All the strains and plasmids used in this study are listed in Table 1. Escherichia coli DH5α and BL21 (DE3) cells were grown in Luria-Bertani (LB) medium containing 10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl at 37°C and pH 7.2. Corynebacterium glutamicum was cultured at 30°C and pH 7.0. The seed medium LB + glucose (LBG) contained 10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl, and 5 g/L glucose. The recombinant colony was grown on LBHIS AGAR medium containing 5 g/L peptone, 5 g/L NaCl, 2.5 g/L yeast extract, 2.5 g/L glucose, 18.5 g/L brain-heart extract, and 91 g/L sorbitol. The fermentation medium contained 20 g/L glucose, 30 g/L corn pulp, 20 g/L (NH₄)₂SO₄, 10 g/L CH₃COONa, 5 g/L urea, 1.34 g/L L-alanine, 2 g/L KH₂PO₄, 1.35 g/L MgSO₄·7H₂O, 0.02 g/L FeSO₄·7H₂O, 0.04 g/L MnSO₄·H₂O, 0.008 g/L nicotinamide, 0.001 g/L biotin, and 0.04 g/L L-leucine. Corynebacterium glutamicum strains are usually grown in medium supplemented with 25 μg/mL kanamycin (50 μg/mL for E. coli).

Illumina MiSeq (https://www.illumina.com.cn) and PacBio RSII (Pacific Biosciences, United States) were used to sequence the genome of C. glutamicum 23604 following gene element prediction of the whole genome sequence (Piwowarek et al., 2020).

pK19mobsacB-tRNA was constructed using the following method. The genome of C. glutamicum 23604 was extracted using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The polymerase chain reaction (PCR) primers used in this study are listed in Table 2. In the first round of PCR, the upstream and downstream regions of the lysine tRNA UUU gene were amplified using primer pairs P1/P2 and P3/P4, with C. glutamicum 23604 genomic DNA as the template. In the second round of PCR, the product of the first round of PCR was used as the template, and P1/P4 was used as the primer pair. The first round of PCR conditions were as follows: pre-degeneration at 95°C for 3 min, 30 cycles of degeneration at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, followed by extension at 72°C for 10 min and holding at 4°C. The second round of PCR conditions were as follows: (1) pre-degeneration at 95°C for 3 min, five cycles of degeneration at 95°C for 30 s, 54°C for 30 s, and pre-extension at 72°C for 90 s, followed by extension at 72°C for 2 min; (2) pre-degeneration at 95°C for 3 min, 30 cycles of degeneration at 95°C for 30 s, 54°C for 30 s, 72°C for 1.5 min, followed by extension at 72°C for 10 min and holding at 4°C. Fragments of a fusion gene, namely, a 1,044 bp fragment of lysine tRNA, were thus obtained. The plasmid pK19mobsacB was digested with the restriction enzymes EcoRI and HindIII (Thermo Fisher Scientific, United States). Seamless cloning was performed using a One Step Cloning Kit (Vazyme Biotech, Nanjing, China), and then the recombinant plasmids were transfected into competent E. coli DH5α cells. Finally, positive recombinant strains were screened by colony PCR, and DNA sequencing was performed.

The Pgit inducible promoter and arginine rare tRNA UCU Parg promoter sequences in the genome of C. glutamicum were found in the National Centre for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/), and the upstream and downstream homologous arm sequences required for homologous double exchange were added to both sides of the sequences. The L-Pgit-R and L-Parg-R gene fragments (GenScript Biotech, Nanjing, China) were obtained and cloned into the EcoRI-and HindIII-digested pK19mobsacB plasmid sites, respectively. The recombinant plasmids were then transfected into competent E. coli DH5α cells. Finally, colony PCR was used to screen for positive clones, and the identity of the fusion fragments was confirmed by sequencing.

In advance, competent C. glutamicum 23604 cells were prepared (Xiao et al., 2020). The competent cells were then given a 10 μL aliquot of DNA before being transferred to a 2 mm electroporation cuvette (Bio-Rad Laboratories, Hercules, California, United States), with the parameters set at 2.2 kV and 5 ms. Following the electroporation, a 6-min heat shock was administered. Then, the plates were incubated for 2 h at 30°C and plated on LBHIS AGAR medium containing 25 μg/mL kanamycin for 1 day.

Two rounds of forward selection for homologous recombination were performed. Selection was first performed under kanamycin resistance conditions to integrate the plasmid into the chromosome. Strains that grew normally under kanamycin resistance conditions were inoculated in LB medium containing 15% sucrose (LBS). After three passes, they were inoculated in LB AGAR medium containing kanamycin or LB AGAR medium containing sucrose, respectively. Strains that grew only on LB AGAR medium containing sucrose were selected. Using the genome as a template, PCR amplification with P1/P4 primers was performed to identify clones carrying the required deletions or undergoing allele exchange. When the lysine tRNA gene was knocked out, the strains that were screened by the second round of sucrose (15%) could not be obtained. Pgit and Parg promoter replacements amplified DNA fragments of 1741 bp and 1,325 bp, respectively.

