Enzymatic Synthesis of Resorcylic Acid Lactones by Cooperation of Fungal Iterative Polyketide Synthases Involved in Hypothemycin Biosynthesis

Zhou, Hui; Qiao, Kangjian; Gao, Zhizeng; Meehan, Michael J.; Li, Jesse W.-H.; Zhao, Xiling; Dorrestein, Pieter C.; Vederas, John C.; Tang, Yi

doi:10.1021/ja100060k

Cited by 82 publications

(125 citation statements)

References 20 publications

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“…Group I also consists of the NRPKSs that are involved in the synthesis of the aromatic portions of the resorcylic acid lactones, including the zearalenone PKS13 (36,37), the hypothemycin Hpm3 (38,39), and the radicicol RDC1/RADS2 (38,40). Primed by different starter units, Group I NRPKSs synthesize a tetraketide backbone and the PT domains perform the regioselective C2-C7 cyclization to yield the aromatic ring.…”

Section: Resultsmentioning

confidence: 99%

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Tang

2010

Journal of Biological Chemistry

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The fungal iterative nonreducing polyketide synthases (NRPKSs) synthesize aromatic polyketides, many of which have important biological activities. The product template domains (PT) embedded in the multidomain NRPKSs mediate the regioselective cyclization of the highly reactive polyketide backbones and dictate the final structures of the products. Understanding the sequence-activity relationships of different PT domains is therefore an important step toward the prediction of polyketide structures from NRPKS sequences and can enable the genome mining of hundreds of cryptic NRPKSs uncovered via genome sequencing. In this work, we first performed phylogenetic analysis of PT domains from NRPKSs of known functions and showed that the PT domains can be classified into five groups, with each group corresponding to a unique product size or cyclization regioselectivity. Group V contains the formerly unverified PT domains that were identified as C6-C11 aldol cyclases. The regioselectivity of PTs from this group were verified by product-based assays using the PT domain excised from the asperthecin AptA NRPKS. When combined with dissociated PKS4 minimal PKS, or replaced the endogenous PKS4 C2-C7 PT domain in a hybrid NRPKS, AptA-PT directed the C6-C11 cyclization of the nonaketide backbone to yield a tetracyclic pyranoanthraquinone 4. Extensive NMR analysis verified that the backbone of 4 was indeed cyclized with the expected regioselectivity. The PT phylogenetic analysis was then expanded to include ϳ100 PT sequences from unverified NRPKSs. Using the assays developed for AptA-PT, the regioselectivities of additional PT domains were investigated and matched to those predicted by the phylogenetic classifications.

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Section: Resultsmentioning

confidence: 99%

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Tang

2010

Journal of Biological Chemistry

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“…TE domains form the BDL macrolactone using the ω-1 alcohol as a nucleophile, but may use alternative nucleophiles such as the C9 enol to yield α-pyrones or water or alcohols from the media to form acyl resorcylic acids (ARAs), acyl dihydroxyphenylacetic acids (ADAs), and their esters (18)(19)(20)(21)(22). iPKSs that produce BDLs in the RAL 14 , RAL 12 , and DAL 12 subclasses have been characterized and reconstituted both in vivo by heterologous expression in yeast and in vitro using isolated recombinant iPKS enzymes (11)(12)(13)(14)(23)(24)(25). Domain exchanges among different BDLSs were used to decipher some of the programming rules of these enzymes and yielded a limited number of structurally diverse unnatural products (3,18,20,21,26,27).…”

Section: Significancementioning

confidence: 99%

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

Zhou

Zhang

et al. 2014

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

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Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.secondary metabolites | fungal genetics

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“…The product template (PT) domain of the nrPKS directs ring closure to yield the resorcinol carboxylate moiety by C-2-C-7 aldol condensation (22)(23)(24), while the thioesterase (TE) domain is responsible for the off-loading of the RAL from the nrPKS by closure of the bridging macrolactone ring (25). RAL synthesis has been characterized in the producer fungi by gene disruptions (14,15,17) and reconstituted both in vivo by heterologous expression in Saccharomyces cerevisiae and in vitro using isolated recombinant PKS enzymes (18,26,27).…”

mentioning

confidence: 99%

Characterization of the Biosynthetic Genes for 10,11-Dehydrocurvularin, a Heat Shock Response-Modulating Anticancer Fungal Polyketide from Aspergillus terreus

Espinosa-Artiles

Schubert

et al. 2013

Appl Environ Microbiol

114

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d 10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta-or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.

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Enzymatic Synthesis of Resorcylic Acid Lactones by Cooperation of Fungal Iterative Polyketide Synthases Involved in Hypothemycin Biosynthesis

Cited by 82 publications

References 20 publications

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Classification, Prediction, and Verification of the Regioselectivity of Fungal Polyketide Synthase Product Template Domains

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

Characterization of the Biosynthetic Genes for 10,11-Dehydrocurvularin, a Heat Shock Response-Modulating Anticancer Fungal Polyketide from Aspergillus terreus

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