KnowVolution: Redesigning enzymes for innovations

Directed evolution has matured in academia and industry to a routinely applied algorithm to tailor enzyme properties1 to match especially demands in synthesis and material science. In order to free directed enzyme evolution from methodological restraints and to efficiently explore its potential, one has to balance time requirements for a directed evolution campaign, the number of generated enzyme variants, and limitations in state of the art screening technologies. For instance, saturation mutagenesis of six amino acid positions in an enzyme, which usually consists of \u3e50 amino acids, yields 64 million (206) different enzyme variants. The latter represents the upper throughput for activity-based screening systems2. In essence, protein engineers have to accept that they will not be able to sample through the theoretical obtainable sequence space of enzyme variants and smarter strategies are required for efficient directed enzyme evolution. The KnowVolution (Knowledge gaining direct evolution)3 approach represents such a directed evolution 2.0 strategy, which identifies in four phase with limited screening efforts, significantly improved enzymes variants and ensures a molecular understanding of improved enzyme properties. Three out of six in a review reported KnowVolution campaigns3 were commercialized by industrial partners; thereby limiting the number of substitutions turned out to be a key prerequisite for maintaining thermal resistance, process stability and selectivity. In addition, directed enzyme evolution by random mutagenesis will be compared to improvements that are obtainable with a variant library that contains all natural possible diversity with ONE amino acid exchange (SSM library)4. The comparison of 3000 mutations from random mutagenesis libraries with the SSM library taught us how many of the natural occurring beneficial positions are obtainable or unobtainable by state of art methodologies in directed evolution and provided first insights on general design principles to improve enzymatic resistance in organic cosolvents4 and ionic liquids4. References: (1) a.Shivange, A. V., Marienhagen, J., Mundhada, H., Schenk, A., Schwaneberg, U. (2009). Curr. Opin. Chem. Biol. 13, 19. b.Ruff, A. J., Dennig, A., Schwaneberg, U. (2013). FEBS J. 280, 2961. (2) a.Körfer, G., Pitzler, C., Vojcic, L., Martinez, R., Schwaneberg, U. (2016). Scientific Reports, 6, 1-12. b.Lülsdorf, N., Pitzler, C., Biggel, M., Martinez, R., Vojcic, L., Schwaneberg, U. (2015). Chem. Commun. 51, 8679. c.Ruff, A. J., Dennig, A., Wirtz, G., Blanusa, M., Schwaneberg, U. (2012). ACS Catalysis 2, 2724. (3) Cheng, F., Zhu, L., Schwaneberg, U. (2015). Chem. Commun. 51, 9760. a.Zhao, J., Frauenkron-Machedjou, V. J., Kardashliev, T., Ruff, A. J., Zhu, L., Bocola, M., Schwaneberg, U. (2017). Appl. Microbiol. Biotechnol., 2017, DOI: 10.1007/s00253-016-8035-1. b.Frauenkron-Machedjou, V. J., Fulton, A., Zhu, L., Bocola, M., Zhu, L., Jaeger, K.-E., Schwaneberg, U. (2015). ChemBioChem, 16, 937-945. c.Zhao, J., Kardashliev, T., Ruff, A. J., Bocola, M., Schwaneberg, U. (2014). Biotechnol. Bioeng. 111, 2380

Optimizing the phosphorus cycle in the sugar beet production process by phytase supplement

