient to boost coupling efficiency and can likely be used in many other P450 evolution campaigns to generate robust P450 catalysts. This work was funded by the China Scholarship Council, No. 201608080082.
Please find the link to this publication here.
Enzyme-compatible dynamic nanoreactors from electrostatically bridged like-charged surfactants and polyelectrolytes
Martin J. Thiele, Mehdi D. Davari, Isabell Hofmann, Melanie König, Carlos G. Lopez, Ljubica Vojcic, Walter Richtering, Ulrich Schwaneberg and Larisa A. Tsarkova, Angewandte Chemie, 2018, DOI: 10.1002/anie.201805021
In the present publication, a discovery of nanoreactors from electrostatically bridged like-charged surfactants and polyelectrolytes was reported. Incorporation of a protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance due to the improved solubilization accessibility for the protease through the interaction with the nanoreactors.
An unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid with like-charged surfactants is reported. Amphiphilic polymer-surfactant complexes with high interfacial activity and a solubilization capacity exceeding that of conventional micelles are formed by bridging with Ca2+ ions. Incorporation of a non-specific protease into such dynamic nanoreactors results in a synergistically enhanced cleaning performance due to improved solubilization of poorly water-soluble immobilized proteins. Competitive interfacial and intermolecular interactions on different time- and length-scales have been resolved using colorimetric analysis, dynamic tensiometry, light scattering, and molecular dynamic simulations. The discovered bridging association mechanism suggests reengineering of surfactant/polymer/enzyme formulations of modern detergents and opens new opportunities in advancing labile delivery systems.
Image: Scheme of an unanticipated mechanism of attractive electrostatic interactions of fully neutralized polyacrylic acid (PAA) with like-charged surfactant SLES. Amphiphilic polymer-surfactant complexes with high interfacial activity and solubilization capacity are formed by bridging with Ca2+ ions. Incorporation of a protease into such dynamic nanoreactors results in an enhanced cleaning performance because of the improved solubilization of poorly water-soluble immobilized proteins.
This research was partially funded by Henkel AG & Co. KGaA, Düsseldorf, Germany, as part of the Henkel Innovation Campus for Advanced Sustainable Technologies (HICAST). Simulations were performed with computing resources granted by JARA-HPC from RWTH Aachen University under projects RWTH0116 and JARA0169. L.A.T. acknowledges financial support by the Russian Foundation for Basic Research (RFBR) according to the research project No 18-53-76005.
A loop engineering strategy improves laccase lcc2 activity in ionic liquid and aqueous solutionCopyright: Bio VI
A. M. Wallraf, H. Liu, L. Zhu, G. Khalfallaha, C. Simons, H. Alibiglou, M. D. Davari and U. Schwaneberg, Green Chemistry, 2018, DOI: 10.1039/C7GC03776G
Importance of a domain-connecting loop in laccase for increased activity in ionic liquid (IL) was identified.
Laccases are involved in lignin degradation. EMIM- and BMIM-based ionic liquids -IL- show excellent solubilization of wooden biomass but impede laccase activity. Protein engineering to improve the activity and resistance of laccases in ILs is promising for lignin valorization for the sustainable production of fuels and bulk high-value chemicals . We report for the first time an efficient semi-rational design with focus on a domain-connecting loop of a laccase lcc2 (loop L1) from Trametes versicolor.
The loop engineering strategy is based on a KnowVolution campaign and can be divided into three steps. Prediction of seven resistance increasing positions out of 37 amino acids in L1 - residues 284-320 - was performed by in silico SSM analysis with FoldX. These seven positions were saturated by SSM and four beneficial positions were subjected to simultaneous SSM using OmniChange. The OmniChange variants OM1 -A285P/A310R/A312E/A318G- and OM3 -A310D/A312P/A318R- showed a 3.9-fold -535.8 ± 36.9 U/mg- and 1.6-fold -216.8 ± 5.3 U/mg increased specific activity in aqueous solution -lcc2 WT, 138.9 ± 6.5 U/mg-, respectively, and up to 8.4-fold increased activity in 35% EMIM EtSO4 and aqueous solution when compared to lcc2 WT. Hydrogen bond pattern analysis revealed that both variants harbor an increased number of hydrogen bonds within the loop and between domains two and three which resulted in increased IL resistance.
