Diversity-Oriented Combinatorial Biosynthesis of Hybrid Polyketide Scaffolds from Azaphilone and Benzenediol Lactone Biosynthons

Bai, Jing; Lu, Yuanyuan; Xu, Ya Ming; Zhang, Wei; Chen, Ming; Lin, Min; Gunatilaka, A. A. Leslie; Xu, Yuquan; Molnár, István

doi:10.1021/acs.orglett.6b00110

Cited by 27 publications

(37 citation statements)

References 27 publications

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“…Considering all of these indicators, the encoding genes of eight putative B. bassiana GTs (SI Appendix, Table S2) were separately expressed in S. cerevisiae BJ5464-NpgA, a host well suited for the expression of fungal enzymes and the reconstitution of fungal polyketide biosynthetic pathways (22)(23)(24)(25). DLD (1) was selected as the model substrate to represent the BDL polyketide family, based on its efficient conversion by B. bassiana ARSEF 2860 to a putative DLD methylglycoside, as verified by HPLC-MS/MS (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S1A 14 (radilarin 17 and lasilarin 18). We also tested nonmacrocyclic BDL congeners, such as isocoumarins (19)(20)(21); acyl-resorcylic acids (ARA) (22)(23)(24); an acyl-dihydroxyphenylacetic acid (ADA) (25); and the benzaldehyde precursor (26) of the azaphilone asperfuranone (22) (SI Appendix, Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

“…S1). BDLs are drug-like fungal secondary metabolites with an astonishing range of bioactivities (22)(23)(24)(25)(26), structurally defined by a 1,3-benzenediol moiety fused to a macrocyclic lactone ring. Based on the carbons involved in this fusion, BDLs can be subdivided into resorcylic acid lactones (RALs, C2-C7 as in 1) and dihydroxyphenylacetic acid lactones (DALs, C3-C8 as in 14).…”

Section: Significancementioning

confidence: 99%

“…We have been developing combinatorial biosynthetic methods in an engineered Saccharomyces cerevisiae production chassis to increase the chemical space accessible by BDL biosynthesis. Thus, we have revealed structural clues that allow morphing RALs and DALs into each other by polyketide synthase (PKS) active site engineering (25); improved the production of unnatural BDL congeners by creating hybrid PKSs (27); and developed PKS subunit shuffling as a practical method to biosynthesize unnatural BDL scaffolds (23) and BDL/azaphilone hybrids (22). Because no glycosylated BDLs are known from fungal producers (26), identifying the B. bassiana GT, and co-opting this enzyme for the total biosynthesis of BDL glycosides in a synthetic biology platform with engineered PKSs, would open up an orthogonal chemical dimension for BDLs.…”

Section: Significancementioning

confidence: 99%

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Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis

Xie

Zhang

Wang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for "sugarcoating" various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase-methyltransferase (GT-MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both- and -glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4--methylglucosides. The resulting "unnatural products" show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis

Xie

Zhang

Wang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…99 In this case the AtCURS2 PKS had to be engineered to contain the AfoE SAT domain (Scheme 13). These experiments clearly show the opportunities for rational production of dened compounds within the DAL/RAL family and are among the most advanced to date in the sphere of natural product biosynthetic engineering in general.…”

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confidence: 99%

Classic fungal natural products in the genomic age: the molecular legacy of Harold Raistrick

Schor

Cox

2018

Nat. Prod. Rep.

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Harold Raistrick was involved in the discovery of many of the most important classes of fungal metabolites during the 20th century. This review focusses on how these discoveries led to developments in isotopic labelling, biomimetic chemistry and the discovery, analysis and exploitation of biosynthetic gene clusters for major classes of fungal metabolites including: alternariol; geodin and metabolites of the emodin pathway; maleidrides; citrinin and the azaphilones; dehydrocurvularin; mycophenolic acid; and the tropolones. Key recent advances in the molecular understanding of these important pathways, including the discovery of biosynthetic gene clusters, the investigation of the molecular and chemical aspects of key biosynthetic steps, and the reengineering of key components of the pathways are reviewed and compared. Finally, discussion of key relationships between metabolites and pathways and the most important recent advances and opportunities for future research directions are given.

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

et al. 2022

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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...

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Diversity-Oriented Combinatorial Biosynthesis of Hybrid Polyketide Scaffolds from Azaphilone and Benzenediol Lactone Biosynthons

Cited by 27 publications

References 27 publications

Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis

Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis

Classic fungal natural products in the genomic age: the molecular legacy of Harold Raistrick

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

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