To determine the expression of tRNA-UUU, RT-qPCR was performed using a fluorescent quantitative PCR instrument (Applied Biosystems, United States) to analyse the expression of tRNA-UUU in wild-type and promoter replacement strains, and the 16s rRNA gene was selected as an internal reference gene for quantification. The experimental methods are shown in the Supplementary Material.

Wild-type strain C. glutamicum 23604 and three colonies of the promoter replacement strain were inoculated into liquid LBG medium and cultured overnight at 200 r/min and 30°C. Sixteen hours later, the seed solution was inoculated in 100 mL fermentation medium at 10%. During the fermentation process, samples were taken from the fermentation medium every 12 h to measure the OD₆₀₀ value and L-lysine concentration. More than three experiments were performed, and the average OD₆₀₀ and L-lysine concentration values were calculated.

To construct pEC-XK99E-enhanced green fluorescent protein (egfpM), pEC-XK99E-enhanced blue fluorescent protein (ebfpM), and pEC-XK99E-mCherryM, the lysine codons in the egfp, ebfp, and mCherry genes were all replaced by the AAA codon. EgfpM, ebfpM, and mCherryM were obtained by gene fragment synthesis (Genscript Biotech, Nanjing, China) and cloned into pEC-XK99E digested with the restriction enzyme EcoRI (Thermo Fisher Scientific, United States) for the expression of different fluorescent proteins. The recombinant plasmids were then transfected into E. coli DH5α competent cells. Finally, positive recombinant strains were identified using colony PCR and DNA sequencing.

Three fluorescent protein granules were extracted and transformed into the preprepared C. glutamicum 23604 receptor cells. The recombinant strains obtained were activated and inoculated into 100 mL LBG medium containing 25 μg/mL kanamycin with an initial OD600 of 0.3. The cells were placed in an incubator with constant temperature oscillation at 30°C and a speed of 200 r/min. After 24 h of culture, the expression of fluorescent protein was induced. Finally, fluorescence intensity was measured using a Biotek Microplate Reader (Biotek Instruments, Inc., Winooski, VA, United States).

pEC-XK99E-egfp ^M was transformed into the strains that replaced the promoter, and the positive recombinant colonies were selected and inoculated into the LBG medium containing 25 μg/mL kanamycin and incubated at 30°C and 200 r/min. Induced expression was performed according to the optimization results of the single factor experiment.

Compared with traditional mutagenesis, the ARTP mutation system can generate a larger gene mutation library. First, the two recombinant strains containing the EGFP ^M screening label were cultured in LBG medium containing 25 μg/mL kanamycin at an OD₆₀₀ of 0.6–0.9, then coated with 10 μL medium uniformly on the stainless-steel minidisc. After exposure to ARTP for 1, 3, 5, 7, and 9 min, the fatality rate was calculated. The optimal ARTP mutants with over 90% treatment lethality were selected and washed with 1 mL LBG medium. Then, 500 μL was inoculated in 50 mL LBG medium and cultured at 30 min and 200 r/min until the OD₆₀₀ reached 1.0. Isopropyl β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.6 mM was added to the medium and incubated at 30°C for 10 h. The 1 mL culture medium was diluted to an OD₆₀₀ of 1.0, and ARTP mutants with strong fluorescence were sorted using flow cytometry under the sort condition of green fluorescence (Mohsin et al., 2015). The cultured cells were washed and resuspended in a potassium phosphate buffer. Then, the cells were analysed using FACS (Beckman Coulter MoFlo XDP, United States), with an excitation line at 488 nm, fluorescence detection at 535 nm, and a sample pressure of 60 psi. The nozzle diameter was set to 70 μm. Sterile, filtered phosphate buffered saline was used as the sheath solution. Data analysis was performed using the Beckman Summit 5.2 software. In the mutant library selection, a gate containing 0.01% of total cells was set up according to the pre-analysis of the mutant library, and cells with higher expression were collected and further fermented in a 96-well plate.

The screened mutants were inoculated into 96-well plates and cultured in LBG medium for 24 h as the seed solution. Meanwhile, C. glutamicum 23604 was inoculated into 96-well plates containing LBG medium using flow cytometry. Seeds of wild-type C. glutamicum 23604 and ARTP mutants were inoculated at 5% into 96-deep-well plates filled with fermentation medium and fermented at 30°C and 600 r/min for 48 h. The content of L-lysine in the fermentation broth was measured at 24 h and 48 h using a biosensor analyzer SBA-40E (Institute of Biology, Shandong Academy of Sciences, Shandong, China) (Geng et al., 2013). The mutant and wild-type strains with the highest L-lysine production obtained in 96-well plates were inoculated in liquid LBG medium to establish three parallel experiments and incubated overnight at 200 r/min and 30°C. After 16 h, the seed solution was inoculated into 100 mL of fermentation medium at 10% inoculum. Following 24 h of fermentation, samples of the fermentation medium were collected at 12 h intervals, and OD₆₀₀ values, as well as L-lysine concentrations, were measured. The average OD₆₀₀ and L-lysine concentration values of more than three experiments were calculated.

