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Enzyme Engineering for Intensified Processes for the Production of Rare Monosaccharides

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Enzyme Engineering for Intensified Processes for the Production of Rare Monosaccharides Research Collection Doctoral Thesis Enzyme engineering for intensified processes for the production of rare monosaccharides Author(s): Bosshart, Andreas Publication Date: 2014 Permanent Link: https://doi.org/10.3929/ethz-a-010252699 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO 22128 Enzyme engineering for intensified processes for the production of rare monosaccharides A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by Andreas Bosshart Master of Science UZH in Biochemistry, University of Zurich born on 29.11.1982 citizen of Fischingen (TG), Switzerland accepted on the recommendation of Prof. Dr. Sven Panke (ETH Zurich, Switzerland), examiner Prof. Dr. Andreas Plückthun (University of Zurich, Switzerland), co-examiner Prof. Dr. Sai Reddy (ETH Zurich, Switzerland), co-examiner 2014 ABSTRACT With the current trend in chemical industry towards more sustainable processes that produce less waste and are less energy-intensive, enzyme-catalyzed reactions are gaining increasing interest due to their striking selectivity and specificity and their ability to operate at ambient conditions (neutral pH, aqueous solution, moderate temperatures). However, biocatalysts considered for industrial application need to provide high reaction rates (i.e., a high specific activity) and operational stability in order to be competitive in economic terms. In order to overcome limitations in such properties, enzyme engineering procedures like directed evolution or rational design have been successfully applied for a wide range of enzymes. This thesis describes the development of a D-tagatose epimerase from Pseudomonas cichorii (PcDTE) into an industrially suitable biocatalyst by directed evolution. D-Tagatose epimerase is arguably the central enzyme in the synthesis of a wide range of rare monosaccharides, carbohydrates that are not available in large amounts from natural resources, but have recently attracted great interest as low-calorie sweetener, chiral building blocks, or active pharmaceutical ingredients. They can be synthesized via simple isomerization or epimerization reactions from readily available bulk sugars as e.g. D-glucose, D-fructose or D-galactose, but these reactions suffer from an unfavorable position of the thermodynamic equilibrium, limiting the yield and making these reactions economically unfeasible. Integration of enzyme- catalyzed reaction and separation of product and reactant, e.g. by continuous chromatography, into one continuous process can offer a highly attractive solution to overcome this limitation. Such an integrated process, however, demands high standards of the biocatalyst in terms of stability, selectivity and specific activity in order to enable an economic process. Thermostability is one of the most frequent limitations in the application of biocatalysts for the synthesis of fine and bulk chemicals, especially at elevated temperatures which are often required in sugar-processing plants to avoid microbial contaminations, to reduce the viscosity and to increase the reaction rate. Systematic screening of the subunit-subunit interface of dimeric PcDTE in a medium-throughput in vitro assay format revealed several mutations that increased the thermostability of PcDTE. Combination of all 9 beneficial sites by iterative saturation mutagenesis (ISM) resulted in variant PcDTE Var8 that had an increase in temperature stability of 21.4°C over wild-type. This variant showed no significant loss in D-fructose conversion over 4 days in a long-term experiment at 50°C, while conversion catalyzed by the WT decreased by 40% in the same time. Next, this variant was taken as basis for improving the specific activity of the enzyme for the reactions D-fructose/D-psicose and L-sorbose/L-tagatose. Saturation mutagenesis of 20 individual residues in the first sphere and 28 residues in the second sphere around the active site revealed 8 mutations that increased activity towards D-fructose in variant IDF8 and 6 mutations that did so for L-sorbose in variant ILS6. IDF8 exhibited an increased specific activity I for D-fructose of 8.6-fold and ILS6 showed a 13.5-fold higher catalytic rate for L-sorbose. The crystal structures of PcDTE Var8, IDF8 and ILS6 in presence of the respective substrates indicated that modifications of the entrance tunnel afforded an increased catalytic rate for IDF8 whereas the increase for ILS6 rather derived from differences in the hydrogen bonding network to substrate and product. The operational performance of further developed variant of IDF8, IDF10-3, was