Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles

Area of Science:

• Synthetic biology
• Biochemistry
• Molecular biology

Background:

• Genetically encoded synthesis of non-natural peptides and depsipeptide macrocycles is challenging.
• Existing methods lack the ability to incorporate diverse non-canonical building blocks efficiently.

Purpose of the Study:

• To develop a system for the direct, genetically encoded synthesis of diverse non-natural peptide and depsipeptide macrocycles.
• To expand the repertoire of non-canonical amino acids and alpha hydroxy acids that can be site-specifically incorporated into proteins.

Main Methods:

• Utilized Syn61Δ3 cells with a synthetic Escherichia coli genome, lacking specific codons and cognate tRNAs.
• Engineered pyrrolysyl-tRNA synthetase/tRNA pairs for co-translational incorporation of non-canonical amino acids and alpha hydroxy acids.
• Developed 49 engineered, mutually orthogonal pairs for decoding distinct non-canonical building blocks and codons.

Main Results:

• Successfully programmed Syn61Δ3 cells for the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each with two non-canonical amino acids.
• Demonstrated co-translational incorporation of diverse aliphatic and aromatic alpha hydroxy acids.
• Achieved encoded synthesis of 12 diverse non-natural depsipeptide macrocycles containing non-canonical side chains and ester bonds.

Conclusions:

• Established a versatile platform for the genetically encoded synthesis of complex non-natural macrocyclic peptides and depsipeptides.
• The engineered orthogonal tRNA synthetase/tRNA pairs significantly expand the scope of unnatural building blocks for protein synthesis.
• This work provides a powerful tool for creating novel biomolecules with tailored properties.