Combinatorial Domain Swaps Provide Insights into the Rules of Fungal Polyketide Synthase Programming and the Rational Synthesis of Non‐Native Aromatic Products

Vagstad, Anna L.; Newman, Adam G.; Storm, Philip A.; Belecki, Katherine; Crawford, Jason M.; Townsend, Craig A.

doi:10.1002/anie.201208550

Cited by 42 publications

(49 citation statements)

References 19 publications

(33 reference statements)

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“…From a matrix of six NR-PKSs enzyme components we have found that ACPs are substantially interchangeable; maintaining SAT–KS pairs is beneficial to overall synthetic efficiency; chain length is largely, but not exclusively, determined by the KS with occasional participation by the PT; PT active sites are unexpectedly accommodating with respect to the chain lengths of the polyketide intermediates presented to them, yet the regiochemistry of their cyclizations is tightly controlled; and, as noted above, the TE is crucial to success. Enzyme-directed cyclizations must outpace spontaneous reaction and TE-mediated editing, 52, 53 but “rules” have emerged from these studies where rapid combinatorial experiments can be used to quickly survey potential heterodomain combinations for assembly into single PKS chimeras for more efficient synthesis. 53 …”

Section: Behaviour and Engineering Of Nr-pkssmentioning

confidence: 99%

Aflatoxin and deconstruction of type I, iterative polyketide synthase function

Townsend

2014

Nat. Prod. Rep.

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In this viewpoint highlights are drawn from a deep analysis of the multifaceted problem of aflatoxin biosynthesis, one of the most highly rearranged polyketide natural products known. Fundamental chemical insights have emerged into how cytochrome P450-mediated skeletal rearrangements occur through probable cationic intermediates and oxidative dearomatizations, which are applicable more widely in natural product catabolism. So to where current experimental methods have failed in our hands, bioinformatic tools and fresh experimental strategies have been developed to identify linker regions in large, polydomain proteins and guide the dissection and reassembly of their component parts.It has been possible to deduce individual catalytic roles, how overall synthesis is coordinated and how these enzymes can be re-engineered in a rational manner to prepare non-natural products. These insights and innovations were often not planned or anticipated, but sprung from the inability to answer fundamental questions. Advances in science can take place by chance favoring the prepared mind, other times by refusing to give up and devising new solutions to address hard questions. Both ways forward played important roles in the investigation of aflatoxin biosynthesis. For these contributions I am pleased to share this special issue of NPR with John Vederas and Tom Simpson, who have been leaders in this field for the last third of a century.

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Section: Behaviour and Engineering Of Nr-pkssmentioning

confidence: 99%

Aflatoxin and deconstruction of type I, iterative polyketide synthase function

Townsend

2014

Nat. Prod. Rep.

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“…The C -terminal His 6 -tagged PksA SAT–KS–MAT tridomain and PT–ACP didomain were over-produced in E. coli and purified by Co 2+ -affinity chromatography as previously described(18). Potential starter units were presented as N -acetylcysteamine (SNAC) thioesters (Figure 1), which were prepared by coupling of N -acetylcysteamine to commercially-available or readily synthesized carboxylic acids (see Supplemental Information).…”

Section: Resultsmentioning

confidence: 99%

“…Reactions of 6 , 8 and 9 were less efficient in naphthopyrone product formation and correspondingly gave increased shunt and truncation products appearing at early retention times in the HPLC chromatogram (Figures S2, S4, S5). Recent deconstruction and domain “swapping” or heterodomain combinatorial experiments have revealed tight control of overall polyketide chain length to largely ± one ketide (C 2 ) extension, while the PT domain exerts strict programming of ring formation despite receiving chain lengths both shorter and longer than wild-type(18, 19). Reactions utilizing the C 8 starter unit 9 exemplify this tight chain length control, as one fewer ketide extension was performed to give a naphthopyrone product with the native C 20 chain length.…”

Section: Resultsmentioning

confidence: 99%

“…Combinatorial assemblies of heterodomains from a selection of NR-PKSs have shown that polyketide chain length is largely controlled by the β-ketoacyl synthase (KS) domain, and the cyclization pattern is determined by the PT domain(18, 19). The enzyme responsible for the biosynthesis of norsolorinic acid anthrone in Aspergillus parasiticus , the precursor to aflatoxin B 1 ( 1 ), PksA(20), accepts an unusual hexanoyl starter unit from a dedicated pair of yeast-like FASs (HexA/B, Scheme 1A)(21–23).…”

