Flavin-dependent halogenases from Cortinarius mushrooms for the regioselective chlorination of anthraquinone pigments

Flavin-dependent halogenases (FDHs) are valuable catalysts for the late-stage diversification of small molecule scaffolds, modulating their biological activities and/or introducing orthogonal functionalities for regiospecific derivatization. This study broadens the repertoire of FDHs by discovering two such enzymes (CkHAL1 and CsHAL2) from Cortinarius mushrooms that chlorinate anthraquinone skeletons from the emodin family when expressed in Saccharomyces cerevisiae. As opposed to the phenolic FDH CcRadH from the ascomycete Chaetomium chiversii that can modify only neutral anthraquinones such as emodin, the substrate range of CkHAL1 and CsHAL2 also extends to acidic pigments such as endocrocin and dermolutein. Correspondingly, our data suggests that Cortinarius and ascomycete FDHs likely arose via parallel evolution. We also identified CsRedE, a Cortinarius flavin reductase (FRD) that efficiently supports CsHal2-mediated catalysis. The FDH-encoding genes are not clustered with those of the FRDs, nor with those of the polyketide synthases or O-methyltransferases for anthraquinone biosynthesis, indicating effective metabolic crosstalk. Furthermore, sequence alignments and docking studies suggested sequence elements and amino acids that may contribute to the regioselectivity and substrate range of the Cortinarius halogenases. The discovery of the FDH and FRD enzymes with substantial sequence divergence from those in Ascomycetes aids the identification of additional such enzymes from basidiomycete fungi, and their utilization for the synthetic biological production of halogenated natural and "unnatural" products. Meanwhile, selective CH functionalization allows tuning of the physicochemical and pharmacokinetic properties of anthraquinone pigments, and may ultimately provide scalable and environmentally more responsible alternatives to synthetic production or extraction of rare analogs from nature.