Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases

Yu, Dayu; Xu, Feng; Gage, David; Zhan, Jixun

doi:10.1039/c3cc42425a

Cited by 22 publications

(33 citation statements)

References 19 publications

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“…Although most of the N‐ and C‐terminal linker regions of PCP1 ybt are strongly disordered (Figure A and Figure S5 in the Supporting Information), there are three exceptions. In the C‐terminal linker, which has recently been shown to be essential for communication between NRPS modules, five residues immediately following α4 interact with the folded core along loop 2. In the N‐terminal linker, a short region approximately 20 residues before α1 displays helical character, as evidenced by chemical shift indexing and weak nuclear Overhauser effect (nOe) crosspeaks.…”

Section: Figuresupporting

confidence: 89%

Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Harden

Frueh

2017

ChemBioChem

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Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We show that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and may guide efforts in engineering NRPSs to obtain new pharmaceuticals.

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Section: Figuresupporting

confidence: 89%

Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Harden

Frueh

2017

ChemBioChem

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“…Whereas the E. coli strains produced low amounts of CDPs with less detectable derivatives, cultivation of A. niger strain MA-hPE-241 (hPE-SYN)w ith supplementation of Lac/ PheLac (10 mm,m olar ratio 1:1) yielded all four expected Lac/ PheLac enniatins ( Figure 2B, 4a-d). The fact that only hexadepsipeptides were found as products of the chimeric CDPSs is as trong indicator that the C-terminal parto ft he protein carries the ring-size determinant,i n agreement with the hypothesis of Yu et al [38] Notably,w hereas ESYN expressed in A. niger resultedi n9 50 mg L À1 enniatin in shake flasks, [37] we obtaineds ignificantly lower (but still exploitable) yields of hybrid CDPs: 4c, 4d and 5b were isolated at 0.5-3.6 mg L À1 ,p robablyb ecause of diminished activity of the hybrid enzymes (possiblys uboptimal interactions of newly arranged NRPS domains). Remarkably,c ompoundsw ith two or three d-Lacs were not detected, and synthesis of these derivatives could not be inducedb ys upplementation with highera mounts of d-Lac.…”

supporting

confidence: 89%

“…Am ore detailed structural characterization of representative derivatives of transgenic A. niger cultures (4c, 4d, 5b)w as performed by NMR spectroscopy (Figures S10 and S11). The fact that only hexadepsipeptides were found as products of the chimeric CDPSs is as trong indicator that the C-terminal parto ft he protein carries the ring-size determinant,i n agreement with the hypothesis of Yu et al [38] Notably,w hereas ESYN expressed in A. niger resultedi n9 50 mg L À1 enniatin in shake flasks, [37] we obtaineds ignificantly lower (but still exploitable) yields of hybrid CDPs: 4c, 4d and 5b were isolated at 0.5-3.6 mg L À1 ,p robablyb ecause of diminished activity of the hybrid enzymes (possiblys uboptimal interactions of newly arranged NRPS domains). Engineering of these hybrid synthetases, codon adaption for A. niger translation, screening for new transformants, andc ultivation optimizationo fA.…”

supporting

confidence: 89%

Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins

et al. 2016

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Non-ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa- and cyclo-octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy-acid-activating module of PF1022 synthetase with the amino-acid-activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity.

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“…Since module 1 of BbBEAS and BbBSLS are exchangeable without affecting the product profiles19, we tested the effects of swapping the C-terminal domains on the product formation. We first constructed an enzyme C 1 -A 1 -T 1 -C 2 -A 2 -MT (BbBEAS) T 2a -T 2b -C 3(BbBSLS) .…”

Section: Resultsmentioning

confidence: 99%

Decoding and reprogramming fungal iterative nonribosomal peptide synthetases

Zhang

et al. 2017

Nat Commun

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Nonribosomal peptide synthetases (NRPSs) assemble a large group of structurally and functionally diverse natural products. While the iterative catalytic mechanism of bacterial NRPSs is known, it remains unclear how fungal NRPSs create products of desired length. Here we show that fungal iterative NRPSs adopt an alternate incorporation strategy. Beauvericin and bassianolide synthetases have the same C1-A1-T1-C2-A2-MT-T2a-T2b-C3 domain organization. During catalysis, C3 and C2 take turns to incorporate the two biosynthetic precursors into the growing depsipeptide chain that swings between T1 and T2a/T2b with C3 cyclizing the chain when it reaches the full length. We reconstruct the total biosynthesis of beauvericin in vitro by reacting C2 and C3 with two SNAC-linked precursors and present a domain swapping approach to reprogramming these enzymes for peptides with altered lengths. These findings highlight the difference between bacterial and fungal NRPS mechanisms and provide a framework for the enzymatic synthesis of non-natural nonribosomal peptides.

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Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases

Cited by 22 publications

References 19 publications

Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Molecular Cross‐Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions

Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins

Decoding and reprogramming fungal iterative nonribosomal peptide synthetases

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