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

Abstract

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.

Keywords: Corynebacterium glutamicum; L-lysine; gene knockout; high flux; rare codon.

FIGURE 1

Genome sequencing analysis. The frequency distribution of amino acid codon usage in the Corynebacterium glutamicum 23604 genome.

FIGURE 2

(A) Low abundance of rare tRNA significantly slowed down protein expression. (B) when the concentration of amino acids in the cell increased, the recognition of the rare codon of tRNA was bound to the corresponding amino acid by amino tRNA synthase, the expression of genes containing the rare codon was restored.

FIGURE 3

Gel electrophoresis of PCR products. (A) PCR gel showing the expected product insert size of 1,325 bp for the L-Parg-R. M, DL2000 DNA marker. (B) PCR gel showing the expected product insert size of 1741 bp for L-Pgit-R. M, DL5000 DNA marker. (C) PCR gel showing the expected product insert size of 548 bp for egfp ^M . M, DL2000 DNA marker.

FIGURE 4

OD₆₀₀ values and L-lysine production in engineered strains of C. glutamicum-Parg and C. glutamicum-Pgit (fermentation medium). Samples were taken from the fermentation medium of C. glutamicum-Parg and C. glutamicum-Pgit every 12 h to measure the OD₆₀₀ value (A) and L-lysine concentration (B). The standard errors are shown as bars.

FIGURE 5

Fluorescence intensities of enhanced green fluorescent protein (EGFP ^M ), enhanced blue fluorescent protein (EBFP ^M ), and mCherry ^M .

FIGURE 6

Atmospheric and room-temperature plasma (ARTP) mutagenesis fatality curve.

FIGURE 7

Flow cytometry analysis results. The ordinate FITC shows the fluorescence intensity of EGFP ^M

FIGURE 8

The L-lysine produced by the wild-type and the mutated strains. (A) Mutants of Corynebacterium glutamicum-Parg. (B) Mutants of Corynebacterium glutamicum-Pgit.

FIGURE 9

OD₆₀₀ values and L-lysine production in engineered strains of C. glutamicum W2 (fermentation medium). During the fermentation process, samples were taken from the fermentation medium every 12 h to measure the OD₆₀₀ value (A) and L-lysine concentration (B) in C. glutamicum 23604 and C. glutamicum W2. The standard errors are shown as bars.