Development of PKS biocatalysts on the example of a multi-enzyme cascade for improved synthesis of antifungal polyketides
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
Polyketide synthases (PKS) are enzymes of the secondary metabolism with great potential for biotechnology. The aim of this project was to advance the establishment of PKS modules as in vitro biocatalysts. As an example, a multi-enzyme cascade with the AmbMod4 module from the ambruticin PKS at the centre was to be developed and used for the formal chemoenzymatic synthesis of jerangolide A. The production of AmbMod4 was to be optimised and a suitable thioesterase (TE) domain for selective release of the module product was to be identified. A C-methyltransferase (CMT) and a cyclase (Zyk) were to be gradually added to this cascade. A range of TE domains were tested in preliminary hydrolysis experiments. As these did not show the desired substrate selectivity, C-terminal TE fusions to AmbMod4 were subsequently used to obtain compact biocatalysts with a higher probability for hydrolysis of the desired product. AmbMod4 produced by heterologous gene expression processed the surrogate of the authentic precursor, SNAC-2-D-5a, only to a very small extent to the desired product 9a. Engineering the module by fusing different TE domains and N-terminal docking domains did not improve this result. Extensive elucidation experiments showed that neither additional contact of AmbMod4 to the neighbouring modules AmbMod3 and AmbMod5 nor to the CMT AmbM led to a relevant improvement of the conversion to 9a. During stepwise reconstitution of the module catalysis, the trifunctional domain AmbDH4 showed its full dehydratase (DH) and enoyl isomerase (EI) activity in the in vitro system, but only very limited epimerase (Epi) activity and was thus identified as a bottleneck. Therefore, the alternative strategy of building the cascade on the basis of module fragments, already formulated in the application, was pursued. New cascades of alcohol dehydrogenases and the heterocyclase AmbDH3 enabled easy access to chiral tetrahydropyranyl thioesters, which served as precursors for conversion by the AmbKS4-AT4 didomain and the AmbKR4-DH4-ACP4 tridomain to the extended -hydroxythioester. Combinations of AmbDH4, the CMT AmbM and PikTE converted the SNAC 4-D-2- enthoate 8a into the desired module product 9a. Towards the end of the project, the module conversion could be increased to 23% by using the TEs MonCII and FosTEII and introducing targeted mutations in the KS4 and DH4 domains.
Publications
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Towards understanding oxygen heterocycle-forming biocatalysts: a selectivity study of the pyran synthase PedPS7. Organic & Biomolecular Chemistry, 20(48), 9645-9649.
Wagner, Lisa; Stang, Jörg; Derra, Sebastian; Hollmann, Tim & Hahn, Frank
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Highly Stereoselective Biocatalytic One-Pot Synthesis of Chiral Saturated Oxygen Heterocycles by Integration of a Biosynthetic Heterocyclase into Multiple-Enzyme Cascades. ACS Catalysis, 14(17), 13420-13428.
Roß-Taschner, Theresa; Derra, Sebastian; Stang, Jörg; Schlotte, Luca; Putratama, Anthony & Hahn, Frank