Entropy analysis indicated that the substitution of alanine at each selected amino acid position A285, A310, A312, and A318 reduced the flexibility of the loop L1. Conservational analysis with ConSurf server showed that the long domain-connecting loop L1 is a conserved feature in fungal laccases and suggests loop engineering as a useful strategy for increasing laccase activity in ILs and aqueous solutions. This work was funded by Deutsche Forschungsgemeinschaft DFG in the frame of the research cluster “ Tailor-Made Fuels from Biomass ” TMFB.
Cavity size engineering of a beta-barrel protein generates efficient biohybrid catalysts for olefin metathesisCopyright: Bio VI
Grimm, A.R.*, Sauer, D.F.*, Davari, M.D., Zhu, L., Bocola, M., Kato, S., Onoda, A., Hayashi, T., Okuda, J., Schwaneberg, U.; ACS Catalysis, 2018, 8, pp 3358–3364, DOI: 10.1021/acscatal.7b03652. (*contributed equally)
The successful application of the presented cavity size engineering strategy to biohybrid catalysis can potentially be transferred to other beta-barrel protein scaffolds to generate cavity sizes that match the sterical demands of synthetic catalysts.
By incorporating a synthetic metal catalyst into a protein scaffold, a biohybrid catalyst is obtained. The protein scaffold can potentially alter the selectivity of the metal catalyst.and solubilizes it in water, while retaining their broad reaction scope. What’s new about this work is that until now, protein scaffolds for synthetic catalysts have mainly been engineered by exchanging individual amino acids. While a lot has been achieved using this conventional strategy, it is limited by the number of amino acids in the protein available for substitution. Here, cavity size engineering of a beta-barrel protein called nitrobindin was performed by duplicating multiple beta-strands to generate an expanded variant.
"Incorporation of a synthetic metal catalyst into a protein scaffold yielded a biohybrid catalyst that combines remarkable performance in aqueous media with the broad reaction scope of organometallic catalysts."
Alexander R. Grimm
It is the first time this has been done for biohybrid catalysis. By duplicating entire beta-strands, it was possible to covalently incorporate bulky catalysts and achieve excellent conversions in a whole series of olefin metathesis reactions – carbon-carbon double bond formation reactions. What’s exciting about this is that the design strategy that was used can potentially be transferred to other beta-barrel protein scaffolds to generate cavity sizes that match the sterical demands of synthetic catalysts. If high-throughput screening is applied to these new variants in the future, it should be possible to explore directed biohybrid catalyst evolution much more efficiently, which will likely further increase performance. This work was made possible through funding from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.
A Whole Cell E. coli Display Platform for Artificial Metalloenzymes: Polyphenylacetylene Production with a Rhodium-Nitrobindin MetalloproteinCopyright: Bio VI
Grimm, A.R., Sauer, D.F., Polen, T., Zhu, L., Hayashi T., Okuda J., Schwaneberg, U. (2018). A whole cell E. coli display platform for artificial metalloenzymes: polyphenylacetylene production with a rhodium-nitrobindin metalloprotein. ACS Catal., 8, 2611-2613.
A Whole Cell E. coli Display Platform for Artificial Metalloenzymes
In the latest publication of our colleageus from the Hybrid Catalysis & High Throughput Screening division, the first bacterial cell surface display-based whole cell biohybrid catalyst, termed ArMt bugs, was generated, characterized and applied to the stereoselective polymerization of phenylacetylene. Whole cell catalysis is very important for the cost-effective production of chemicals by biotechnological means. Despite the promising application of whole cells to biohybrid catalysis, researchers worldwide had to face the inactivation of their valuable synthetic metal catalysts within cells due to abundant thiols.
The authors therefore armed the surface of their Escherichia coli whole cells with rhodium-nitrobindin biohybrid catalysts via an esterase-based autotransporter to separate the catalysts from inhibiting cellular compounds. A high turnover number of 39,000,000 per cell in the polymerization of phenylacetylene and potential applications in directed evolution and high-throughput screening make the ArMt bugs an attractive platform for bioorthogonal reactions. This work was made possible through funding from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.
