Abstract
The translation of rare codons relies on their corresponding rare tRNAs, which could not be fully charged under amino acid starvation. Theoretically, disrupted or retarded translation caused by the lack of charged rare tRNAs can be partially restored by feeding or intracellular synthesis of the corresponding amino acids. Inspired by this assumption, we develop a screening or selection system for obtaining overproducers of a target amino acid by replacing its common codons with the corresponding synonymous rare alternative in the coding sequence of selected reporter proteins or antibiotic-resistant markers. Results show that integration of rare codons can inhibit gene translations in a frequency-dependent manner. As a proof-of-concept, Escherichia coli strains overproducing l-leucine, l-arginine or l-serine are successfully selected from random mutation libraries. The system is also applied to Corynebacterium glutamicum to screen out l-arginine overproducers. This strategy sheds new light on obtaining and understanding amino acid overproduction strains.
Highlights
The translation of rare codons relies on their corresponding rare tRNAs, which could not be fully charged under amino acid starvation
We show that the protein expressions from the rare codon-rich gene derivatives are dramatically increased by feeding or enhanced intracellular synthesis of the corresponding amino acid
One is a selection system based on rare codon-rich antibiotic resistance genes
Summary
The translation of rare codons relies on their corresponding rare tRNAs, which could not be fully charged under amino acid starvation. Disrupted or retarded translation caused by the lack of charged rare tRNAs can be partially restored by feeding or intracellular synthesis of the corresponding amino acids. Inspired by this assumption, we develop a screening or selection system for obtaining overproducers of a target amino acid by replacing its common codons with the corresponding synonymous rare alternative in the coding sequence of selected reporter proteins or antibiotic-resistant markers. Cells overproducing an amino acid might produce enough functional proteins to survive the stresses from the analogue of that amino acid[7] and could be selected. It is of great need to develop alternative approaches that provide accuracy, sensitivity, and high-throughput simultaneously
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