Statistical analysis was performed using IBM SPSS Statistics 29 (IBM SPSS, Turkey). One-way ANOVA was used to analyse the significance of mean difference of samples. The significance of differences was estimated with 0.05 level of confidence.

Genome sequencing and analysis showed that some codons were rare codons of Corynebacterium glutamate (Figure 1). For example, in the genome of C. glutamicum 23604, six codons encoded arginine, among which AGA was used less than 4%, while two codons, AAA and AAG, encoded lysine, with a frequency distribution of 40% and 60%, respectively. There was only one tRNA with the anticodon UUU, and two tRNAs with the anticodon CUU, all of which were single-copy genes. In addition, the frequency of the natural rare arginine codon AGA was very low, only 4%. Therefore, there was no natural rare L-lysine codon in C. glutamicum 23604. Consequently, this codon cannot be used for the screening of L-lysine high-yielding strains. In this study, the strategy of artificial construction of the rare codon was adopted. The translation of the rare codon is affected by the corresponding rare tRNA, and the regulation of the relevant translation level is particularly significant when the corresponding amino acid is deficient in cells. However, when the concentration of amino acids in the cell is increased to allow the rare codon recognition of tRNA to bind to the corresponding amino acid under the action of aminoyl tRNA synthase, genes containing the rare codon can be translated and expressed (Figure 2). In this study, tRNA with the UUU anticodon was constructed to enable the recognition of the rare artificial lysine codon AAA.

Plasmid pK19mobsacB was used to construct the knockout vector, and tRNA deletion strains with anticodon UUU were expected to be obtained through double exchange homologous recombination. The positive strain from the first round of homologous recombination was screened using kanamycin. The strains that successfully underwent the first homologous recombination were inoculated in LBG medium for culture, and then coated on LBS AGAR medium after gradient dilution. None of the strains grew, so the strains that underwent the second homologous recombination failed to be successfully screened. The results showed that C. glutamicum 23604 cannot be dependent on a tRNA whose only codon is CUU.

The knockout vector was constructed using the pK19mobsacB plasmid, and tRNA promoter replacement strains with Parg and Pgit promoters were obtained by double-swap homologous recombination. The positive strains, named C. glutamicum-Parg and C. glutamicum-Pgit, were verified using PCR and electrophoresis (Figures 3A,B). The expression level of tRNA-UUU in the wild type and promoter replacement strains was shown in Supplementary Figure S2, in which the expression level of tRNA-UUU in C. glutamicum-Parg was significantly decreased (p < 0.01). The fermentation results showed that the production of L-lysine in the final fermentation broth was reduced by 2.3% and 3.1% and that the biomass was reduced by 3.3% and 2.99% in the two promoter replacement strains, C. glutamicum-Parg and C. glutamicum-Pgit, respectively, compared to those of the wild-type strain C. glutamicum 23604 (Figure 4). From the above statistical analysis results, it can be seen that the L-lysine yield of the two promoter replacement strains was significantly different compared to the wild type strain (p < 0.05).

After induction, among the three fluorescent proteins (Figure 5), EGFP ^M had the strongest fluorescence intensity. Therefore, EGFP ^M was finally selected as the fluorescence screening marker. The fluorescent protein expression vector pEC-XK99E-egfp ^M was transformed into a promoter replacement strain, which was identified as positive by PCR and electrophoresis (Figure 3C).

As the flow cytometry sorting system can perform ultra-high-speed sorting and purification of specific cells, it was used to sort the mutants treated with ARTP for 7 min (Figure 6). The ratio of the total number of screened cells to the total cell number was approximately 1/100000. As shown in the analysis and screening results in Figure 7, some cells showed strong fluorescence. Single cells with a stronger fluorescence signal were sorted and inoculated in 96-well plates.

The results of the fermentation of mutants in 96-well plates are shown in Figure 8. Fifty-five and 31 strains were obtained from the mutants of C. glutamicum-Parg and C. glutamicum-Pgit, with screening efficiencies of 38/55 and 4/31, respectively. Among them, the screening efficiency of the C. glutamicum-Parg mutant reached 69%. Compared with the L-lysine yield of C. glutamicum 23604, that of C. glutamicum W2 was significantly increased by 9.7% (16.87 g. L^-1, p < 0.05). Furthermore, the biomass of C. glutamicum W2 was increased by 3.4%, compared to that of the wild-type C. glutamicum 23604 (Figure 9).