TABLE 1.
Strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristics or sequence | Source, reference, or purpose |
|---|---|---|
| Strains | ||
| E. coli DH5α | supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 17 |
| WT C. glutamicum | WT strain ATCC 13032, biotin-auxotrophic | American Type Culture Collection |
| C. glutamicum DM1729 | pyc(P458S) hom(V59A) lysC(T311I), derived from WT C. glutamicum | For construction, see Table S1 in the supplemental material |
| C. glutamicum DM1729 ΔilvB | Strain DM1729 with deletion of ilvB, encoding the large subunit of the acetolactate synthase (AHAS) | This work |
| C. glutamicum DM1729 ΔC-T ilvN | Strain DM1729 with deletion of the last 83 amino acids of the C-terminal domain of ilvN, encoding the small subunit of the AHAS | This work |
| C. glutamicum DM1933 | Δpck pyc(P458S) hom(V59A), 2 copies of lysC(T311I), 2 copies of asd, 2 copies of dapA, 2 copies of dapB, 2 copies of ddh, 2 copies of lysA, 2 copies of lysE derived from WT C. glutamicum | For construction, see Table S1 in the supplemental material |
| C. glutamicum DM1933 ΔilvB | Strain DM1933 with deletion of ilvB, encoding the large subunit of the AHAS | This work |
| C. glutamicum DM1933 ΔC-T ilvN | Strain DM1933 with deletion of the last 83 amino acids of the C-terminal domain of ilvN, encoding the small subunit of the AHAS | This work |
| Plasmids | ||
| pK19mobsacB | Kmr, mobilizable (oriT), oriV | 44 |
| pK19mobsacB ΔilvB | pK19mobsacB carrying a truncated ilvB gene | 32 |
| pK19mobsacB ΔC-T ilvN | pK18mobsacB carrying a truncated ilvN gene (shortened by 249 bp) | This work |
| Oligonucleotides | ||
| P1 | 5′-CCCAAGCTTGCTGTTTCCAGATGACCAACC-3′ | Primer for deletion of ΔC-T ilvN |
| P2 | 5′-GGCGATAGTGGTCTCTTCATCAAGTCGCACGACTTTGAGC-3′ | Primer for deletion of ΔC-T ilvN; crossover overlap underlined |
| P3 | 5′-GAAGAGACCACTATCGCCACAGCAATTAATCTGATTGC-3′ | Primer for deletion of ΔC-T ilvN; crossover overlap underlined |
| P4 | 5′-CGCGGATCCCGTTCAGGTTTGGCTCGATG-3′ | Primer for deletion of ΔC-T ilvN and to verify ilvB deletion |
| PD1 | 5′-CCAAGATGGCTAATTCTGACGTCACC-3′ | Primer to verify ΔC-T ilvN deletion |
| PD2 | 5′-GACTAGTCACATTTATGCAGCAGGTGC-3′ | Primer to verify ΔC-T ilvN deletion |
| PilvB | 5′-GCAACAGACATCTGTCGC-3′ | Primer to verify ilvB deletion |