Didepside Formation by the Nonreducing Polyketide Synthase Preu6 of Preussia isomera Requires Interaction of Starter Acyl Transferase and Thioesterase Domains

Abstract

Orsellinic acid (OA) derivatives are produced by filamentous fungi using nonreducing polyketide synthases (nrPKSs). The chain-releasing thioesterase (TE) domains of such nrPKSs were proposed to also catalyze dimerization to yield didepsides, such as lecanoric acid. Here, we use combinatorial domain exchanges, domain dissections and reconstitutions to reveal that the TE domain of the lecanoric acid synthase Preu6 of Preussia isomera must collaborate with the starter acyl transferase (SAT) domain from the same nrPKS. We show that artificial SAT-TE fusion proteins are highly effective catalysts and reprogram the ketide homologation chassis to form didepsides. We also demonstrate that dissected SAT and TE domains of Preu6 physically interact, and SAT and TE domains of OA-synthesizing nrPKSs may co-evolve. Our work highlights an unexpected domain-domain interaction in nrPKSs that must be considered for the combinatorial biosynthesis of unnatural didepsides, depsidones, and diphenyl ethers.Derivatives of orsellinic acid (OA, 1) form a large class of structurally diverse polyketides widely distributed in plants, lichens, algae, fungi and bacteria. [1] OA polymers, that is, depsides, depsidones, and diphenyl ethers, feature ester or ether linkages. [2,3] These linkages may form by oxidative coupling or oxidative rearrangements catalyzed by tailoring enzymes after the release of the polyketide core from the nonreducing polyketide synthase (nrPKS) enzyme. [4][5][6] Alter-natively, such linkages result from nrPKS-catalyzed transformations of nascent, acyl carrier protein-bound intermediates. [7,8] Among the ester-linked OA dimers, lecanoric acid (2), a lichen metabolite also produced by filamentous fungi, displays various biological activities, including histidine decarboxylase inhibitory, [9] radical scavenging, [10,11] and antifungal activities. [12] Three nrPKSs from different fungi have been shown to yield the didepside 2, all without the involvement of post-PKS tailoring enzymes. These nrPKSs (AN7909 from Aspergillus nidulans, [7,8] CPUR_07425 from Claviceps purpurea, [13] and Preu6 from Preussia isomera, [12] ) share the same domain architecture, that is, SAT (starter acyl transferase)-KS (ketoacyl synthase)-AT (acyl transferase)-PT (product template)-ACP1 and 2 (acyl carrier protein 1 and 2)-TE (thioesterase) (Figure S1, Table S1).When expressed in Saccharomyces cerevisiae, AN7909 affords the diaryl ether diorcinolic acid (3) with only trace amounts of 2. [7,8] The dissected AN7909-TE efficiently converts the N-acetylcysteamine thioester of 2 (2-SNAC) into 3 and 1-SNAC into 1 in vitro (Figure 1A). [8] This confirms that AN7909-TE not only releases both 3 and 1, but also catalyzes a Smiles rearrangement to the diaryl ether 3. [8] However, AN7909-TE converts 1-SNAC into 3 with low efficiency in vitro; [8] therefore, its role in the in vivo formation of the ester 2 remains to be further elucidated.In contrast, overexpression of CPUR_07425 in C. purpurea yielded 2 and its ethyl ester, with only trace amounts of diaryl et...

The Depside Derivative Pericodepside Inhibits Cancer Cell Metastasis and Proliferation by Suppressing Epithelial–Mesenchymal Transition

“…Advances in biotechnology and genetic engineering have facilitated the biosynthesis of several depsides, making their production more economically feasible. − At the same time, the production of certain depsides and their derivatives by biosynthesis may provide an environmentally friendly and sustainable alternative to conventional chemical synthesis methods. Moreover, improvements in the biosynthesis of these compounds will facilitate their ultimate large-scale industrial production and use.…”