A bacterial strain with a unique quadruplet codon specifying non-native amino acids

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetics

Background:

• The genetic code can be expanded to include non-canonical amino acids using unique codons.
• Current methods often utilize stop codons or quadruplet codons, each with limitations in efficiency and specificity.
• Quadruplet codons face challenges due to competing recognition by endogenous cellular machinery.

Purpose of the Study:

• To enhance the efficiency of quadruplet codon-mediated suppression for incorporating non-native amino acids.
• To investigate the use of a Methanocaldococcus jannaschii-derived tRNA/aminoacyl-tRNA synthetase pair in a recoded bacterial strain.
• To demonstrate efficient quadruplet codon usage by mitigating competing triplet codon recognition.

Main Methods:

• Utilized a genomically recoded E. coli strain (E. coli C321) with abolished amber stop codon (UAG) mediated termination.
• Introduced a Methanocaldococcus jannaschii-derived frame-shift suppressor tRNA/aminoacyl-tRNA synthetase pair.
• Assessed the suppression efficiency of UAGN quadruplet codons in the recoded bacterial system.

Main Results:

• The Methanocaldococcus jannaschii-derived system significantly enhanced UAGN suppression efficiency in the recoded E. coli strain.
• Elimination of competing triplet codon recognition was key to improving quadruplet codon performance.
• Demonstrated successful incorporation of non-native amino acids using efficient quadruplet codons.

Conclusions:

• Efficient quadruplet codons for non-native amino acid incorporation can be achieved by minimizing interference from endogenous translation machinery.
• This approach offers a promising strategy for expanding the genetic code beyond current limitations.
• The study highlights the potential of engineered tRNA/synthetase pairs in synthetic biology applications.