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Exploring and engineering the substrate specificity of transaminases

  • In 2010, the identification of 17 novel (R)-ATAs represented a breakthrough for the biocatalytic asymmetric synthesis of chiral amines, because only one (R)-ATA was described before. These novel ATAs were identified in a bioinformatic approach by studying the substrate acceptance of BCATs and DATAs to deduce the unknown substrate coordination of (R)-ATAs. Article I describes an alternative approach for the identification of (R)-ATA activity by reengineering the substrate- recognition site of α-AATs. While the engineering of the eBCAT led to the formation of an initial (R)-amine acceptance only, the (R)-ATA activity was successfully introduced in the DATA scaffold. These results demonstrate the transformation of an α-AAT in a moderately active (R)-ATA for the first time and highlight the evolutionary relationship between α-AATs and ATAs. Despite the availability of different ATAs nowadays, their substrate spectrum is limited due to the natural composition of their active sites. Several protein-engineering studies showed the widening of the substrate spectrum and the acceptance of bulky substrates by screening large mutant libraries to identify beneficial variants. In Article II, we developed an in silico engineering approach for amine transaminases to improve the conversion of bulky substrates and to reduce the number of variants to be tested in the laboratory. The resulting double-mutants of the (S)-ATA from C. violaceum displayed a >200-fold improved activity towards the bulky benchmark substrate. These variants expand the available biocatalytic toolbox for the synthesis of bulky amines, and the developed framework paves the way for rational protein-engineering protocols. By studying unconventional transaminase substrates, we explored the potential of the available in- house transaminase toolbox in Articles III, IV, V, and VI. In Article III, we showed the transamination of a β-keto ester, leading to the synthesis of β-phenylalanine. The described cascade in Article IV enables the synthesis of amino carbohydrates. In addition, Article V describes an enzymatic cascade for the synthesis of amino fatty acids, which was extended in Article VI to obtain fatty amines. The findings of this thesis clearly contribute to the understanding of the substrate scope and specificity of amine transaminases and expand the application of this versatile biocatalyst beyond classical ketone substrates.
  • Die Dissertation behandelt die Veränderung von Transaminasen mittels Protein Engineering, um neue Erkenntnisse der evolutionären Entwicklung und der Struktur-Aktivitätsbeziehung dieser Enzyme zu erhalten. Ferner wurde die Synthese ausgewählter Amine untersucht, um die Anwendungsgebiete von Transaminasen in der Biokatalyse zu erweitern.

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Author: Moritz Voß
Title Additional (German):Untersuchung und Veränderung der Substratspezifität von Transaminasen
Referee:Prof. Dr. Uwe T. Bornscheuer, Prof. Dr. Per Berglund
Advisor:Prof. Dr. Uwe T. Bornscheuer
Document Type:Doctoral Thesis
Year of Completion:2020
Date of first Publication:2020/07/06
Granting Institution:Universität Greifswald, Mathematisch-Naturwissenschaftliche Fakultät
Date of final exam:2020/07/03
Release Date:2020/07/06
Tag:Biocatalysis, Protein Engineering, Transaminases
GND Keyword:Biokatalyse
Faculties:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie und Biochemie
DDC class:500 Naturwissenschaften und Mathematik / 540 Chemie