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Understanding and Exploiting Fungal Bisorbicillinoid Biosynthesis

Subject Area Biological and Biomimetic Chemistry
Term from 2017 to 2021
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 388965482
 
Final Report Year 2022

Final Report Abstract

The biosynthetic pathways towards the polyketides terrein and (dimeric) sorbicillinoids, such as the Diels-Alder derived bisorbicillinol, were investigated. Both pathways were reconstituted step by step in Aspergillus oryzae and the key biosynthetic transformations were adressed through a combination of in vivo and in vitro studies. Co-expression of three genes from the sorbicillinoid BGC of Trichoderma reesei reconstituted the biosynthetic pathway up to the highly reactive sorbicillinol that was effectively reduced in the heterologous host A. oryzae. Dimeric sorbicillinoids were exclusively produced upon presence of a fourth gene encoding the FMO TrSorD. Further feeding studies confirmed that TrSorD is an unprecedented FMO that independently catalyzes both epoxidation and intermolecular dimerization of sorbicillinol with itself or other suitable molecules, following a Diels-Alder or Michael-like mechanism. The non- homologous FMO PcSorD from Penicillium chrysogenum plays the same catalytic role as TrSorD, and furthermore oxidizes sorbicillinol into oxosorbicillinol. These results answer the long-standing question for the involvement of an in vivo catalyst during the dimerization of sorbicillinol. Mutational studies indicated that covalent flavinylation is essential for enzyme activity of both TrSorD and PcSorD. Previous work showed that terrein is formed via an oxidative decarboxylation/ ring contraction sequence of the precursor 6-hydroxymellein. The production of 6-hydroxymellein was established in Aspergillus oryzae NSAR1 by co-expression of two genes encoding the non- reducing polyketide synthase (PKS) TerA and the PKS-like multidomain protein TerB. Deliberate point mutations of catalytic key residues within both proteins revealed that TerB is a novel type of of trans-acting enzyme. The catalytically active ketoreductase domain of TerB reduces the growing polyketide intermediate that is tethered to the PKS TerA; the catalytically inactive dehydratase domain of TerB appears to mediate protein-protein interactions with TerA. The flavin-dependent monooxygenase (FMO) TerC was demonstrated to open the lactone ring of 6-hydroxymellein via oxidative decarboxylation using a combination of heterologous expression in A. oryzae and in vitro assays. The chemically unstable aromatic product is hydroxylated by the second FMO TerD in vitro, but further reconstitution of the pathway was not successful. Heterologous expression of a homologous biosynthetic gene cluster (BGC) from a marine Roussoella sp. yielded a related aromatic intermediate. Although the ring contraction was not observed, the pivotal lactone opening during the biosynthesis of terrein and related metabolites was experimentally elucidated for the first time. The biosynthesis of other secondary metabolites in T. reesei was also investigated. A PKS-NRPS biosynthetic gene cluster was expressed in Aspergillus oryzae and shown to be responsible for the biosynthesis of the potent antifungal metabolite ilicicolin H. Although the biosynthesis of this compound was recently also described by Tang and coworkers for the first time, our work resulted in the production of several new congeners.

Publications

  • Diels-Alder Reactions During the Biosynthesis of Sorbicillinoids, Angew. Chem. Int Ed., 2020, 59, 5816–5822
    Kahlert, L.; Bassiony, E. F.; Cox, R. J.; Skellam, E. J.
    (See online at https://doi.org/10.1002/anie.201915486)
  • The Same but Different: Multiple Functions of the Fungal Flavin Dependent Monooxygenase SorD from Penicillium Chrysogenum, Chem. Commun. 2020, 56, 10934–10937
    Kahlert, L.; Cox, R. J.; Skellam, E. J.
    (See online at https://doi.org/10.1039/d0cc03203d)
  • Biosynthesis of 6-Hydroxymellein Requires a Collaborating Polyketide Synthase-like Enzyme, Angew. Chem. Int. Ed., 2021, 60, 11423–11429
    Kahlert, L.; Villanueva, M.; Cox, R. J.; Skellam, E. J.
    (See online at https://doi.org/10.1002/anie.202100969)
  • Early Oxidative Transformations During the Biosynthesis of Terrein and Related Natural Products, Chem. Eur. J., 2021, 27, 11895–11903
    Kahlert, L.; Bernardi, D.; Hauser, M.; Ióca, L. P.; Berlinck, R. G. S.; Skellam, E. J.; Cox, R. J.
    (See online at https://doi.org/10.1002/chem.202101447)
  • Total Mycosynthesis: Rational Bioconstruction and Bioengineering of Fungal Natural Products, Synthesis, 2021, 53, 2381–2394
    Kahlert, L.; Schotte, C.; Cox, R. J.
    (See online at https://doi.org/10.1055/a-1401-2716)
  • Trichoderma Reesei Contains a Biosynthetic Gene Cluster That Encodes the Antifungal Agent Ilicicolin H, J. Fungi, 2021, 7, 1034
    Shenouda, M. L.; Ambilika, M.; Cox, R. J.
    (See online at https://doi.org/10.3390/jof7121034)
 
 

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