Phosphate stewardship and ultimately recycling is one of the great challenges of humankind. Depleting phosphorus (P) resources demand new strategies for an efficient use of this essential nutrient. Therefore, especially phosphorus cycles in agriculture have to be closed. Against this backdrop we propose a new value chain to recover phosphate from plant waste material in sugar production. The approach is based on naturally occurring enzymes that free the phosphate bound in an organic form (mainly phytate in sugar beet slices). Thereby, the currently implemented value chain of phosphate rock mining, production of phosphoric acid, chemical synthesis of polyphosphates, and after use phosphate disposal into waste water, rivers and finally into the ocean will be extended and in the long run disrupted. In sugar production processes this could be achieved by supplementing thermally resistant phytases to leach the phosphate bound as phytate form sugar beet slices. With this procedure the P concentration in sugar beet slices and the export of phosphorous with fodder to areas with high animal density and in consequence P-excess in fields will be reduced. Instead, isolated phosphorus will be transferred into spent lime and subsequently back to the sugar beet fields. The BioSC collaboration project PhytaPhoS assesses and evaluates the potential of P recovery employing phytase, its feasibility and economic approaches from lab scale to field application. Phytases are extremely highly active phosphatases (\u3e1000 U/mg), mobilizing inorganic phosphate from plant based phytate, which is a natural plant phosphate reservoir [1]. Aim in the project is improving specific activity and thermal resistance of a selected phytase by directed evolution [2,3,4] and optimizing the phytase production by employing a signal peptide library from Bacillus subtilis [5]. Please click Additional Files below to see the full abstract

Особенности применения автодинных КВЧ модулей для сканирующей зондовой микроволновой микроскопии

Представлен анализ структуры КВЧ микроскопа, конструкции зондового элемента и использования КВЧ автодинных модулей как варианта реализации микроволной сканирующей микроскопии. Показано существенное упрощение реализации КВЧ микроскопа при использовании автодинных датчиков

High-Yield Synthesis of Enantiopure 1,2-Amino Alcohols from L-Phenylalanine via Linear and Divergent Enzymatic Cascades

[Image: see text] Enantiomerically pure 1,2-amino alcohols are important compounds due to their biological activities and wide applications in chemical synthesis. In this work, we present two multienzyme pathways for the conversion of l-phenylalanine into either 2-phenylglycinol or phenylethanolamine in the enantiomerically pure form. Both pathways start with the two-pot sequential four-step conversion of l-phenylalanine into styrene via subsequent deamination, decarboxylation, enantioselective epoxidation, and enantioselective hydrolysis. For instance, after optimization, the multienzyme process could convert 507 mg of l-phenylalanine into (R)-1-phenyl-1,2-diol in an overall isolated yield of 75% and >99% ee. The opposite enantiomer, (S)-1-phenyl-1,2-diol, was also obtained in a 70% yield and 98–99% ee following the same approach. At this stage, two divergent routes were developed to convert the chiral diols into either 2-phenylglycinol or phenylethanolamine. The former route consisted of a one-pot concurrent interconnected two-step cascade in which the diol intermediate was oxidized to 2-hydroxy-acetophenone by an alcohol dehydrogenase and then aminated by a transaminase to give enantiomerically pure 2-phenylglycinol. Notably, the addition of an alanine dehydrogenase enabled the connection of the two steps and made the overall process redox-self-sufficient. Thus, (S)-phenylglycinol was isolated in an 81% yield and >99.4% ee starting from ca. 100 mg of the diol intermediate. The second route consisted of a one-pot concurrent two-step cascade in which the oxidative and reductive steps were not interconnected. In this case, the diol intermediate was oxidized to either (S)- or (R)-2-hydroxy-2-phenylacetaldehyde by an alcohol oxidase and then aminated by an amine dehydrogenase to give the enantiomerically pure phenylethanolamine. The addition of a formate dehydrogenase and sodium formate was required to provide the reducing equivalents for the reductive amination step. Thus, (R)-phenylethanolamine was isolated in a 92% yield and >99.9% ee starting from ca. 100 mg of the diol intermediate. In summary, l-phenylalanine was converted into enantiomerically pure 2-phenylglycinol and phenylethanolamine in overall yields of 61% and 69%, respectively. This work exemplifies how linear and divergent enzyme cascades can enable the synthesis of high-value chiral molecules such as amino alcohols from a renewable material such as l-phenylalanine with high atom economy and improved sustainability

Postoperatives zerebrales Autoregulationsmonitoring mittels Cerebral Oximetry Index (COx) zur Definition individueller Blutdruckgrenzen bei Säuglingen mit angeborenen Fehlbildungen des Herzens

