AUTHOR=Yang Cuiping , Peng Zehao , Yang Lu , Du Bowen , Guo Chuanzhuang , Sui Songsen , Wang Jianbin , Li Junlin , Wang Junqing , Li Nan 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1194511

DOI=10.3389/fbioe.2023.1194511

ISSN=2296-4185

ABSTRACT=<p><bold>Background:</bold> 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.</p><p><bold>Methods:</bold> We constructed the rare L-lysine codon AAA by corresponding tRNA promoter replacement in <italic>C. glutamicum</italic>. 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 <italic>Corynebacterium glutamicum</italic> 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.</p><p><bold>Results:</bold> 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.</p><p><bold>Conclusion:</bold> The application of artificially constructed rare codons in this study represents an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.</p>