TABLE 1.
Bacterial strains, plasmids, and primers
| Strain, plasmid, or primer | Relevant characteristic(s)c | Reference or source |
|---|---|---|
| E. coli strains | ||
| LY168 | frdA::(Zm frg celYEc FRT) ΔldhA::FRT ΔadhE::(Zm frg estZPp FRT) ΔackA::FRT rrlE::(pdc adhA adhB FRT) lacY::FRT ΔmgsA::FRT | 13, 44, 45 |
| LY180 | ΔfrdBC::(Zm frg celYEc) ΔldhA::(Zm frg casABKo) adhE::(Zm frg estZPp FRT) ΔackA::FRT rrlE::(pdc adhA adhB FRT) ΔmgsA::FRT | This study |
| EMFR9 | LY180 improved for furfural tolerance | This study |
| EMFR9 ΔyqhD | EMFR9 ΔyqhD::kan | This study |
| EMFR9 ΔdkgA | EMFR9 ΔdkgA::cat sacB | This study |
| EMFR9 ΔyqhD ΔdkgA | EMFR9 ΔyqhD::kan, ΔdkgA::cat sacB | This study |
| BL21(λDE3) | F−ompT gal dcm lon hsdSB(rB− mB−) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) | Promega (Madison, WI) |
| TOP10F′ | F′{lacIq Tn10 (Tetr)} mcriA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL endA1 nupG | Invitrogen (Carlsbad, CA) |
| Plasmidsa | ||
| pCR 2.1 TOPO | bla kan lacZ Plac | Invitrogen (Carlsbad, CA) |
| pLOI4301 | yqhD gene in pCR 2.1 TOPO | This study |
| pLOI4302 | yjjN gene in pCR 2.1 TOPO | This study |
| pLOI4303 | dkgA gene in pCR 2.1 TOPO | This study |
| pLOI4304 | yqfA gene in pCR 2.1 TOPO | This study |
| pLOI4305 | yajO gene in pCR 2.1 TOPO | This study |
| pLOI4306 | ydhU gene in pCR 2.1 TOPO | This study |
| pLOI4307 | ydhV gene in pCR 2.1 TOPO | This study |
| pLOI4308 | ygcW gene in pCR 2.1 TOPO | This study |
| pLOI4309 | nemA gene in pCR 2.1 TOPO | This study |
| pLOI4310 | yjgB gene in pCR 2.1 TOPO | This study |
| pLOI4311 | ydhS gene in pCR 2.1 TOPO | This study |
| pLOI4312 | ydhY gene in pCR 2.1 TOPO | This study |
| pLOI4313 | His-tagged yqhD in pET15b | This study |
| PLOI4314 | His-tagged dkgA in pET15b | This study |
| pET15b | T7 promoter, bla, His-tagged vector | Novagen (Madison, WI) |
| pKD4 | FRT kan FRT | 5 |
| PKD46 | Para bla Red recombinase (γ,β,exo) | 5 |
| Primersb | ||
| yqhD cloning | For-ACATCAGGCAGATCGTTCTC | This study |
| Rev-CCACAGCTTAGTGGTGATGA | ||
| yjjN cloning | For-GGAGAGCCGAATCATGTCTA | This study |
| Rev-CCGGAACCTGTCTCAACCAA | ||
| dkgA cloning | For-GCCTGCTCCGGTGAGTTCAT | This study |
| Rev-CCGGCTCTGCATGATGATGT | ||
| yqfA cloning | For-GCTGGAGAGGTATACATGTG | This study |
| Rev-GCCGTATTCGCTCGAAGAGT | ||
| yajO cloning | For-CCGCAGCACATGCAACTTGA | This study |