Mit steigender Lebenserwartung der Patienten mit angeborenen Herzfehlern, die sich bereits im Neugeborenenalter schweren kardialen Eingriffen unterziehen müssen, rücken zunehmend neurologische Langzeitfolgen perioperativer Dysreguationszustände und damit die Entwicklung neuroprotektiver Strategien in den Fokus der Forschung. Die vorliegende Arbeit befasst sich mit der Frage, ob bei Säuglingen mit angeborenen Fehlbildungen des Herzens im Lebensalter bis 12 Monate in den ersten 24h postoperativ nach erfolgter Korrektur oder Palliation des Vitiums unter Einsatz der Herzlungenmaschine individuelle Blutdruckbereiche definierbar sind, in denen die Funktion der zerebralen Autoregulation intakt und damit das Risiko zerebraler Hypo- und Hyperoxien reduziert ist. Darüber hinaus sollte evaluiert werden, ob die ggf. ermittelten Blutdruckwerte mit Literaturwerten bzw. bisherigen Empfehlungen zum postoperativen Blutdruckmanagement übereinstimmen. In der vorliegenden, monozentrischen, prospektiven Studie wurden bei 36 Säuglingen nahinfrarotspektroskopisch die Autoregulationsfähigkeit als Cerebral Oximetry Index (COx) sowie mittels invasiver Messung der mittlere arterielle Blutdruck (MAP) ermittelt. Der optimale Blutdruck (MAPopt), die obere und untere Blutdruckgrenze (ULA, LLA) mit noch erhaltender zerebraler Autoregulation wurden mittels erstellter U-Kurve visuell bestimmt. Es konnte gezeigt werden, dass eine Definition individueller Autoregulationsgrenzen im postoperativen Setting möglich ist, ein MAPopt konnte bei fast allen Probanden, ein LLA und ULA in den meisten Fällen ermittelt werden. In rund 13% der untersuchten Zeit unterschritt der MAP das LLA. Die zentrale Beobachtung des vorliegenden Studie ist, dass das LLA oberhalb der bisher publizierten empfohlenen Blutdruckgrenzen lag, und damit der Hinweis darauf besteht, dass Patienten unter Einhaltung der bisher geltenden Empfehlungen einem Risiko zerebraler Hypoxien bzw. Ischämien ausgesetzt sein könnten. Palliierte Patienten wiesen zwar eine niedrigere zerebrale Sauerstoffsättigung auf, zeigten sich aber nicht häufiger dysautoreguliert. Das Ziel künftiger Studien sollte sein, zu evaluieren, inwieweit das neurologische Outcome durch die Funktion der zerebralen Autoregulation beeinflusst werden kann und ob sich ggf. durch Manipulation einzelner Einflussfaktoren neue zerebrale Läsionen verhindern lassen

KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding

The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm2 on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents

Appart

Appart es una app con la que se creará la NFT más colaborativa del planeta. Una mega escultura digital compuesta de Tótems 3D creados por los participantes / artistas con la herramienta de creación de la misma app . Una obra de arte fractal, y un museo en si misma. La GobalNFT. Con Appart, se pretende crear un espacio en el que democratizar el arte (tanto para usuario como para espectador), generar igualdad de oportunidad y valor, y demostrar que la tecnologia puede ser una buena herramienta para llevar a cabo una democracia real, más transparente, y descentralizada. En general, todo el proyecto gira en torno el arte y la democracia en varios de sus sentidos y/o aplicaciones: es un proyecto humanista y tecnológico.Appart és una app amb què es crearà la NFT més col·laborativa del planeta. Una mega escultura digital composta de Tòtems 3D creats pels participants/artistes amb l’eina de creació de la mateixa app. Una obra d’art fractal i un museu en si mateixa. La GlobalNFT. Amb Appart, es pretén crear un espai on democratitzar l’art (tant per a usuari com per a espectador), generar igualtat d’oportunitat i valor, i demostrar que la tecnologia pot ser una bona eina per dur a terme una democràcia real, més transparent , i descentralitzada. En general, tot el projecte gira al voltant de l’art i la democràcia en diversos sentits i/o aplicacions: és un projecte humanista i tecnològic.Appart is an app that will create the most collaborative NFT on the planet. A mega digital sculpture composed of 3D Tótems created by the participants / artists with the creation tool of the same app. A work of fractal art, and a museum in itself. The GlobalNFT. With Appart, the aim is to create a space in which to democratize art (both for users and viewers), generate equality of opportunity and value, and demonstrate that technology can be a good tool to carry out a real, more transparent democracy. , and decentralized. In general, the entire project revolves around art and democracy in several of its meanings and/or applications: it is a humanistic and technological project

