Table 1.
Strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Relevant characteristic(s) or sequence | Source, reference, or purpose |
|---|---|---|
| Strains | ||
| E. coli DH5α | F− ϕ80lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17 (rK− mK+) supE44 thi-1 gyrA96 relA1 phoA | 62 |
| C. glutamicum WT | Wild-type strain ATCC 13032, biotin auxotrophic | American Type Culture Collection |
| C. glutamicum ΔaceE | C. glutamicum WT with deletion of the E1p gene (aceE) of the pyruvate dehydrogenase complex | 24 |
| C. glutamicum DM1800 | l-Lysine producer; pycP485S, lysCT311I, derived from C. glutamicum WT | 63 |
| C. glutamicum DM1800 aceE A16 | C. glutamicum DM1800 in which the native aceE promoter was replaced by the dapA-A16 promoter | This work |
| C. glutamicum DM1933 | l-Lysine producer; Δpck, pycP458S; homV59A; 2× lysCT311I, 2× asd, 2× dapA, 2× dapB, 2× ddh, 2× lysA, 2× lysE, derived from C. glutamicum WT | 64 |
| C. glutamicum DM1933 aceE A16 | C. glutamicum DM1933 in which the native aceE promoter was replaced by the dapA-A16 promoter | This work |
| C. glutamicum aceE L1, A23, A25, A16 | C. glutamicum WT in which the native aceE promoter was replaced by the dapA-L1, -A23, -A25, or -A16 promoter, respectively | This work |
| C. glutamicum aceE A16 Δpqo | C. glutamicum aceE A16 with deleted pqo gene, encoding pyruvate:quinone oxidoreductase | This work |
| C. glutamicum aceE A16 Δpqo Δppc | C. glutamicum aceE A16 Δpqo with deleted ppc gene, encoding phosphoenolpyruvate carboxylase | This work |
| C. glutamicum aceE A16 Δpqo Δppc ΔilvE | C. glutamicum aceE A16 Δpqo Δppc with deleted ilvE gene, encoding transaminase B | This work |
| Plasmids | ||
| pK18/19mobsacB | Kmr, mobilizable (oriT), oriV | 40 |
| pK18mobsacBaceE-rAc | pK18mobsacB carrying a truncated promoter region of the aceE gene, encoding the E1 subunit of the PDHC | This work |
| pK18mobsacB PaceE dapA-L1, -A23, -A25, -A16 | pK19mobsacB PaceE carrying the dapA-L1, -A23, -A25, or -A16 promoter, respectively | This work |
| pK19mobsacB Δppc | pK19mobsacB carrying a truncated ppc gene, encoding phosphoenolpyruvate carboxylase | This work |
| pK19mobsacB Δpqo | pK19mobsacB carrying a truncated pqo gene, encoding pyruvate:quinone oxidoreductase | 41 |
| pK19mobsacB ΔilvE | pK19mobsacB carrying a truncated ilvE gene, encoding transaminase B | 61 |
| pK19mobsacB dPgltA540-PdapA-L1, -A23, -A25, -A16 | pK19mobsacB carrying a truncated promoter region of the gltA gene and the dapA-L1, -A23, -A25, or -A16 promoter, respectively | 60 |
| pJC4 ilvBNCD | Kanr; plasmid carrying the ilvBNCD genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and dihydroxyacid dehydratase | 65 |
| pJC4 ilvBNCE | Kanr; plasmid carrying the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B | 22 |
| Oligonucleotides | ||
| dapAfow | 5′-AACTGCAGAACCAATGCATTGGTTCTGCAGTTATCACA CCC-3′ | Amplification of dapA promoters, NsiI site underlined |
| dapArev2 | 5′-GGGAATTCCATATGAGGCTCCTTTTAAATCGAGCGGCT CCGGTCTTAGCTGTTAAACC-3′ | Amplification of dapA promoters/verification of promoter exchange, NdeI site underlined |
| ace1 | 5′-CCCAAGCTTGCACATTACCGTCCAACC-3′ | Sequencing of dapA promoters/verification of promoter exchange |
| ace2 | 5′-CGCGGATCCCGACGGTAACGCTTCTCC-3′ | Sequencing of dapA promoters |
| ilvE1 | 5′-GCGTTGACTGATTCTTGGTC-3′ | Primer to verify deletion of ilvE (11) |
| ilvE2 | 5′-CGAGTTCGATGGAATCTTCG-3′ | Primer to verify deletion of ilvE (11) |
| pqodel1 | 5′-AAGGAATTCGTTTTCGAGGCGACCAGACAG-3′ | Primer to verify deletion of pqo (41) |
| pqodel4 | 5′-TGGCACAAGCTTGTTAAGCGCTCGCGGTCAATG-3′ | Primer to verify deletion of pqo (41) |
| ppc1 | 5′-CCCAAGCTTGAGTTGCGCAGCGCAGTG-3′ | Primer for deletion of ppc, HindIII site underlined |
| ppc2 | 5′-GTGCTGCGCAATGCTGAGGGCATTAGAGCAGTGGATT GG-3′ | Primer for deletion of ppc, crossover overlap underlined |
| ppc3 | 5′-CCTCAGCATTGCGCAGCACATCGGCCACAGCTTCTGC-3′ | Primer for deletion of ppc, crossover overlap underlined |
| ppc4 | 5′-CGCGGATCCCGATGACATCAGGTTCCTC-3′ | Primer for deletion of ppc, BamHI site underlined |
| ppcdel1 | 5′-GGAATAGACTCGCTCGGC-3′ | Primer to verify deletion of ppc |
| ppcdel2 | 5′-GTGAACAGGCTCTCGATGC-3′ | Primer to verify deletion of ppc |
| aceEup-fw | 5′-CGGGATCCCGACCCAATGCGTACCGATGTG-3′ | Primer for deletion of aceE promoter region, BamHI site underlined |
| aceE-intrev | 5′-GCGCTAGCGCCACCATCGGAGGTGTTGTTC-3′ | Primer for deletion of aceE promoter region, NheI site underlined |
| aceE-rAC-soeleft | 5′-TTGATCGGCCATATGTATTATGCATCTCTCACGTTTGACG CGAATCG-3′ | Primer for deletion of aceE promoter region, crossover overlap underlined, NsiI and NdeI in italic |
| aceE-rAC-soeright | 5′-CAAACGTGAGAGATGCATAATACATATGGCCGATCAAGC AAAACTTGG-3′ | Primer for deletion of aceE promoter region, crossover overlap underlined, NsiI and NdeI in italic |