Multiplexing of Combinatorial Chemistry in Antimycin Biosynthesis: Expansion of Molecular Diversity and Utility

Yan, Yan; Chen, Jing; Zhang, Lihan; Zheng, Qingfei; Han, Ying; Zhang, Hua; Zhang, Daozhong; Awakawa, Takayoshi; Abe, Ikuro; Liu, Wen

doi:10.1002/anie.201305569

Cited by 78 publications

(116 citation statements)

References 30 publications

(3 reference statements)

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“…Alternatively, precursor-directed biosynthesis (PDB) may be employed to produce azide-or alkyne-labeled natural products based on the promiscuity of biosynthetic machinery. Mainly used as a tool to introduce structural diversity, PDB has allowed us and other researchers to generate labeled natural products 3,[6][7][8] . However, the coexistence of diffusible precursors and final products 9 with the same chemical handle introduces significant background in the PDB production system, making it incompatible with in situ bioorthogonal chemical transformations.…”

Section: Introductionmentioning

confidence: 99%

De novo biosynthesis of terminal alkyne-labeled natural products

2014

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Abstract:The terminal alkyne is a functionality widely used in organic synthesis, pharmaceutical science, material science, and bioorthogonal chemistry. This functionality is also found in acetylenic natural products, but the underlying biosynthetic pathways for its formation are not well understood. Here we report the characterization of the first carrier proteindependent terminal alkyne biosynthetic machinery in microbes. We further demonstrate that this enzymatic machinery can be exploited for the in situ generation and incorporation of terminal alkynes into two natural product scaffolds in E. coli. These results highlight the prospect for tagging major classes of natural products, including polyketides and polyketide/non-ribosomal peptide hybrids, using biosynthetic pathway engineering.2 Natural products are important small molecules widely used as drugs, pesticides, herbicides, and biological probes. Tagging natural products with a unique chemical handle enables the visualization, enrichment, quantification, and mode of action study of natural products through bioorthogonal chemistry [1][2][3][4] . One prevalent bioorthogonal reaction is the triazole-forming azide-alkyne [3+2] cycloaddition, often referred to as "click" chemistry 5 . This reaction has enabled selective imaging and study of azide-or alkyne-labeled glycans, proteins, nucleic acids and lipids. Despite the success with macromolecules, the labeling of natural products has not been adequately explored. It is often challenging to obtain tagged natural products through total synthesis because of their structural complexity, or through semi-synthesis because of their chemical lability and the limited supply of most natural products. Alternatively, precursor-directed biosynthesis (PDB) may be employed to produce azide-or alkyne-labeled natural products based on the promiscuity of biosynthetic machinery. Mainly used as a tool to introduce structural diversity, PDB has allowed us and other researchers to generate labeled natural products 3,[6][7][8] . However, the coexistence of diffusible precursors and final products 9 with the same chemical handle introduces significant background in the PDB production system, making it incompatible with in situ bioorthogonal chemical transformations. We report here the de novo biosynthesis of alkyne-labeled natural products without the feeding of alkynoic precursors by characterizing and engineering the novel terminal alkyne synthetic machinery that living systems offer.Many acetylenic natural products contain a terminal alkyne functionality, which seems to be crucial for their bioactivity (Fig. 1) 10,11 . Terminal alkynes can be formed by acetylenases, a special family of desaturases that catalyze O 2 -dependent dehydrogenation of C-C bonds in a diiron dependent mechanism 12 . Several membrane-bound acetylenases have recently been 3 identified from plants, insects, fungi, and bacteria 4,10,[13][14][15][16][17] , but all of these reports have been limited to bioinformatics or in vivo studies through mutagenesis or ...

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Section: Introductionmentioning

confidence: 99%

De novo biosynthesis of terminal alkyne-labeled natural products

2014

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“…The recombinant enzyme exhibited AntE activity (Yan et al, 2013) and the typical yield was about 5 mg per litre of culture. The purified His 6 -tag-fused AntE migrated as a single band with a molecular mass of 47 kDa on SDS-PAGE, which agrees well with the calculated value of 46.7 kDa for His 6 -tag-fused AntE.…”

Section: Resultsmentioning

confidence: 99%

“…Our previous in vivo and in vitro analyses of AntE demonstrated the extraordinary promiscuity in its substrate recognition, resulting in the production of a number of unnatural extender units such as heptynoylmalonyl-CoA, cyclohexanepropanoylmalonyl-CoA and chloropentanoylmalonyl-CoA, as well as various natural alkylmalonyl-CoA extender units, which enabled us to produce a series of unnatural antimycin derivatives ( Fig. 1b; Yan et al, 2013). These observations unambiguously demonstrated the promise of AntE as an enzyme for supplying non-natural extender units in PKS engineering.…”

Section: Introductionmentioning

confidence: 99%

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Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase fromStreptomycessp. NRRL 2288

et al. 2014

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AntE from Streptomyces sp. NRRL 2288 is a crotonyl-CoA carboxylase/ reductase that catalyzes the reductive carboxylation of various , -unsaturated acyl-CoAs to provide the building block at the C7 position for antimycin A biosynthesis. Recombinant AntE expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 76.4, b = 96.7, c = 129.6 Å , = = = 90.0 . A diffraction data set was collected at the KEK Photon Factory to 2.29 Å resolution.

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“…成方面也取得突破, 合成了一系列结构新颖的类似物 [13,14] . 另一方面, 随着蛋白质研究技术的发展, 很多与次生代谢产物生物合成相关的调节酶被克隆表达并鉴定, 其晶体结构被解析, 结构和功能的关系得以深入研究.…”

Section: 最近国内学者利用组合合成策略在抗霉素 (Antimycin)、苯二酚内酯(Bunclassified

Combinatorial Synthesis of Flavonoids and 4-Hydroxy-δ-lactones by Plant-Originated Enzymes

莫婷¹,

王娟²,

高博闻³

et al. 2015

Chin. J. Org. Chem.

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4-Coumarate coenzyme A ligase (4CL) and chalcone synthase (CHS), the two pivotal enzymes involved in the biosynthesis of flavonoids were applied to combinatorially synthesize varied unnatural natural products. As result, 25 unnatural natural products including eight flavonoid analogues, 16 4-hydroxy-δ-lactones and one benzalacetone were synthesized by 4CL and CHS. The synthetic methodology would be useful for the construction of library of structurally diverse of small molecules.

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Multiplexing of Combinatorial Chemistry in Antimycin Biosynthesis: Expansion of Molecular Diversity and Utility

Cited by 78 publications

References 30 publications

De novo biosynthesis of terminal alkyne-labeled natural products

De novo biosynthesis of terminal alkyne-labeled natural products

Crystallization and preliminary X-ray diffraction analysis of AntE, a crotonyl-CoA carboxylase/reductase fromStreptomycessp. NRRL 2288

Combinatorial Synthesis of Flavonoids and 4-Hydroxy-δ-lactones by Plant-Originated Enzymes

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