Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

Koch, Nikolaj G.; Goettig, Peter; Rappsilber, Juri; Budiša, Nediljko

doi:10.3390/ijms222011194

Cited by 17 publications

(28 citation statements)

References 72 publications

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“…Over the past two decades, genetic code expansion has evolved into a powerful technique to genetically introduce non-canonical amino acids (ncAAs) into proteins and peptides ( Liu and Schultz, 2010 ; Mukai et al, 2017 ; de la Torre and Chin, 2021 ). More than 200 ncAAs with a broad repertoire of chemistries and new-to-nature functionalities have been incorporated by various routes ( Wan et al, 2014 ; Dumas et al, 2015 ; Koch et al, 2021 ; Pagar et al, 2021 ). The most commonly used method is site-specific stop codon suppression (SCS) in vivo , using bacterial hosts.…”

Section: Introductionmentioning

confidence: 99%

Engineered bacterial host for genetic encoding of physiologically stable protein nitration

Koch

Baumann²,

Nickling³

et al. 2022

Front. Mol. Biosci.

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Across scales, many biological phenomena, such as protein folding or bioadhesion and cohesion, rely on synergistic effects of different amino acid side chains at multiple positions in the protein sequence. These are often fine-tuned by post-translational modifications that introduce additional chemical properties. Several PTMs can now be genetically encoded and precisely installed at single and multiple sites by genetic code expansion. Protein nitration is a PTM of particular interest because it has been associated with several diseases. However, even when these nitro groups are directly incorporated into proteins, they are often physiologically reduced during or shortly after protein production. We have solved this problem by using an engineered Escherichia coli host strain. Six genes that are associated with nitroreductase activity were removed from the genome in a simple and robust manner. The result is a bacterial expression host that can stably produce proteins and peptides containing nitro groups, especially when these are amenable to modification. To demonstrate the applicability of this strain, we used this host for several applications. One of these was the multisite incorporation of a photocaged 3,4-dihydroxyphenylalanine derivative into Elastin-Like Polypeptides. For this non-canonical amino acid and several other photocaged ncAAs, the nitro group is critical for photocleavability. Accordingly, our approach also enhances the production of biomolecules containing photocaged tyrosine in the form of ortho-nitrobenzyl-tyrosine. We envision our engineered host as an efficient tool for the production of custom designed proteins, peptides or biomaterials for various applications ranging from research in cell biology to large-scale production in biotechnology.

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Section: Introductionmentioning

confidence: 99%

Engineered bacterial host for genetic encoding of physiologically stable protein nitration

Koch

Baumann²,

Nickling³

et al. 2022

Front. Mol. Biosci.

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“…For example, Atsushi et al designed Candidatus methanomethylophilus alvus PylRS (CMaPylRS) variants to incorporate Nε-benzyloxycarbonyl-L-lysine (ZLys) derivatives into proteins based on the alignment of the sequences of homologous PylRSs from different species [ 33 ]. Additionally, the mutation residues of previously developed homologous PylRS variants are frequently employed as a guide when generating new Methanosarcina mazei PylRS (MmPylRS) or Methanosarcina barkeri (MbPylRS) variants [ 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids

Liang

et al. 2023

Biomolecules

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In the field of genetic code expansion (GCE), improvements in the efficiency of noncanonical amino acid (ncAA) incorporation have received continuous attention. By analyzing the reported gene sequences of giant virus species, we noticed some sequence differences at the tRNA binding interface. On the basis of the structural and activity differences between Methanococcus jannaschii Tyrosyl-tRNA Synthetase (MjTyrRS) and mimivirus Tyrosyl-tRNA Synthetase (MVTyrRS), we found that the size of the anticodon-recognized loop of MjTyrRS influences its suppression activity regarding triplet and specific quadruplet codons. Therefore, three MjTyrRS mutants with loop minimization were designed. The suppression of wild-type MjTyrRS loop-minimized mutants increased by 1.8–4.3-fold, and the MjTyrRS variants enhanced the activity of the incorporation of ncAAs by 15–150% through loop minimization. In addition, for specific quadruplet codons, the loop minimization of MjTyrRS also improves the suppression efficiency. These results suggest that loop minimization of MjTyrRS may provide a general strategy for the efficient synthesis of ncAAs-containing proteins.

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“…The advancements in the genetic code expansion (GCE) allow enhanced protein properties by introducing unique functional groups beyond nature’s limited building blocks ( Wang and Schultz, 2004 ; Young and Schultz, 2018 ). To this end, a series of orthogonal amino-acyl transfer RNA (tRNA) synthetase (aaRS)/tRNA pairs have been developed to encode distinct non-canonical amino acids (ncAAs) in vivo ( Chin, 2014 , 2017 ; Young and Schultz, 2018 ; Tseng et al, 2020 ; Koch et al, 2021 ; Lee et al, 2021 ). Over the last 2 decades, more than 200 ncAAs have been genetically encoded in prokaryotes and eukaryotes ( Pagar et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

et al. 2022

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Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme’s active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resolution of the 1-phenylpropan-1-amine was performed using benzaldehyde as an amino acceptor, which is cheaper than pyruvate. Our results highlight the utility of ncAAs for designing enzymes with enhanced functionality beyond the limit of 20 canonical amino acids.

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Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains

Cited by 17 publications

References 72 publications

Engineered bacterial host for genetic encoding of physiologically stable protein nitration

Engineered bacterial host for genetic encoding of physiologically stable protein nitration

Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

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