determined in an enzyme-membrane reactor at different temperatures in presence of the respective substrate and confirmed that these final variants exhibited an up to 40-fold higher total turnover numbers compared to WT PcDTE. Finally, to reduce the screening effort for the directed evolution of PcDTE, a growth-based selection system was developed that was expected to facilitate the discovery of variants with improved specific activity significantly. Therefore, a de novo metabolic pathway from D-allose via D-psicose and D-fructose to fructose 6-phosphate was established by introduction of the enzymes L-rhamnose isomerase, D-tagatose epimerase and fructokinase. Additionally, five genes or complete operons of the E. coli host were deleted that were found to interfere with the proposed metabolic pathway. Growth of the assembled selection system on D-allose as sole carbon source could demonstrate the principle feasibility of the approach, but consistent failures of the selections suggest that either a pathway intermediate had a toxic effect on the selection host or that the catalytic activity of one of the pathway enzymes had too low activity to effectively satisfy the flux requirement for growth on D-allose. II ZUSAMMENFASSUNG Der Fokus der chemischen Industrie liegt zunehmend auf neuen Prozessen, die weniger Abfall produzieren, weniger Energie verbrauchen und weniger gefährliche oder giftige Lösungsmittel benötigen. Enzym-katalysierte Reaktionen können hier einen entscheidenden Beitrag leisten, sowohl wegen ihrer erstaunlichen Spezifität und Selektivität, als auch wegen ihrer Fähigkeit, Reaktionen in wässriger Lösung, bei beinahe neutralem pH und moderaten Temperaturen zu katalysieren. Um sowohl prozesstechnischen als auch ökonomischen Ansprüchen zu genügen, muss ein Biokatalysator jedoch bestimmte Anforderungen erfüllen, wie beispielsweise eine seht gute spezifische Aktivität und Selektivität, als auch genügend Prozessstabilität gewährleisten. Durch Protein-Engineering lassen sich praktisch alle dieser Parameter an die Prozessbedürfnisse anpassen. In dieser Arbeit wird die Entwicklung einer D-Tagatoseepimerase vom Bakterium Pseudomonas cichorii (PcDTE) behandelt, die durch gerichtete Evolution zu einem Biokatalysator verändert wurde, der prozessbedingte Anforderungen erfüllen kann. D-Tagatoseepimerase ist das zentrale Enzym in der Synthese von seltenen Zuckern, die in letzter Zeit grosses Interesse geweckt haben durch ihre potentielle Rolle als kalorienarme Süssstoffe, als chirale Bausteine für die Synthese von Arzneistoffen oder direkt als pharmazeutisch aktive Stoffe. Diese seltenen Zucker kommen in der Natur nur in Spuren vor, lassen sich aber durch einfache enzymatische Isomerisierungsreaktionen aus günstig verfügbaren Zuckern wie D-Glukose, D-Fruktose oder D-Galaktose herstellen. Das Hauptproblem bei der Synthese dieser Stoffe durch enzymatische Katalyse ist das thermodynamische Gleichgewicht der Iso- oder Epimerisierungsreaktionen, was dazu führt, dass die Reaktionen unvollständig ablaufen und somit nur eine relativ geringe Ausbeute ermöglichen. Durch eine Integration der enzymatischen Reaktion mit einer direkt anschliessenden Trennung des Produkts von Ausgangs- und Zwischenprodukten und einem Recycling des Ausgangsstoffs zurück in die Reaktion, lässt sich dieses Hindernis jedoch effektiv überwinden. Andererseits stellt eine solche Integration hohe Anforderungen an den Biokatalysator bezüglich Selektivität, spezifische Aktivität und vor allem Thermostabilität, die indirekt – als Stellvertreter für Prozessstabilität allgemein – und in diesem Fall auch direkt (siehe unten) von Bedeutung ist. Ungenügende Prozess- und/oder Thermostabilität ist eine der häufigsten Limitationen bei der Anwendung von Biokatalysatoren in der Synthese von Fein- und Massenchemikalien. Oftmals werden erhöhte Temperaturen benötigt, speziell in der Zuckerindustrie, um Kontaminationen durch Mikroorganismen zu verhindern, die Viskosität der Zuckerlösung zu reduzieren oder um die Reaktionsrate zu erhöhen. Das Wildtypenzym PcDTE zeigte eine ungenügende Stabilität bei solchen Bedingungen. Eine systematische Durchmusterung der Kontaktflächen der beiden Untereinheiten des dimeren Enzyms PcDTE förderte mehrere Mutationen zutage, die eine erhöhte Thermostabilität des Enzyms bewirkten, vermutlich indem sie die Trennung der III Untereinheiten als den die Enzyminaktivierung einleitenden Schritt hinauszögern. Die Kombination von optimalen Mutationen in allen neun identifizierten Positionen durch iterative 20 Sättigungsmutagenese resultierte in einer finalen Variante namens PcDTE Var8, die einen T50 - Wert (die Temperatur, bei der nach einer Inkubation von 20 min nur noch 50% der ursprünglichen Aktivität gemessen werden kann) von 87°C erreichte, was einem
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