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.
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
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.
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.
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.