Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

McDaniel, Robert; Thamchaipenet, Arinthip; Gustafsson, Claes; Fu, Huimin; Betlach, Melanie C; Betlach, Mary C.; Ashley, Gary W.

doi:10.1073/pnas.96.5.1846

Cited by 442 publications

(356 citation statements)

References 27 publications

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“…In addition, exploring new heterologous hosts for polyketide production would also be important. Combinatorial biosynthesis of polyketides has only been reported in Streptomyces coelicolor 27 and E. coli, 28 with a limited success in terms of product titers compared to their wild-type counterparts. By utilizing insights learned from fundamental analysis of type I PKSs, we hope to contribute to the achievable goal of exploitation of this attractive enzyme as a platform for novel molecule production.…”

Section: Resultsmentioning

confidence: 99%

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

2016

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Section: Resultsmentioning

confidence: 99%

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

2016

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“…The potential diversity of macrocyclic compounds attainable by TE catalysis is further amplified by the fact that a myriad of macrocyclization reactions are performed by TE domains from different NRPS and polyketide synthetase assembly lines (2). The utility of such terminal TE domains can be exploited in efforts to engineer synthetases to generate novel compounds (28)(29)(30)(31). Additionally, it is feasible that these small (25-35 kDa) enzymatic domains in isolation could be used in the generation of diverse libraries of macrocyclic compounds with possible biological activity in areas distinct from the natural synthetase product.…”

Section: Discussionmentioning

confidence: 99%

The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides

Kohli

Takagi

Walsh

2002

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

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Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and N ␣ -methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to ␣IIb␤3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.M acrocyclic structure is a desirable feature for biologically or pharmaceutically active compounds. Macrocyclization stabilizes products from degradation and decreases the conformational flexibility of a molecule compared with its linear analog by constraining it to a biologically active conformation (1). In the biosynthesis of macrocyclic natural products, such as the macrolactam antibiotic tyrocidine, the macrolactone antifungal fengycin, and the macrolactone siderophore enterobactin by nonribosomal peptide synthetases (NRPSs), a C-terminal thioesterase (TE) domain introduces this constraint by catalyzing cyclization of a linear precursor (2, 3). Analogous cyclases in polyketide synthetase assemblies generate erythromycin and ketolide antibiotics as well as other bioactive macrolactones (4, 5).We have recently shown that several 28-to 35-kDa C-terminal TE domains excised from megadalton NRPS subunit proteins retain autonomous macrocyclization activity with peptide thioester substrates that mimic their natural protein-bound peptide phosphopantetheinyl substrates (6, 7). As a representative example, TycC TE, the C-terminal TE domain from the synthetase responsible for the biosynthesis of tyrocidine, macrocyclizes a soluble decapeptide thioester with the native peptide sequence of the antibiotic tyrocidine A (Fig. ...

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“…Therefore, it is possible that it arises by the loss of the C-2-C-5 portion of fragment h 5 . The remaining fragment, m/z 123, has the formula C 6 H 12 ONa ϩ , and could be generated by a plethora of routes, so will not be considered further.…”

Section: Stutt Ebmentioning

confidence: 99%

“…Particular interest is focused upon producing polyketide libraries through combinatorial genetic manipulation. To avoid producing an unmanageable number of compounds, such libraries are often limited to aglycones [5] which also have simpler structures and are thus easier to analyze.…”

mentioning

confidence: 99%

“…The large number of samples that may be expected from combinatorial experiments will require a faster, more sensitive analysis for preliminary screening and structure determination [5][6][7]. A more rapid method of molecular characterization may be high performance liquid chromatography (HPLC) followed by tandem (MS-MS) or sequential mass spectrometry (MS n ), but for this to be of use some understanding of the fragmentation reactions of macrolide antibiotics is required.…”

mentioning

confidence: 99%

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Structural elucidation studies on 14- and 16-membered macrolide aglycones by accurate-mass electrospray sequential mass spectrometry

Roddis

Gates

Roddis

et al. 2002

J. Am. Soc. Mass Spectrom.

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Collision induced dissociation sequential mass spectrometry was used to investigate the fragmentation of the heptaketide macrolide aglycones, 6-deoxyerythronolide B (6-dEB), erythronolide B (EB), and acetate-starter EB (Ac-EB). The fragmentations of two previously reported octaketide analogs produced by "stuttering" of the erythromycin polyketide synthase, stuttered-6-dEB and acetate-starter stuttered-6-dEB were also studied. The accuracy with which the mass of each fragment was measured allowed it to be attributed to an unambiguous formula. Most of the experiments were repeated using samples dissolved in deuterated solvents. These data were then used to deduce plausible fragmentation pathways of the five compounds which were shown to have a high degree of similarity. Preliminary fragmentation analysis of a novel octaketide analog was performed and the structure was predicted as stuttered EB. Subsequent scale-up of the bacterial fermentations, followed by isolation and characterization by nuclear magnetic resonance spectroscopy confirmed this prediction. Further fragmentation experiments were then performed on this compound, which provided further evidence of the similarity of the fragmentation schemes. These results demonstrate the utility of collision induced dissociation sequential mass spectrometry analysis in the preliminary screening of bacterial fermentations for new polyketides. These studies were performed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. (J Am Soc Mass Spectrom 2002, 13, 862-874)

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Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

Cited by 442 publications

References 27 publications

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides

Structural elucidation studies on 14- and 16-membered macrolide aglycones by accurate-mass electrospray sequential mass spectrometry

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