Section: Introductionmentioning

confidence: 99%

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Starter Unit Flexibility for Engineered Product Synthesis by the Nonreducing Polyketide Synthase PksA

et al. 2015

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Non-reducing polyketide synthases (NR-PKSs) are unique among PKSs in their domain structure, notably including a starter unit:acyl-carrier protein (ACP) transacylase (SAT) domain that selects an acyl group as the primer for biosynthesis, most commonly acetyl-CoA from central metabolism. This clan of mega-enzymes resembles fatty acid synthases (FASs) by both sharing their central chain elongation steps and their capacity for iterative catalysis. In this mode of synthesis, catalytic domains involved in chain extension exhibit substrate plasticity to accommodate growing chains as small as two carbons to 20 or more. PksA is the NR-PKS central to the biosynthesis of the mycotoxin aflatoxin B1 and whose SAT domain accepts an unusual hexanoyl starter from a dedicated yeast-like FAS. Explored in this paper is the ability of PksA to utilize a selection of potential starter units as substrates to initiate and sustain extension and cyclization to on-target, programmed polyketide synthesis. Most of these starter units were successfully accepted and properly processed by PksA to achieve biosynthesis of the predicted naphthopyrone product. Analysis of the on-target and derailment products revealed trends of tolerance by individual PksA domains to alternative starter units. In addition, natural and unnatural variants of the active site cysteine were examined and found to be capable of biosynthesis, suggesting possible direct loading of starter units onto the β-ketoacyl synthase (KS) domain. In light of the data assembled here, the predictable synthesis of un-natural products by NR-PKSs is more fully defined.

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“…Possibilities can be envisioned where their intrinsic substrate flexibility can be exploited. What is lost in binding affinity may well be compensated for by high effective concentration provided by carrier-protein mediated intraprotein transfer [18].…”

Section: Discussionmentioning

confidence: 99%

Deconstruction of Iterative Polyketide Synthases

Townsend¹

2014

New Chemistry and New Opportunities From the Expanding Protein Universe

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The enormous progress of the last 25 years in natural products biochemistry has been dominated by investigation of giant microbial enzymes comprised of organized catalytic domains that function according to two distinct paradigms. Each is "programmed" to sequentially assemble simple precursor molecules over multiple steps without diffusible intermediates to synthesize highly elaborated products. Both synthetic strategies rely upon carrier proteins to hold and shuttle extending intermediates among catalytic domains in an ordered fashion. Collectively these megaproteins are impressive "supramolecular machines" exemplifying protein catalysis far more sophisticated than the classical view of "one gene, one enzyme, one reaction" [1], and are of central importance to the creation of small-molecule drugs for human and animal health. Assembly-line and iterative catalysis in natural product biosynthesisThe application of molecular biological techniques to natural product biosynthetic problems in the mid 1980's ushered in the modern era of research in the field. Seminal events include isolation of the penicillin gene cluster and characterization of the non-ribosomal peptide synthetase (NRPS) ACVS that assembles the classical Arnstein L,L,D-tripeptide precursor of the antibiotic [2], Fig. 1. Biochemical experiments on isolated wild-type NRPS enzymes had already established a linear mode of accreting ATP-dependent synthesis [3], but now isolation of the corresponding biosynthetic genes and their translation to domains of recognized function in loosely repeated sets, or "modules," underscored the exquisitely organized nature of the "assembly line" synthetic program executed by these large enzymes. The end of 1990 marked the watershed co-discovery of genes encoding three giant modular polyketide synthases (PKSs) that synthesize the 6-deoxyerythronolide core of the macrolide antibiotic erythromycin [4,5]. Together deoxyerythronolide B synthases (DEBS)1-3, totaling ca. 10 6 Da, opened the door to a second paradigm of assembly line megaenzymes whose synthetic program could be "read" from the N -to C-terminus.The parallel universes of NRPS and modular PKS enzymes in their canonical forms (there are many exceptions and variations on the theme) are extravagant extensions of the classical view of enzyme function enunciated 70 years ago [1]; that New Chemistry and New Opportunities from the Expanding Protein Universe Downloaded from www.worldscientific.com by NANYANG TECHNOLOGICAL UNIVERSITY on 08/20/15. For personal use only.

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Combinatorial Domain Swaps Provide Insights into the Rules of Fungal Polyketide Synthase Programming and the Rational Synthesis of Non‐Native Aromatic Products

Cited by 42 publications

References 19 publications

Aflatoxin and deconstruction of type I, iterative polyketide synthase function

Aflatoxin and deconstruction of type I, iterative polyketide synthase function

Starter Unit Flexibility for Engineered Product Synthesis by the Nonreducing Polyketide Synthase PksA

Deconstruction of Iterative Polyketide Synthases

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