An enzymatic route to α-tocopherol synthons: Aromatic hydroxylation of pseudocumene and mesitylene with P450 BM3Copyright: Bio VI
Dennig*, A., Weingartner*, A. M., Kardashliev, T., Müller, C. A., Tassano, E., Schürmann, M., Ruff, A. J., Schwaneberg, U. (2017). Chemistry - European Journal , first published online: October 9 2017, DOI: 10.1002/chem.201703647
P450 BM3 variants for the production of key phenolic building blocks for α-tocopherol synthesis
Alternative enzymatic routes for vitamin E synthesis are important to match increasing demands. Here, we show the first direct aromatic hydroxylation of pseudocumene and mesitylene in water, with O2 and under mild reaction conditions to access five key phenolic α -tocopherol synthons including a direct route to trimethylhydroquinone, TMHQ. The P450 BM3 wild-type catalyzed a 94% selective aromatic hydroxylation of mesitylene, whereas pseudocumene was hydroxylated to a large extent ranging from 46-64% on benzylic positions. Site-saturation mutagenesis generated a new P450 BM3 mutant, named “variant M3”, with a 3 to 8fold increased coupling efficiency and 75 to 230-fold increased activity for pseudocumene and mesitylene conversion. Additional π - π interactions introduced by mutation A330F improved not only productivity and coupling efficiency, but also a 61 to 75% selectivity toward aromatic hydroxylation of pseudocumene. Detailed product pattern analysis, substrate docking and mechanistic considerations support the hypothesis that pseudocumene binds in an inverted orientation in the active site of P450 BM3 WT as compared to P450 BM3 variant M3 to allow this change in chemo-selectivity.
Under continuous NADPH-recycling the novel P450 BM3 variant M3 was able to produce the key tocopherol precursor TMHQ at 35% selectivity at concentrations up to 0.18 mg ml-1 directly from pseudocumene, which is a significant step toward more sustainable synthesis of vitamin E. The reaction can be performed in one-pot without the need for intermediate purification or additional catalysts. In case of mesitylene over-oxidation leads to dearomatization and formation of a valuable p-quinol synthon that can directly serve as educt for synthesis of TMHQ. This study provides an enzymatic route to key phenolic synthons for α-tocopherols and first catalytic and mechanistic insights into direct aromatic hydroxylation and dearomatization of trimethylbenzenes with O2.
Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3-HydroxyalkanoatesCopyright: Bio VI
Yunus Ensari, Gaurao V. Dhoke, Mehdi D. Davari, Marco Bocola, Anna Joëlle Ruff, Ulrich Schwaneberg, Chemistry – A European Journal 2017, 23, 51, Issue, 12636-12645.
Enlargment of substrate binding pocket of cpADH5 led the conversion of medium chain methyl 3-hydroxyalkanoates and inverted enantiopreference for short chain methyl 3-hydroxyalkanoates.
Selective oxidation of primary or secondary alcohols to carbonyl compounds, i.e. aldehydes and ketones, is a key reaction in organic synthesis and industry. Oxidation of alcohol to ketones can be performed efficiently by a great number of chemical oxidation methods. Selectivity, undesired by-product formation, substrate scope and conversion rates are still major challenges in oxidation of alcohols. Alcohol dehydrogenases are promising alternatives to metal catalysts and perform reversible oxidations of primary or secondary alcohols to their corresponding aldehydes or ketones.
We hope that the enzymatic oxidation of 3-hydroxy-FAMEs will open novel synthetic routes to attractive building block in the synthesis of pheromones and b-lactam antibiotics.
Gaurao V. Dhoke
Ketone-functionalized fatty acid derivatives are versatile compounds in organic chemistry, are used as intermediates and building blocks in the production of pharmaceutically active compounds, detergents, and so on.
This study reports the first reengineered Candida parapsilosis alcohol dehydrogenase - cpADH5 - for the efficient oxidation of methyl 3-hydroxyhexanoate and methyl 3-hydroxyoctanoate, which were not converted by the cpADH5 WT, to their corresponding ketones. In this study, we mutated and redesigned the substrate binding pocket of cpADH5 in order to extend its substrate scope toward medium-chain 3-hydroxy fatty acid methyl esters – FAME- which contain 6--12 carbon atoms in their alkyl chains. Furthermore, we describe the inverted enantiopreference for the oxidation of methyl 3-hydroxybutyrate which was observed for our engineered cpADH5 variant.
Research highlight: Micro delivery service for fertilizersCopyright: Wiley-VCH
Plants can absorb nutrients through their leaves as well as their roots. However, foliar fertilization over an extended period is difficult. In the journal Angewandte Chemie, German researchers have now introduced an efficient delivery system for micronutrients based on biohybrid microgels. Special peptides anchor the "microcontainers" onto the leaf surface while binding sites inside ensure gradual release of the "cargo".
