Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coliStrain Produces a New-to-nature Bioactive Lantibiotic ContainingThienopyrrole-Alanine

Kuthning, Anja; Durkin, Patrick; Oehm, Stefan; Hoesl, Michael Georg; Budiša, Nediljko; Süssmuth, Roderich D.

doi:10.1038/srep33447

Cited by 32 publications

(22 citation statements)

References 64 publications

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“…The lichenicidin bioengineering systems were then employed to: (a) Determine the impact of Ser, Thr and Cys residues on the bioactivity of lichenicidin (Caetano et al, ), (b) to insert noncanonical amino acid residues into the lichenicidin sequence and thus, expand its structural diversity (Oldach et al, ), and (c) to elucidate the flexibility of the posttranslational modification machinery, using chimeric genes lichenicidin‐haloduracin (Caetano, Barbosa, Möesker, Süssmuth, & Mendo, ). More recently, an improved system for lichenicidin production in E. coli was developed to ease Bliα and Bliβ purification (Kuthning, Mösker, & Süssmuth, ); the same vectors were employed for total in vivo insertion of toxic noncanonical amino acids using an evolutionarily adapted E. coli host strain (Kuthning et al, ).…”

Section: Introductionmentioning

confidence: 99%

Lichenicidin rational site‐directed mutagenesis library: A tool to generate bioengineered lantibiotics

Barbosa

Caetano

Mösker

et al. 2019

Biotech & Bioengineering

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Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram‐negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two‐peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user‐friendly method that can be scaled up to screen large libraries of bioengineered two‐peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure–activity relationship studies carried out for other lantibiotics.

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Section: Introductionmentioning

confidence: 99%

Lichenicidin rational site‐directed mutagenesis library: A tool to generate bioengineered lantibiotics

Barbosa

Caetano

Mösker

et al. 2019

Biotech & Bioengineering

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“…For residue-specific incorporation, production strains could be streamlined to ncAA incorporation as shown recently for an E. coli strain adapted to L -β-(thieno[3,2- b ]pyrrolyl)alanine used to produce the correspondingly modified lantibiotic lichenicidin ( Kuthning et al, 2016 ). As an alternative to recombinant production, orthogonal translation can be introduced into native producers, as shown for B. cereus ( Luo et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

Prospects of In vivo Incorporation of Non-canonical Amino Acids for the Chemical Diversification of Antimicrobial Peptides

et al. 2017

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The incorporation of non-canonical amino acids (ncAA) is an elegant way for the chemical diversification of recombinantly produced antimicrobial peptides (AMPs). Residue- and site-specific installation methods in several bacterial production hosts hold great promise for the generation of new-to-nature AMPs, and can contribute to tackle the ongoing emergence of antibiotic resistance in pathogens. Especially from a pharmacological point of view, desirable improvements span pH and protease resistance, solubility, oral availability and circulation half-life. Although the primary focus of this report is on ribosomally synthesized and post-translationally modified peptides (RiPPs), we have included selected cases of peptides produced by solid phase peptide synthesis to comparatively show the potential and impact of ncAA introduction. Generally speaking, the introduction of ncAAs in recombinant AMPs delivers novel levels of chemical diversification. Cotranslationally incorporated, they can take part in AMP biogenesis either through direction interaction with elements of the post-translational modification (PTM) machinery or as untargeted sites with unique physicochemical properties and chemical handles for further modification. Together with genetic libraries, genome mining and processing by PTM machineries, ncAAs present not a mere addition to this process, but a highly diverse pool of building blocks to significantly broaden the chemical space of this valuable class of molecules. This perspective summarizes new developments of ncAA containing peptides. Challenges to be resolved in order to reach large-scale pharmaceutical production of these promising compounds and prospects for future developments are discussed.

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“…Bacterial strains incorporating ncAAs based on the selective pressure incorporation method (Figure 1 a ) are auxotrophic for the amino acid being substituted (87) and thus are dependent on the substituting ncAA in the absence of the canonical amino acid (47, 65). One interesting example is the B. subtilis HR23 strain that was adapted to use 4-fluorotryptophan and can no longer grow without 4-fluorotryptophan even in the presence of Trp (84, 156), indicating that the functions of some essential proteins became sensitive to this ncAA (156).…”

Section: Stabilizing Expanded Genetic Codesmentioning

confidence: 99%

Rewriting the Genetic Code

Mukai

Lajoie

Englert

et al. 2017

Annu. Rev. Microbiol.

137

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The genetic code—the language used by cells to translate their genomes into proteins that perform many cellular functions—is highly conserved throughout natural life. Rewriting the genetic code could lead to new biological functions such as expanding protein chemistries with noncanonical amino acids (ncAAs) and genetically isolating synthetic organisms from natural organisms and viruses. It has long been possible to transiently produce proteins bearing ncAAs, but stabilizing an expanded genetic code for sustained function in vivo requires an integrated approach: creating recoded genomes and introducing new translation machinery that function together without compromising viability or clashing with endogenous pathways. In this review, we discuss design considerations and technologies for expanding the genetic code. The knowledge obtained by rewriting the genetic code will deepen our understanding of how genomes are designed and how the canonical genetic code evolved.

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Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coliStrain Produces a New-to-nature Bioactive Lantibiotic ContainingThienopyrrole-Alanine

Cited by 32 publications

References 64 publications

Lichenicidin rational site‐directed mutagenesis library: A tool to generate bioengineered lantibiotics

Lichenicidin rational site‐directed mutagenesis library: A tool to generate bioengineered lantibiotics

Prospects of In vivo Incorporation of Non-canonical Amino Acids for the Chemical Diversification of Antimicrobial Peptides

Rewriting the Genetic Code

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