Continuous directed evolution of aminoacyl-tRNA synthetases

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Orthogonal aminoacyl-tRNA synthetases (AARSs) are crucial for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins.
• Traditional directed evolution methods often yield AARSs with diminished activity and selectivity compared to wild-type enzymes.
• Efficient methods are needed to rapidly evolve AARSs with improved enzymatic properties for enhanced protein engineering.

Purpose of the Study:

• To develop and apply phage-assisted continuous evolution (PACE) for rapid generation of highly active and selective orthogonal AARSs.
• To improve the enzymatic efficiency and specificity of AARSs for site-specific ncAA incorporation.
• To demonstrate the utility of PACE in advancing orthogonal translation systems.

Main Methods:

• Designed PACE selections to achieve hundreds of generations of directed evolution for orthogonal AARSs.
• Evolved a chimeric Methanosarcina spp. pyrrolysyl-tRNA synthetase (PylRS) using PACE.
• Applied simultaneous positive and negative selection PACE to enhance the selectivity of a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase variant.

Main Results:

• PACE improved the enzymatic efficiency (kcat/KMtRNA) of a chimeric PylRS by 45-fold.
• Transplantation of evolved mutations increased yields of proteins with ncAAs by up to 9.7-fold.
• PACE enhanced the selectivity of a tyrosyl-tRNA synthetase variant for site-specific p-iodo-L-phenylalanine incorporation.

Conclusions:

• PACE is a highly effective method for rapidly evolving orthogonal AARSs with superior activity and specificity.
• The evolved AARSs expand the toolkit for orthogonal translation systems and protein engineering.
• This work establishes PACE as a powerful platform for optimizing enzymes used in synthetic biology.