| Rev-ATGGCGCTGCCGACCAATGA | ||
| ydhU cloning | For-CCGCATCTGTATCGCCGGTT | This study |
| Rev-GCCGATGCGAGCATGATTCGT | ||
| ydhV cloning | For-ATTATCGAGTGGAAAGATAT | This study |
| Rev-CGTAGTCTCCGTTCTGCTTA | ||
| ygcW cloning | For-ACCTTTCTTTTTTTTTGCCT | This study |
| Rev-TTACGACCGCTGCCGGAATC | ||
| nemA cloning | For-TTATTGCGACGCCTGCCGTT | This study |
| Rev-GTTCAATCACCGCTTCTTCG | ||
| yjgB cloning | For-CCTGCCATGCTCTACACTTC | This study |
| Rev-CTGGTTAGATGGCGACTATG | ||
| ydhS cloning | For-AACTTATCTGATAACACTAA | This study |
| Rev-CCAACAGCGGCGACAATGTA | ||
| ydhY cloning | For-TCAGGCTGCTGAATTGTCAG | This study |
| Rev-GGCACCAGATCCAGTTAATG | ||
| Deletion of yqhD | For-GTTCTCTGCCCTCATATTGGCCCAGCAAAGGGAGCAAGTAGTGTAGGCTGGAGCTGCTTC | This study |
| Rev-GACGAAATGCCCGAAAACGAA AGTTTGAGGCGTAAAAAGCCATATGAATATCCTCCTTA | ||
| Deletion of dkgA | Outward 1-ACGGTTGGATTAGCCATACG | This study |
| Outward 2-GACCAGTTCGGCGGCTAACA | ||
| For-GCCTGCTCCGGTGAGTTCAT | ||
| Rev-CCGGCTCTGCATGATGATGT | ||
| yqhD cloning into pET15b | For-TGACTCTCGAGATGAACAACTTTAATCTGCA | This study |
| Rev-AGTCAGGATCCTTAGCGGGCGGCTTCGTATA | ||
| dkgA cloning into pET15b | For-ATATGCCTCGAGATGGCTAATCCAACCGTTAT | This study |
| Rev- CCGATAGGATCCTTAGCCGCCGAACTGGTCAGG | ||
| Sequencing yqhD | yqhD_for1 CGGCGAGGTACTGGTGAC | This study |
| yqhD_rev1 CATGTTAGCCGCCGAACT | ||
| yqhD_seq1 TCATGTTGGCTTCTGCCG | ||
| yqhD_seq2 GCGCAATCGCTGGTTTAC | ||
| yqhD_seq3 GTTCCGATGATGAGCGTATTG | ||
| yqhD_seq4 AGGCGTTTTCGATCAGAAAG | ||
| Sequencing dkgA | dkgA_for1 CCAGCAACCGGTTCAGAAT | This study |
| dkgA_rev1 AACGCGTGAAAATAGCGACT | ||
| dkgA_seq1 GCGGTAAAGAGATTAAAAGCGC | ||
| dkgA_seq2 TATGGCTAATCCAACCGTTATTAAG | ||
| dkgA_seq3 CCCGCCCGTTGTTACTCT | ||
| Sequencing of yqfA | pcr_for CCATCCGCGACGAGTCTGAA | This study |
| pcr_rev GGTGAAGCGGAACTGAACAA | ||
| seq1 CCATCCGCGACGAGTCTGAA | ||
| seq2 CGACGCTCTATCACGCCATT | ||
| Sequencing of yjjN | pcr_for TGCGCTGTTTAAGATCGCT | This study |
| pcr_rev CATGATTGCCTTCTCGGG | ||
| seq1 ACTGAGATGATCTCAAGCGATTG | ||
| seq2 GGAAACAACGCGAGATACCT | ||
| seq3 CCACGCTGGCAGAAACCTA |
a
The genes inserted into pCR 2.1 TOPO include a native ribosomal binding site gene and a transcriptional terminator. Expression is from the plasmid promoter (Plac).
b
Orientation of genes cloned into pCR 2.1 TOPO was verified by PCR analysis.
c
Data for primers are 5′-to-3′ sequences.