A tRNAModification-based strategy for Identifying amiNo acid Overproducers (AMINO)

Area of Science:

• Biotechnology
• Synthetic Biology
• Metabolic Engineering

Background:

• The multi-billion-dollar amino acid market demands high-performance microbial production systems.
• Current screening methods lack universality for diverse amino acid production.
• Engineering transfer RNA (tRNA) offers a potential avenue to modulate amino acid biosynthesis.

Purpose of the Study:

• To develop a general screening strategy for identifying microbial overproducers of both proteinogenic and non-proteinogenic amino acids.
• To establish a selection system based on engineered tRNAs and marker genes for amino acid overproduction.
• To demonstrate the applicability of this system for screening L-tryptophan overproducers.

Main Methods:

• Modification of tRNA structure to influence aminoacylation levels by aminoacyl-tRNA synthetases.
• Development of a selection system employing engineered tRNAs and marker genes.
• Application of growth-based and fluorescence-activated cell sorting (FACS)-based screening on random mutation libraries.
• Utilizing Escherichia coli and Corynebacterium glutamicum as microbial hosts.

Main Results:

• Successfully screened overproducers for five specific amino acids, including L-tryptophan.
• Demonstrated the efficacy of the developed selection system in both Escherichia coli and Corynebacterium glutamicum.
• Validated the system's capability for identifying amino acid overproducing strains through growth-based and FACS-based methods.

Conclusions:

• A universal strategy for screening amino acid overproducers has been established.
• The system is applicable to both proteinogenic and non-proteinogenic amino acids.
• This approach can be utilized in both recoded and non-recoded microbial hosts for enhanced amino acid production.