Foliar fertilization is already commonly used in areas such as viniculture, when the leaves on the vines turn yellow due to a mineral deficiency. Yet, despite the use of detergents, adhesives, and humectants, controlled delivery of nutrients through foliar fertilization over several weeks is nearly impossible to achieve. Up to 80% of the nutrients are washed away, winding up in the soil and being converted into forms that the plant cannot use. In addition, they can be washed into bodies of water and cause environmental problems. An additional problem is that strong sunlight evaporates the water out of the applied fertilizer solution. This results in a high salt concentration that draws water out of the leaf and causes burn damage.
A team from DWI-Leibniz Institute for Interactive Materials in Aachen, RWTH Aachen University, and the University of Bonn has now developed a foliar fertilization system based on biocompatible microgels that adhere selectively to leaves for a long period and slowly deliver nutrients in a controlled fashion. Microgels are tiny particles of cross-linked macromolecules that can bind water and other molecules, such as fertilizers very efficiently.
Led by Ulrich Schwaneberg, Andrij Pich and Felix Jakob, the researchers equipped the interiors of gel particles with binding sites modeled on the iron-binding proteins of bacteria. These ensure that the iron ions are released slowly. The microgels are loaded with an iron-containing solution at a pH of 3. When the pH rises to 7, the microgels shrink, releasing water and binding the iron.
The surface of the gel particles is equipped with anchor peptides from lactic acid bacteria. These bind securely to leaf surfaces to hinder rinsing away of the microgels. The water in the gel provides an aqueous microenvironment that allows the iron to diffuse into the leaves. Yellow leaves of iron-deficient cucumber plants rapidly turned green in spots where the new foliar fertilizer was applied.
By incorporating different binding sites, the microgel "containers" can be loaded with a multitude of other metal ions or agents. A controlled delivery of agents as required would minimize the applied quantities as well as the release of fertilizers and pesticides into the environment. Low production costs, high levels of loading, easy application, and adjustable adhesive properties should make broad industrial applications possible. The goal is to make self-regulating delivery systems for sustainable agriculture.
Richard A. Meurer et al, Biofunctional Microgel-Based Fertilizers for Controlled Foliar Delivery of Nutrients to Plants, Angewandte Chemie International Edition (2017). DOI: 10.1002/anie.201701620
Generation of building blocks for synthesis of filter membranesCopyright: Bio VI
The iron transporter FhuA - Ferric hydroxamate uptake protein component A is a channel protein produced by the bacterium Escherichia coli which was tailored for the generation of synthetic membranes using FhuA proteins and polymers. The FhuA protein forms a permeable channel with a uniform pore size from 2.5 to 3.0 nm and a barrel-like structure. Lysine residues were specifically located in a rim on the outer surface of the channel above the transmembrane region for the attachment of polymer chains. This design enabled the grafting of PNIPAAm polymer chains from the outer FhuA channel surface. In the future, the synthesized building blocks of the FhuA channel proteins and polymer chains will be used for generation of hybrid membranes for nanofiltration processes. These generated membranes can be applied in the downstream processing as molecular sieves for the separation of different components which is essential for synthesizing sweeteners, pesticides and pharmaceuticals.
In vitro flow cytometry-based screening platform for cellulase engineeringCopyright: Bio VI
Screening technologies are of pivotal importance for tailoring biocatalysts in directed evolution, as millions of mutant enzyme variants could be generated in every trial. Hence, ultrahigh throughput screening techniques have been developed in order to still complete such trials in a reasonable amount of time. These techniques are well capable of analyzing up to 107 events per hour and thus can analyze the complete coverage of a generated protein sequence with high efficiency.
This technology becomes even more powerful if it is coupled with a cell-free enzyme expression technique. This expression method enables the experimentator to reduce diversity loss when transforming mutant libraries into expression hosts, to design enzymes of animal or human origin or even perform directed evolution of toxic enzymes.
The first ever combination of such a cell-free compartmentalization platform with a flow cytometry-based screening has been achieved in the InVitroFlow technology and successfully applied to directed evolution of cellulose enzymes.
Screening through the PLICable promoter toolbox enhances protein production in Escherichia coliCopyright: Bio VI
Escherichia coli is a common host for recombinant protein production in which product titers are highly dependent on the employed expression system. Thereby, promoters are a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed. It enables the identification of the most suitable promoter out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) for high level protein production in a single cloning step and after a screening experiment.