Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum

Background: L-lysine is widely used in the feed, food, and pharmaceutical industries, and screening for high L-lysine-producing strains has become a key goal for the industry.Methods: We constructed the rare L-lysine codon AAA by corresponding tRNA promoter replacement in C. glutamicum. Additionally, a screening marker related to the intracellular L-lysine content was constructed by converting all L-lysine codons of enhanced green fluorescent protein (EGFP) into the artificial rare codon AAA. The artificial EGFP was then ligated into pEC-XK99E and transformed into competent Corynebacterium glutamicum 23604 cells with the rare L-lysine codon. After atmospheric and room-temperature plasma mutation and induction culture, 55 mutants (0.01% of total cells) with stronger fluorescence were sorted using flow cytometry, and further screened by fermentation in a 96-deep-well plate and 500 mL shaker.Results: The fermentation results showed that the L-lysine production was increased by up to 9.7% in the mutant strains with higher fluorescence intensities, and that the highest screening positive rate was 69%, compared with that in the wild-type strain.Conclusion: The application of artificially constructed rare codons in this study represents an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.

Automated summary

In this study, high L-lysine-producing strains were screened by constructing the artificial rare codon AAA in C. glutamicum. Fermentation results showed that the mutant strains with higher fluorescence intensities had an increase of up to 9.7% in L-lysine production, and the highest screening positive rate was 69%. Therefore, the application of artificially constructed rare codons provides an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.