TABLE 1.
Strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristic(s) or sequence | Source, reference, or use |
|---|---|---|
| E. coli DH5α | F− φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk− mk+) phoA supE44 thi gyrA96 relA1 λ− | Invitrogen |
| C. glutamicum strains | ||
| WT | Wild-type strain ATCC 13032 | American Type Culture Collection |
| WT(pEKEx5) | WT carrying plasmid pEKEx5 | This study |
| WT(pEKEx5_odx) | WT carrying odx on plasmid pEKEx5 | This study |
| WT(pEKEx2_odx) | WT carrying odx on plasmid pEKEx2 | This study |
| WT Δodx | odx deletion mutant of WT | This study |
| WT Δpyk | pyk deletion mutant of WT | 33 |
| WT Δpyk(pEKEx5) | pyk deletion mutant of WT carrying plasmid pEKEx5 | This study |
| WT Δpyk(pEKEx5_odx) | pyk deletion mutant of WT carrying odx on plasmid pEKEx5 | This study |
| DM1729 | l-Lysine-producing strain, pyc(P458S) hom(V59A) lysC(T311I) | 16 |
| DM1730 | l-Lysine-producing strain, pyc(P458S) hom(V59A) lysC(T311I) Δpck | B. Bathe, Evonik Degussa AG |
| DM1730(pEKEx5) | DM1730 carrying plasmid pEKEx5 | This study |
| DM1730(pEKEx5_odx) | DM1730 carrying odx on plasmid pEKEx5 | This study |
| DM1730 Δodx | odx deletion mutant of DM1730 | This study |
| Plasmids | ||
| pK19mobsacB | Vector for allelic exchange in C. glutamicum, KmroriV oriT sacB lacZα | 50 |
| pK19mobsacB_Δodx | pK19mobsacB carrying the odx gene with internal 679-bp deletion | This study |
| pEKEx5 | Expression vector for use in E. coli and C. glutamicum, Spr Ptac His6 | 30 |
| pEKEx5_odx | pEKEx5 carrying structural odx gene | This study |
| pEKEx2 | Expression vector used in E. coli and C. glutamicum, Kmr Ptac | 15 |
| pEKEx2_odx | pEKEx5 carrying structural odx gene with gap ribosomal binding site | This study |
| Oligonucleotides (primers) | ||
| cg1458DelFrag1For | 5′-GCGGATCCCACCGGCATCAAATTGTGTC-3′ | Deletion in odx, BamHI site (underlined) |
| cg1458DelFrag1Rev | 5′-GGAATGGAGTATGGAAGTTGGCACGGGCGGTGAGGTTAG-3′ | Deletion in odx, artificial crossover overlap (underlined) |
| cg1458DelFrag2For | 5′-CCAACTTCCATACTCCATTCCCATCGGCAAGCTGGGCAAC-3′ | Deletion in odx, artificial crossover overlap (underlined) |
| cg1458DelFrag2Rev | 5′-GCGGATCCTTGCCTTGAGCACAATGTC-3′ | Deletion in odx, BamHI site (underlined) |
| cg1458_oe_for | 5′-CGGGATCCATGCGTTTTGGACGA-3′ | Amplification of odx, BamHI site (underlined) |
| cg1458_oe_rev | 5′-GCGTCGACTTAGGCGTCCACAAC-3′ | Amplification of odx, SalI site (underlined) |
| cg1458ForGapPEx2 | 5′-GCGTCGACAGGAGAACAAACATGCGTTTTGGACGAATTGC-3′ | Amplification of odx, SalI site (underlined) and gap ribosome binding site (bold type) |
| cg1458RevEx2 | 5′-CGGGATCCTTAGGCGTCCACAACTG-3′ | Amplification of odx, BamHI site (underlined) |
| Opcg1458For | 5′-CGAAGGTGAGCTCGCAGTAG-3′ | Analysis of transcriptional organization of odx |
| Opcg1459Rev | 5′-GATGCCGGTGGTGACGTATC-3′ | Analysis of transcriptional organization of odx |
| Opcg1459ForPos | 5′-ATGGATCACCACCATCAC-3′ | Analysis of transcriptional organization of odx |
| Opcg1459RevPos | 5′-TTTCCGACAGCCACTTTG-3′ | Analysis of transcriptional organization of odx |
| Opcg1459RT | 5′-TCGTTGCCTTGAGCACAATG-3′ | Analysis of transcriptional organization of odx |
| SP1_odx | 5′-AAGGAAGGGATTCGGATTGG-3′ | Analysis of transcriptional start site of odx |
| SP2_odx | 5′-AGTGATTCTGCGGACTTC-3′ | Analysis of transcriptional start site of odx |
| SP3_odx | 5′-ATCGGCGTAGTTACGGCCAATC-3′ | Analysis of transcriptional start site of odx |