Table 1

Strains, plasmids, and oligonucleotides used in this study

Strain, plasmid, or oligonucleotideRelevant characteristic(s) or sequenceSource, reference, or purpose
Strains
    E. coli DH5αF ϕ80lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17 (rK mK+) supE44 thi-1 gyrA96 relA1 phoA62
    C. glutamicum WTWild-type strain ATCC 13032, biotin auxotrophicAmerican Type Culture Collection
    C. glutamicum ΔaceEC. glutamicum WT with deletion of the E1p gene (aceE) of the pyruvate dehydrogenase complex24
    C. glutamicum DM1800l-Lysine producer; pycP485S, lysCT311I, derived from C. glutamicum WT63
    C. glutamicum DM1800 aceE A16C. glutamicum DM1800 in which the native aceE promoter was replaced by the dapA-A16 promoterThis work
    C. glutamicum DM1933l-Lysine producer; Δpck, pycP458S; homV59A; 2× lysCT311I, 2× asd, 2× dapA, 2× dapB, 2× ddh, 2× lysA, 2× lysE, derived from C. glutamicum WT64
    C. glutamicum DM1933 aceE A16C. glutamicum DM1933 in which the native aceE promoter was replaced by the dapA-A16 promoterThis work
    C. glutamicum aceE L1, A23, A25, A16C. glutamicum WT in which the native aceE promoter was replaced by the dapA-L1, -A23, -A25, or -A16 promoter, respectivelyThis work
    C. glutamicum aceE A16 ΔpqoC. glutamicum aceE A16 with deleted pqo gene, encoding pyruvate:quinone oxidoreductaseThis work
    C. glutamicum aceE A16 Δpqo ΔppcC. glutamicum aceE A16 Δpqo with deleted ppc gene, encoding phosphoenolpyruvate carboxylaseThis work
    C. glutamicum aceE A16 Δpqo Δppc ΔilvEC. glutamicum aceE A16 Δpqo Δppc with deleted ilvE gene, encoding transaminase BThis work
Plasmids
    pK18/19mobsacBKmr, mobilizable (oriT), oriV40
    pK18mobsacBaceE-rAcpK18mobsacB carrying a truncated promoter region of the aceE gene, encoding the E1 subunit of the PDHCThis work
    pK18mobsacB PaceE dapA-L1, -A23, -A25, -A16pK19mobsacB PaceE carrying the dapA-L1, -A23, -A25, or -A16 promoter, respectivelyThis work
    pK19mobsacB ΔppcpK19mobsacB carrying a truncated ppc gene, encoding phosphoenolpyruvate carboxylaseThis work
    pK19mobsacB ΔpqopK19mobsacB carrying a truncated pqo gene, encoding pyruvate:quinone oxidoreductase41
    pK19mobsacB ΔilvEpK19mobsacB carrying a truncated ilvE gene, encoding transaminase B61
    pK19mobsacB dPgltA540-PdapA-L1, -A23, -A25, -A16pK19mobsacB carrying a truncated promoter region of the gltA gene and the dapA-L1, -A23, -A25, or -A16 promoter, respectively60
    pJC4 ilvBNCDKanr; plasmid carrying the ilvBNCD genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and dihydroxyacid dehydratase65
    pJC4 ilvBNCEKanr; plasmid carrying the ilvBNCE genes encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B22
Oligonucleotides
    dapAfow5′-AACTGCAGAACCAATGCATTGGTTCTGCAGTTATCACA CCC-3′Amplification of dapA promoters, NsiI site underlined
    dapArev25′-GGGAATTCCATATGAGGCTCCTTTTAAATCGAGCGGCT CCGGTCTTAGCTGTTAAACC-3′Amplification of dapA promoters/verification of promoter exchange, NdeI site underlined
    ace15′-CCCAAGCTTGCACATTACCGTCCAACC-3′Sequencing of dapA promoters/verification of promoter exchange
    ace25′-CGCGGATCCCGACGGTAACGCTTCTCC-3′Sequencing of dapA promoters
    ilvE15′-GCGTTGACTGATTCTTGGTC-3′Primer to verify deletion of ilvE (11)
    ilvE25′-CGAGTTCGATGGAATCTTCG-3′Primer to verify deletion of ilvE (11)
    pqodel15′-AAGGAATTCGTTTTCGAGGCGACCAGACAG-3′Primer to verify deletion of pqo (41)
    pqodel45′-TGGCACAAGCTTGTTAAGCGCTCGCGGTCAATG-3′Primer to verify deletion of pqo (41)
    ppc15′-CCCAAGCTTGAGTTGCGCAGCGCAGTG-3′Primer for deletion of ppc, HindIII site underlined
    ppc25′-GTGCTGCGCAATGCTGAGGGCATTAGAGCAGTGGATT GG-3′Primer for deletion of ppc, crossover overlap underlined
    ppc35′-CCTCAGCATTGCGCAGCACATCGGCCACAGCTTCTGC-3′Primer for deletion of ppc, crossover overlap underlined
    ppc45′-CGCGGATCCCGATGACATCAGGTTCCTC-3′Primer for deletion of ppc, BamHI site underlined
    ppcdel15′-GGAATAGACTCGCTCGGC-3′Primer to verify deletion of ppc
    ppcdel25′-GTGAACAGGCTCTCGATGC-3′Primer to verify deletion of ppc
    aceEup-fw5′-CGGGATCCCGACCCAATGCGTACCGATGTG-3′Primer for deletion of aceE promoter region, BamHI site underlined
    aceE-intrev5′-GCGCTAGCGCCACCATCGGAGGTGTTGTTC-3′Primer for deletion of aceE promoter region, NheI site underlined
    aceE-rAC-soeleft5′-TTGATCGGCCATATGTATTATGCATCTCTCACGTTTGACG CGAATCG-3′Primer for deletion of aceE promoter region, crossover overlap underlined, NsiI and NdeI in italic
    aceE-rAC-soeright5′-CAAACGTGAGAGATGCATAATACATATGGCCGATCAAGC AAAACTTGG-3′Primer for deletion of aceE promoter region, crossover overlap underlined, NsiI and NdeI in italic