Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs.
• The Methanosarcina mazei pyrrolysyl-tRNA synthetase/tRNA (MmPylRS/MmPyltRNA) pair is widely used but lacks orthogonality with new pairs.
• Discovering new orthogonal pairs is crucial for advancing genetic code expansion.

Purpose of the Study:

• To engineer novel aaRS/tRNA pairs for genetic code expansion.
• To achieve orthogonality with endogenous systems and the established MmPylRS/MmPyltRNA pair.
• To demonstrate the simultaneous function of multiple distinct pairs for incorporating various non-canonical amino acids.

Main Methods:

• Engineering pyrrolysyl-tRNA synthetase (PylRS) variants lacking the N-terminal domain (ΔNPylRS).
• Creating PylRS/tRNA_CUA pairs orthogonal to endogenous systems and the MmPylRS/MmPyltRNA pair.
• Utilizing site-directed mutagenesis to reprogram ncAA specificity.

Main Results:

• Several ΔNPylRS/^PyltRNA_CUA pairs demonstrated activity and orthogonality in Escherichia coli.
• New PylRS/^PyltRNA pairs were generated, exhibiting mutual orthogonality with the MmPylRS/MmPyltRNA pair.
• Transplanting mutations successfully reprogrammed ncAA specificity in the novel PylRS pairs.
• Distinct PylRS/^PyltRNA-derived pairs were shown to function concurrently, decoding different codons and incorporating distinct ncAAs.

Conclusions:

• Novel PylRS/^PyltRNA pairs offer a valuable alternative for genetic code expansion.
• These engineered pairs expand the toolkit for orthogonal protein synthesis.
• The ability to use multiple distinct pairs simultaneously facilitates complex protein engineering and synthetic biology applications.