Minimally Invasive Mutagenesis Gives Rise to a Biosynthetic Polyketide Library

Kushnir, Susanna; Sundermann, Uschi; Yahiaoui, Samir; Brockmeyer, Andreas; Janning, Petra; Schulz, Frank

doi:10.1002/anie.201202438

Cited by 53 publications

(35 citation statements)

References 36 publications

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“…In particular, the docking domains between MilA1 and the following multienzyme, AveA2, still matched well. Efforts were carried out to minimize the deleterious perturbations to the protein-protein interactions introduced by the manipulations of domain/module swapping (38). PKS gene replacement is a reliable strategy to guarantee that the catalytic domains remain in their native environments.…”

Section: Discussionmentioning

confidence: 99%

Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

Huang

Chen²,

Zhang

et al. 2015

Appl Environ Microbiol

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c Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.A vermectins (AVMs) are a series of 16-membered macrocyclic lactone derivatives with potent anthelmintic and insecticidal properties (1, 2). Ivermectin (IVM), a hydrogenated product of AVM B1, has been one of the best-selling antiparasitics since 1981 (3). However, as insect resistance to AVM (4) and IVM (5-7) is on the rise, alternatives or new products with enhanced potency and expanded spectra of activity are urgently needed.Milbemycins (MBMs) are another group of 16-membered macrolides that share similar structures with AVMs. The insecticidal activity of milbemectin (a mixture of MBMs A3 and A4) against some parasites is higher than those of AVM and IVM, while the toxicity is significantly lower (8).The structural differences among AVM, IVM, and MBM are in C-25, C-22-23, and C-13 ( Fig. 1) (9). IVM differs from AVM in C-22-23 (the former has a saturated bond, whereas the latter has a double bond in that position), but its toxicity is lower than that of AVM (10), implying that the single bond in C-22-23 should be the better structure in terms of safety. The major structural difference between IVM and MBM is a bisoleandrosyloxy substituent attached at C-13 of IVM, whereas that position is unsubstituted in MBM. Also, there are different alkyl substituents at C-25: in IVM, the substituent can be isopropyl or secondary butyl, while in MBM, it can be methyl or ethyl. However, MBM is more potent against some parasites and is safer than IVM, suggesting that one or both of the moieties in C-25 and C-13 of MBM is (are) the better structure(s) in these ways than that (those) of IVM. Another conclusion is that the two oleandroses in C-13 are important to the antiparasitic activity of IVM, since their removal will reduce the activity significantly, about 30-fold (10). Based on the curr...

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Section: Discussionmentioning

confidence: 99%

Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

Huang

Chen²,

Zhang

et al. 2015

Appl Environ Microbiol

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“…3). SLIC has been applied to the combinatorial biosynthesis of premonensin derivatives 57 . Mutated fragments of β-carbon processing domains of type I monensin PKS were rapidly cloned into the temperature-sensitive shuttle vector using SLIC and were introduced through in-place homologous recombination into the chromosomal monensin PKS genes of Streptomyces cinnamonensis A495 leading to a library of 22 premonensin redox derivatives with a few ( 29 – 31) (Fig.…”

Section: Synthetic Biology To the Rescuementioning

confidence: 99%

Reinvigorating natural product combinatorial biosynthesis with synthetic biology

2015

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Natural products continue to play a pivotal role in drug discovery efforts and in understanding human health. The ability to extend nature’s chemistry through combinatorial biosynthesis – altering functional groups, regiochemistry, and scaffold backbones through manipulation of biosynthetic enzymes – offers unique opportunities to create natural product analogues. Incorporating emerging synthetic biology techniques has the potential to further accelerate the refinement of combinatorial biosynthesis as a robust platform for the diversification of natural chemical drug leads. Two decades after the field originated, we discuss the current limitations, realities, and the state of the art of combinatorial biosynthesis, including the engineering of substrate specificity of biosynthetic enzymes and the development heterologous expression systems for biosynthetic pathways. We also propose a new perspective for the combinatorial biosynthesis of natural products that could reinvigorate drug discovery by using synthetic biology in combination with synthetic chemistry.

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“…36 According to the recruited starter units, the loading ATs are classified into two types. A conserved arginine in the active site indicates a KSQ-type loading AT that recruits a dicarboxylic acid starter unit while the absence of arginine in the corresponding position suggests an Ave-type loading AT that accepts a monocarboxylic acid starter unit.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Structural and Functional Analysis of the Loading Acyltransferase from Avermectin Modular Polyketide Synthase

Wang

et al. 2015

ACS Chem. Biol.

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The loading acyltransferase (AT) domains of modular polyketide synthases (PKSs) control the choice of starter units incorporated into polyketides and are therefore attractive targets for the engineering of modular PKSs. Here, we report the structural and biochemical characterizations of the loading AT from avermectin modular PKS, which accepts more than 40 carboxylic acids as alternative starter units for the biosynthesis of a series of congeners. This first structural analysis of loading ATs from modular PKSs revealed the molecular basis for the relaxed substrate specificity. Residues important for substrate binding and discrimination were predicted by modeling a substrate into the active site. A mutant with altered specificity toward a panel of synthetic substrate mimics was generated by site-directed mutagenesis of the active site residues. The hydrolysis of the N-acetylcysteamine thioesters of racemic 2-methylbutyric acid confirmed the stereospecificity of the avermectin loading AT for an S configuration at the C-2 position of the substrate. Together, these results set the stage for region-specific modification of polyketides through active site engineering of loading AT domains of modular PKSs.

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Minimally Invasive Mutagenesis Gives Rise to a Biosynthetic Polyketide Library

Abstract: Not in the public domain: Site-directed mutagenesis of megasynthases was the key to the generation of a library of polyketides in bacteria. Redox derivatizations are used to change the bioactivity profile of the compounds.

Cited by 53 publications

References 36 publications

Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

Reinvigorating natural product combinatorial biosynthesis with synthetic biology

Structural and Functional Analysis of the Loading Acyltransferase from Avermectin Modular Polyketide Synthase

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