Directed Evolution of an Fe(II)-Dependent Halogenase for Asymmetric C(sp3)-H Chlorination

Within this thesis, the chlorinase Wi-WelO15 from Westiella intricata UH strain HT-29-1 was explored for its evolvability towards non-natural substrates and its biocatalytic applicability. Organohalogens play an important role as pharmaceuticals and agricultural chemicals or synthetic intermediat...

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Bibliographic Details
Main Author: Düwel, Sabine
Contributors: Höbenreich, Sabrina (Dr. rer. nat.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2018
Chemie
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Summary:Within this thesis, the chlorinase Wi-WelO15 from Westiella intricata UH strain HT-29-1 was explored for its evolvability towards non-natural substrates and its biocatalytic applicability. Organohalogens play an important role as pharmaceuticals and agricultural chemicals or synthetic intermediates. However selective aliphatic chlorination under mild and non-hazardous conditions at complex molecules is still a formidable challenge in chemical and enzymatic catalysis. A new class of aliphatic halogenases, which carries a great potential as useful biocatalysts for late stage chlorination, was discovered in 2014. The enzyme class belongs to a big superfamily of non-heme iron oxygenases. A previously found subclass of aliphatic halogenases are dependent on an acyl-carrier protein, in contrast to that, the new subclass is able chlorinate freestanding substrates. Synthetic methods, available to chemists, can be imagined as a toolbox which is used to create molecules of interest. If this class of halogenases can be engineered to chlorinate selectively non-natural substrates and can be produced and used under convenient conditions in the lab, they can be added to the synthesis toolbox enabling late-stage chlorination at complex molecules. Within this thesis, Wi-welO15 and Hw-welO15 (from Hapalosiphon welwitschii UH IC-52-3) were amplified from genomic DNA, and inserted into a high copy plasmid, resulting in great amounts of 60-80 mg enzyme/L expression culture by heterologous expression in E. coli. Purification of the halogenase was achieved by immobilized metal affinity chromatography and if necessary, also size exclusion chromatography. Crystallization of the starting variant Wi-0 for directed evolution was achieved and a structure of the enzyme could be solved by molecular replacement. To test the evolvability of the halogenases towards non-natural substrates, a lead molecule was chosen that contains a ketone instead of the naturally occurring isonitrile functionality, which was not converted by both halogenases, with initial reaction conditions. First random mutagenesis of Hw-WelO15, based on a model created by SwissModel, was pursued while no structural information was available. After we solved the structure of Wi-0, directed evolution via semi-rational and rational mutagenesis was applied to evolve the halogenase in 4 generations to accept the chosen lead molecule. A panel of chlorinases was tested as lysates as well as isolated enzymes. Hereby the best variants revealed up to 87% product formation under optimized conditions while <1% product formation was present with Wi-0. Modifications at the lead structure were partially accepted and in total five non-natural substrates were chlorinated with >99.9% regio- and diastereoselectivity and chemoselectivities of up to 96-99%. In addition, transformation of a range of seven substrates were transformed into complex mixtures of novel products. Hydroxylation by aliphatic halogenases is described, if non-native substrates are used. One variant found in the directed evolution already has a decreased chlorination selectivity and produces 32% hydroxylated side product. With the goal to increase the hydroxylation selectivity, several variants were created. Because these catalysts completely lost their activity, instead the monooxygenase P450BM3 was used to produce four new, oxygenated products of the lead structure. Our discovered halogenases from different states of the engineering process were characterized for their kinetic properties, revealing that the best variant Wi-12 has a 93-fold improvement in comparison to Wi-2, which has the lowest measurable kcat/Km . The two variants with highest product formation each have their own kinetic profile. While Wi-11 is most active with a kcat of 0.64 min-1, Wi-12 has the lowest Km of 20 μM for the lead substrate. Furthermore, the stability of the halogenases was examined and stabilized variants proposed by the PROSS server created. As read out, melting temperatures were determined revealing a high thermal stability for the wild types with Tm of 62 °C for Hw-WelO15 and 54 °C for Wi- WelO15. Created variants generally showed an improve in thermal stability and combination of stability with activity improving mutations resulted in a melting temperature of 61 °C for Wi-5P3. Incubation of purified enzymes in buffer at different temperatures with subsequent SDS-PAGE analysis showed that after incubation at 65 °C for 4 h, still soluble enzyme of the stabilized variants remains. Activity assays at increased temperatures or solvent ratios, showed a residual activity of 90% at 45 °C with Wi-5P3 and 85% residual activity in the presence of 5% ethanol.
Physical Description:324 Pages
DOI:https://doi.org/10.17192/z2018.0519