From natural to artificial metalloproteins – Challenges and opportunities

Organometallic catalysts are used in a wide range of industrially important reactions. By immobilizing such catalysts to a protein (Fig. 1), we can bring them into aqueous media, fine-tune activities and selectivities and overcome the challenges associated with trace metal removal in the product fraction [1, 2]. Trace metal removal is particularly important for the synthesis of pharmaceutical compounds. Typically, the transition metal content should be below 10 ppm. We have employed two types of metal catalysts, a Ru-based Grubbs-Hoveyda-type catalyst for olefin metathesis and a Rh-catalyst for phenylacetylene polymerization. These catalysts were covalently attached to either nitrobindin (NB) [3] or ferric hydroxamate uptake protein component: A (FhuA) [4] β-barrel proteins, yielding biohybrid catalysts (also denoted artificial metalloproteins) that can be immersed in aqueous reaction media either in their free form or immobilized to bacterial cells. Moreover, we could show that the metal catalysts can be immobilized on surfaces consisting of silica or polypropylene via peptide-based adhesion promoters, thereby enabling “green” surface immobilization strategies with the potential of catalyst recycling [5]. Either strategy yielded highly active catalysts that show great promise for single or sequential one-pot reactions. Separation of products and the catalysts was readily achieved by extraction. With the potential to tune reaction efficiencies and selectivities by modifying either the metal catalyst or the protein surrounding, biohybrid catalysts bear great potential to amend or even substitute the repertoire of reactions available by organic synthesis and, likewise, biocatalysis. Please click Additional Files below to see the full abstract

Molecular understanding of sterically controlled compound release through an engineered channel protein (FhuA)

<p>Abstract</p> <p>Background</p> <p>Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Δ1-160; Onaca <it>et al</it>., 2008). Compound fluxes within the FhuA Δ1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.</p> <p>Results</p> <p>In total, 12 FhuA Δ1-160 variants were generated to gain insights on sterically controlled compound fluxes: Subset A) six FhuA Δ1-160 variants in which one of the six lysines in the interior of FhuA Δ1-160 was substituted to alanine and Subset B) six FhuA Δ1-160 variants in which only one lysine inside the barrel was not changed to alanine. Translocation efficiencies were quantified with the colorimetric TMB (3,3',5,5'-tetramethylbenzidine) detection system employing horseradish peroxidase (HRP). Investigation of the six subset A variants identified position K556A as sterically important. The K556A substitution increases TMB diffusion from 15 to 97 [nM]/s and reaches nearly the TMB diffusion value of the unlabeled FhuA Δ1-160 (102 [nM]/s). The prominent role of position K556 is confirmed by the corresponding subset B variant which contains only the K556 lysine in the interior of the barrel. Pyridyl labeling of K556 reduces TMB translocation to 16 [nM]/s reaching nearly background levels in liposomes (13 [nM]/s). A first B-factor analysis based on MD simulations confirmed that position K556 is the least fluctuating lysine among the six in the channel interior of FhuA Δ1-160 and therefore well suited for controlling compound fluxes through steric hindrance.</p> <p>Conclusions</p> <p>A FhuA Δ1-160 based reduction triggered release system has been shown to control the compound flux by the presence of only one inner channel sterical hindrance based on 3-(2-pyridyldithio)propionic-acid labeling (amino acid position K556). As a consequence, the release kinetic can be modulated by introducing an opportune number of hindrances. The FhuA Δ1-160 channel embedded in liposomes can be advanced to a universal and compound independent release system which allows a size selective compound release through rationally re-engineered channels.</p