Recoded organisms engineered to depend on synthetic amino acids

Area of Science:

• Synthetic biology
• Genetic engineering
• Microbiology

Background:

• Genetically modified organisms (GMOs) offer industrial benefits but require robust biosafety measures.
• Existing biocontainment strategies face limitations like cross-feeding and genetic mutations.
• There is a need for advanced genetic isolation and biocontainment for GMOs.

Purpose of the Study:

• To engineer genomically recoded organisms (GROs) with enhanced biocontainment.
• To establish synthetic auxotrophy dependent on exogenously supplied synthetic amino acids (sAAs).
• To improve genetic isolation and prevent horizontal gene transfer in engineered microbes.

Main Methods:

• Engineered an Escherichia coli strain with an orthogonal translation system for synthetic amino acids (sAAs).
• Utilized multiplex automated genome engineering to introduce TAG codons into 22 essential genes.
• Developed sAA-dependent auxotrophic strains with multiple essential gene modifications.

Main Results:

• Created robust sAA-dependent GROs with undetectable escape frequencies after extensive culturing.
• Demonstrated that engineered auxotrophies were not rescued by cross-feeding in environmental assays.
• Achieved significant improvement in biocontainment compared to existing methods.

Conclusions:

• Genomically recoded organisms (GROs) dependent on synthetic amino acids offer enhanced biocontainment.
• These GROs provide genetic isolation, impeding horizontal gene transfer.
• Engineered auxotrophy advances orthogonal barriers for safe application of GMOs in open systems.