Hemiacetal-less rapamycin derivatives

Engineering polyketide synthases is one of the most promising ways of producing a variety of polyketide derivatives. Exploring the undiscovered chemical space of this medicinally important class of middle molecular weight natural products will aid in the development of improved drugs in the future. In previous work, we established methodology designated ‘module editing’ to precisely manipulate polyketide synthase genes cloned in a bacterial artificial chromosome. Here, in the course of investigating the engineering capacity of the rapamycin PKS, novel rapamycin derivatives 14, which lack the hemiacetal moiety, were produced through the heterologous expression of engineered variants of the rapamycin PKS.

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Fig: Domain organizations of PKSs edited to produce hemiacetal-less rapamycin derivatives. The circles represent each domain and are colored based on the traditional module boundary (start before KS and end after ACP). The polyketide substructure proposed to be biosynthesised by each engineered PKS is shown. For clarity, modules 5 to 9 of RapB are omitted. We achieved ΔM14 and ΔM13-14 by setting the editing point at the KR-ACP linker region, whereas the KS-AT linker region was used for ΔM11-12. Unlike the wild-type product of RapC, the designed intermediates are supposed to lack the hydroxyl or the ketone for forming the six-membered hemiacetal ring. KS ketosyntase, AT acyltransferase,ACP acyl carrier protein, KR ketoreductase, DH dehydratase, DHL dehydratase-like (inactive), ER enoylreductase. Dashed box corresponds to the partial structures proposed to be biosynthesized by each construct.

Three kinds of module deletions in the polyketide synthase RapC were designed, and the genetically engineered vectors were prepared by the in vitro module editing technique. Streptomyces avermitilis SUKA34 transformed with these edited PKSs produced new rapamycin derivatives. The planar structures of 14 established based on 1D and 2D NMR, ESI–TOF–MS and UV spectra revealed that 2 and 3 had skeletons well-matched to the designs, but 1 and 4 did not. The observations provide important insights into the mechanisms of the later steps of rapamycin skeletal formation as well as the ketone-forming oxygenase RapJ.

Kudo, K., Nishimura, T., Kozone, I. et al. Hemiacetal-less rapamycin derivatives designed and produced by genetic engineering of a type I polyketide synthase. Sci Rep 11, 9944 (2021). https://doi.org/10.1038/s41598-021-88583-z

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