Codon Randomization for Rapid Exploration of Chemical Space in Thiopeptide Antibiotic Variants

Young, Travis S.; Dorrestein, Pieter C.; Walsh, Christopher T.

doi:10.1016/j.chembiol.2012.10.013

Cited by 78 publications

(101 citation statements)

References 51 publications

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“…(14–17) Biosynthetic engineering of thiopeptides has rapidly emerged as an effective strategy to provide analogs that could ultimately support improved pharmacological and pharmacokinetic parameters. (18–24)…”

mentioning

confidence: 99%

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Zhang

Kelly

2015

ACS Chem. Biol.

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Thiopeptides are posttranslationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery’s tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogs. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched, hydrophobic residues, are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogs were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

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confidence: 99%

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Zhang

Kelly

2015

ACS Chem. Biol.

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“…We have been among the groups that have reported gene clusters for the ribosome-targeting 26-membered macrocycle class (thiocillins) (28) and the EF-Tutargeting 29-membered macrocycles (GE37468) (17,18). In this study, we have turned to the 35-membered macrocycle scaffold and identified a candidate biosynthetic gene cluster for berninamycin from a draft genome sequence of the known producer Streptomyces bernensis UC 5144.…”

mentioning

confidence: 99%

The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds

Malcolmson

Young

Ruby

et al. 2013

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

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Berninamycin is a member of the pyridine-containing thiopeptide class of antibiotics that undergoes massive posttranslational modifications from ribosomally generated preproteins. Berninamycin has a 2-oxazolyl-3-thiazolyl-pyridine core embedded in a 35-atom macrocycle rather than typical trithiazolylpyridine cores embedded in 26-atom and 29-atom peptide macrocycles. We describe the cloning of an 11-gene berninamycin cluster from Streptomyces bernensis UC 5144, its heterologous expression in Streptomyces lividans TK24 and Streptomyces venezuelae ATCC 10712, and detection of variant and incompletely processed scaffolds. Posttranslational maturation in S. lividans of both the wild-type berninamycin prepeptide (BerA) and also a T3A mutant generates macrocyclic compounds as well as linear variants, which have failed to form the pyridine and the macrocycle. Expression of the gene cluster in S. venezuelae generates a variant of the 35-atom skeleton of berninamycin, containing a methyloxazoline in the place of a methyloxazole within the macrocyclic framework.heterologous expression | linear precursors | prepeptide gene replacement

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“…This method may be generalized by using one of several "photo-caged" unnatural amino acids that have been developed to incorporate natural amino acids that are protected from posttranslational modification in similar fashion and subsequently postbiosynthetically deprotected to yield novel thiopeptides not otherwise expressible in vivo. Although no mutants were found with enhanced activity in this study, new chemical functionalities provided by ncAAs to thiopeptide biosynthesis offer the potential to screen a diverse chemical space for mutants with increased activity in the future (8,13).…”

Section: Discussionmentioning

confidence: 69%

“…Several methods have been used to produce thiopeptide variants, including biosynthetic pathway engineering (7,8), semisynthesis (9), and total chemical synthesis (10), with the latter providing the greatest control over chemical structure. However, the total synthesis of variants in sufficient quantities for mechanistic or therapeutic purposes is often time-consuming and costly.…”

mentioning

confidence: 99%

Recombinant thiopeptides containing noncanonical amino acids

Luo

Zambaldo

Liu

et al. 2016

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

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Thiopeptides are a subclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs) with complex molecular architectures and an array of biological activities, including potent antimicrobial activity. Here we report the generation of thiopeptides containing noncanonical amino acids (ncAAs) by introducing orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs into a thiocillin producer strain of Bacillus cereus. We demonstrate that thiopeptide variants containing ncAAs with bioorthogonal chemical reactivity can be further postbiosynthetically modified with biophysical probes, including fluorophores and photo-cross-linkers. This work allows the site-specific incorporation of ncAAs into thiopeptides to increase their structural diversity and probe their biological activity; similar approaches can likely be applied to other classes of RiPPs.noncanonical amino acid | biosynthesis | natural products | thiopeptides | antibiotic

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Codon Randomization for Rapid Exploration of Chemical Space in Thiopeptide Antibiotic Variants

Cited by 78 publications

References 51 publications

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A

The posttranslational modification cascade to the thiopeptide berninamycin generates linear forms and altered macrocyclic scaffolds

Recombinant thiopeptides containing noncanonical amino acids

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