Engineering posttranslational proofreading to discriminate nonstandard amino acids

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Nonstandard amino acid (nsAA) incorporation diversifies protein function but faces challenges due to aminoacyl-tRNA synthetase polyspecificity.
• Accurate evaluation of nsAA incorporation is crucial for protein engineering and biological process investigation.

Purpose of the Study:

• To develop a novel quality control system for accurate and rapid evaluation of nsAA incorporation.
• To engineer a specific nsAA incorporation machinery for enhanced biocontainment and growth of engineered bacteria.

Main Methods:

• Hijacking the N-end rule pathway for a posttranslational proofreading system based on protein degradation.
• Evolving a tyrosyl-tRNA synthetase (TyrRS) variant and tRNA^Tyr for biphenylalanine incorporation specificity.
• Engineering the ClpS protein to adapt the N-end rule pathway for various nsAAs.

Main Results:

• Proteins with desired N-terminal nsAAs showed significantly longer half-lives, enabling effective proofreading.
• The evolved machinery improved biphenylalanine incorporation specificity in vitro and in vivo.
• Genetically engineered *Escherichia coli* strains dependent on biphenylalanine exhibited enhanced biocontainment and growth.

Conclusions:

• The posttranslational proofreading system offers an alternative paradigm for molecular recognition and accurate expansion of the genetic code.
• This system enhances the fidelity of nsAA incorporation, advancing protein engineering capabilities.
• The N-end rule pathway can be rationally engineered for broad applicability in nsAA incorporation quality control.