Catalytic Relationships Between Type I and Type II Iterative Polyketide Synthases: The Aspergillus parasiticus Norsolorinic Acid Synthase.

Ma, Yue; Smith, Leah H.; Cox, Russell J.; Beltrán-Álvarez, Pedro; Arthur, Christopher J.; Frs, Thomas J. Simpson

doi:10.1002/chin.200704197

Cited by 4 publications

(8 citation statements)

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“…2A. The ACP domain of PKS13 was verified to be holo and was likely phosphopantetheinylated by the E. coli holo-ACP synthase, as reported for other nonreducing fungal PKS ACP domains (4,16). To assay PKS13 activities, we supplied the purified enzyme with malonyl-CoA as the extender unit and 11-hydroxyundecanyl-S-acetylcysteamine 5 as the starter unit.…”

Section: Resultsmentioning

confidence: 82%

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae

Zhou

Zhan

Watanabe

et al. 2008

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

103

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Resorcylic acid lactones represent a unique class of fungal polyketides and display a wide range of biological activities, such as nanomolar inhibitors of Hsp90 and MAP kinase. The biosynthesis of these compounds is proposed to involve two fungal polyketide synthases (PKS) that function collaboratively to yield a 14-membered macrolactone with a resorcylate core. We report here the reconstitution of Gibberella zeae PKS13, which is the nonreducing PKS associated with zearalenone biosynthesis. Using a small molecule mimic of the natural hexaketide starter unit, we reconstituted the entire repertoire of PKS13 activities in vitro, including starter-unit selection, iterative condensation, regioselective C2-C7 cyclization, and macrolactone formation. PKS13 synthesized both natural 14-membered and previously uncharacterized 16-membered resorcylic acid lactones, indicating relaxed control in both iterative elongation and macrocyclization. PKS13 exhibited broad starter-unit specificities toward fatty acyl-CoAs ranging in sizes between C6 and C16 and displayed the highest activity toward decanoyl-CoA. PKS13 was shown to be active in Escherichia coli and synthesized numerous alkyl pyrones and alkyl resorcylic esters without exogenously supplied precursors. We demonstrated that PKS13 can interact with E. coli fatty acid biosynthetic machinery and can be primed with fatty-acyl ACPp at low-micromolar concentrations. PKS13 synthesized new polyketides in E. coli when the culture was supplemented with synthetic precursors, showcasing its utility in precursor-directed biosynthesis. PKS13 is therefore a highly versatile polyketide macrolactone synthase that is useful in the engineered biosynthesis of polyketides, including resorcylic acid lactones that are not found in nature.ketosynthase ͉ resorcylic acid ͉ starter unit ͉ Hsp90 ͉ thioesterase

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Section: Resultsmentioning

confidence: 82%

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae

Zhou

Zhan

Watanabe

et al. 2008

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

103

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“…Reconstitution of the catalytic domains as individual enzymes has been successful in analyzing the biochemical and structural features of animal FAS [13] and bacterial type I PKS, including the KS-AT [14,15], KR [16], ACP [17], and thioesterase domains [18][19][20] the UWA algorithm to clone and identify the function of the starter unit:ACP transacylase domain from norsolorinic acid synthase (NSAS) involved in aflatoxin biosynthesis [22]. Recently, Ma et al [23] demonstrated that the standalone NSAS ACP domain can function as the ACP component of a bacterial minimal PKS in vitro.…”

Section: Domains [3]mentioning

confidence: 99%

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

Tang

2007

The FEBS Journal

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The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.

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“…1 A). These domains include the starter-unit:ACP acyltransferase (SAT) involved in starter unit selection (9), KS, malonyl-CoA:ACP transferase (MAT) for extender unit loading (10), product-template (PT) (11), ACP, and thioesterase/claisen-cyclase (TE/CLC) (12). The juxtaposition of these domains enables the megasynthase to initiate, extend, and cyclize the polyketide backbone into multicyclic, fungal-specific products (8).…”

mentioning

confidence: 99%

“…We have recently demonstrated that full-length fungal megasynthases can be expressed and reconstituted in E. coli, hence making this family an attractive machinery for E. coli-based biosynthesis of bacterial aromatic polyketides (13). However, despite the analogous iterative Claisen-like condensations catalyzed by two families of PKSs during chain elongation (10), cyclization modes of the poly-␤-ketone backbones are drastically different (14,15) (Fig. 1).…”

mentioning

confidence: 99%

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

Zhang

Tang

2008

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

105

106

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Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKSWJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKSWJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.cyclase ͉ ketosynthase ͉ megasynthase

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Catalytic Relationships Between Type I and Type II Iterative Polyketide Synthases: The Aspergillus parasiticus Norsolorinic Acid Synthase.

Cited by 4 publications

References 1 publication

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae

A polyketide macrolactone synthase from the filamentous fungus Gibberella zeae

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli

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