Engineering Translation Components Improve Incorporation of Exotic Amino Acids

Katoh, Takayuki; Suga, Hiroaki

doi:10.3390/ijms20030522

Cited by 16 publications

(16 citation statements)

References 62 publications

(83 reference statements)

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“…iSAT can in the future be combined with ribosome display, in a platform call RISE, to engineer the ribosome for altered chemical properties. Utilization of iSAT with flexizyme-aminoacylated tRNAs would be necessary for the incorporation of amino acids without aaRSs. ,− …”

Section: Discussionmentioning

confidence: 99%

In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins

et al. 2021

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Efforts to expand the scope of ribosome-mediated polymerization to incorporate noncanonical amino acids (ncAAs) into peptides and proteins hold promise for creating new classes of enzymes, therapeutics, and materials. Recently, the integrated synthesis, assembly, and translation (iSAT) system was established to construct functional ribosomes in cell-free systems. However, the iSAT system has not been shown to be compatible with genetic code expansion. Here, to address this gap, we develop an iSAT platform capable of manufacturing pure proteins with site-specifically incorporated ncAAs. We first establish an iSAT platform based on extracts from genomically recoded Escherichia coli lacking release factor 1 (RF-1). This permits complete reassignment of the amber codon translation function. Next, we optimize orthogonal translation system components to demonstrate the benefits of genomic RF-1 deletion on incorporation of ncAAs into proteins. Using our optimized platform, we demonstrate high-level, multi-site incorporation of p-acetyl-phenylalanine (pAcF) and p-azido-phenylalanine into superfolder green fluorescent protein (sfGFP). Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.

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Section: Discussionmentioning

confidence: 99%

In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins

et al. 2021

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“…The flexible in vitro translation (FIT) system comprises the integration of a reconstituted E. coli translation system and NAA-tRNAs prepared by flexizymes (artificially evolved ribozymes able to catalyze the aminoacylation of tRNA with a variety of nonproteinogenic amino acids (NAAs). FIT facilitates expression of various peptides containing NAAs from designed mRNA templates according to the newly designated genetic table by means of genetic code reprogramming (Figure 2A) [3,25,[33][34][35]. Therefore, the advantage of the FIT system as a cyclization method is the incorporation of more NAAs as the backbone macrocyclic peptides [1,36].…”

Section: Flexible In Vitro Translation (Fit)mentioning

confidence: 99%

Biosynthetic Strategies for Macrocyclic Peptides

Wang¹,

Khojasteh²,

Su³

2021

Molecules

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Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.

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“…Owing to the broad tolerance of flexizyme for tRNAs and amino acids, including NAAs, various NAA-tRNAs can be prepared. The combination of a reconstituted E. coli translation system and NAA-tRNAs prepared by flexizyme is referred to as the flexible in vitro translation (FIT) system, which enables the synthesis of various peptides containing NAAs by means of genetic code reprogramming (Yamagishi et al, 2011;Katoh et al, 2017;Passioura et al, 2018a;Katoh and Suga, 2019).…”

Section: Genetic Code Manipulation Technologies For Incorporation Of mentioning

confidence: 99%

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies

et al. 2020

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Backbone macrocyclic structures are often found in diverse bioactive peptides and contribute to greater conformational rigidity, peptidase resistance, and potential membrane permeability compared to their linear counterparts. Therefore, such peptide scaffolds are an attractive platform for drug-discovery endeavors. Recent advances in synthetic methods for backbone macrocyclic peptides have enabled the discovery of novel peptide drug candidates against diverse targets. Here, we overview recent technical advancements in the synthetic methods including 1) enzymatic synthesis, 2) chemical synthesis, 3) split-intein circular ligation of peptides and proteins (SICLOPPS), and 4) in vitro translation system combined with genetic code reprogramming. We also discuss screening methodologies compatible with those synthetic methodologies, such as one-beads one-compound (OBOC) screening compatible with the synthetic method 2, cell-based assay compatible with 3, limiting-dilution PCR and mRNA display compatible with 4.

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Engineering Translation Components Improve Incorporation of Exotic Amino Acids

Cited by 16 publications

References 62 publications

In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins

In vitro-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins

Biosynthetic Strategies for Macrocyclic Peptides

Methodologies for Backbone Macrocyclic Peptide Synthesis Compatible With Screening Technologies

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