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...

Mechanism Behind the Programmed Biosynthesis of Heterotrimeric Fungal Depside Thielavin A

“…The diaryl ether‐forming PKS, AN7909, uses the thioesterase (TE) domain for depside bond formation (Figure 1A), [3a] whereas the starter‐unit acyltransferase (SAT) domain performs esterification in DrcA, [3b] resulting in the synthesis of the heterodimeric depside CJ‐20,557 (Figure 1B). A similar SAT‐catalyzed depside bond synthesis has been observed in the reaction with Preu6 to yield lecanoric acid [3c] . The biosynthesis of nornidulin involves two PKSs, collaborating to afford unguidepside A (Figure 1C).…”

Genome Mining and Biosynthetic Reconstitution of Fungal Depsidone Mollicellins Reveal a Dual Functional Cytochrome P450 for Ether Formation

“…The remaining genes in the moll BGC encode a transcription factor (mollA), a flavinbinding oxidoreductase (mollB), a decarboxylase (mollC), a cytochrome P450 monooxygenase (mollD), and an aromatic prenyltransferase (mollF) (Table S1). Further phylogenetic analysis reveals that MollE is most homologous to DrcA, 15 Atr1, 13 Preu6, 16 and DepH 17 (Figure S3), a group of NR-PKSs that were recently reported for the biosynthesis of depsides. A homologous cytochrome P450 monooxygenase of DepG has been identified to catalyze the ether formation in the biosynthesis of the depsidone nornidulin.…”

“…13,14 Remarkably, the esterification reaction to form the depside CJ-20577 was biochemically confirmed to be catalyzed by the starter acyl transferase (SAT) domain of the NR-PKS DrcA, 15 and ester bond formation in lecanoric acid was catalyzed by the interaction of SAT and the thioesterase (TE) domain of the NR-PKS Preu6 (Figure S1). 16 Recently, after releasing the depside from the TE domain of NR-PKS DepH, a dedicated cytochrome P450 DepG was identified to generate an ether bond to form the depsidone 17 (Figure 1B and Figure S1).…”

“…Heterologous expression studies demonstrated that a single NR-PKS is responsible for catalyzing the formation of the aromatic subunits and the subsequent esterification reaction to generate the depsides atranorin and lecanoric acid in the lichens Stereocaulon alpinum and Pseudevernia furfuracea , respectively. , Remarkably, the esterification reaction to form the depside CJ-20577 was biochemically confirmed to be catalyzed by the starter acyl transferase (SAT) domain of the NR-PKS DrcA, and ester bond formation in lecanoric acid was catalyzed by the interaction of SAT and the thioesterase (TE) domain of the NR-PKS Preu6 (Figure S1). Recently, after releasing the depside from the TE domain of NR-PKS DepH, a dedicated cytochrome P450 DepG was identified to generate an ether bond to form the depsidone (Figure B and Figure S1).…”

Targeted Discovery of Glycosylated Natural Products by Tailoring Enzyme-Guided Genome Mining and MS-Based Metabolome Analysis

“…We recently also isolated 1 in the filamentous fungi Aspergillus ustus CGMCC3.3904. Although the biosynthesis of fungal depsides has been discussed very recently, little is known about the GTs naturally responsible for their glycosylation. Herein, we identified the first fungal C -glycosyltransferase AuCGT, which was related to the biosynthesis of stromemycin from A.…”