TABLE 2.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Description, genotype, or sequence | Reference |
|---|---|---|
| Strains | ||
| MG1655 | F− λ−ilvG rfb-50 rph-1 | 29 |
| MG1655 ΔadhE | MG1655 ΔadhE::FRT-Kan-FRT; adhE deletion mutant in MG1655 | This study |
| MG1655 ΔsdhB | MG1655 ΔsdhB::FRT-Kan-FRT; sdhB deletion mutant of MG1655 | This study |
| LS5218 | fadR601 atoC(Con)2 | 59 |
| MG1655 fadR* | MG1655 evolved for rapid growth on decanoic acid | This study |
| MG1655 fadR* atoC(Con) | MG1655 fadR* transduced with the atoC(Con) gene of LS5218 | This study |
| MG1655 fadR* atoC(Con) ΔadhE | MG1655 fadR* atoC(Con) ΔadhE::FRT-Kan-FRT; adhE deletion mutant of MG1655 fadR* atoC(Con) | This study |
| MG1655 fadR* atoC(Con) ΔsucA | MG1655 fadR* atoC(Con) ΔsucA::FRT-Kan-FRT; sucA deletion mutant of MG1655 fadR* atoC(Con) | This study |
| MG1655 fadR* atoC(Con) ΔsdhB | MG1655 fadR* atoC(Con) ΔsdhB::FRT-Kan-FRT; sdhB deletion mutant of MG1655 fadR* atoC(Con) | This study |
| MG1655 fadR* atoC(Con) ΔsucD | MG1655 fadR* atoC(Con) ΔsucD::FRT-Kan-FRT; sucD deletion mutant of MG1655 fadR* atoC(Con) | This study |
| Plasmids | ||
| pTH.adhE* | E. coli adhE mutant (E568K) under the control of Ptrc (Ampr, oriR pBR322) | This study |
| pZS.crt.bcd.etfAB.hbd | C. acetobutylicum butyryl-CoA synthesis operon (crt, bcd, etfAB, hbd) under the control of PLtetO-1 (Tetr, oriR SC101*, cat) | This study |
| pTH.atoB.adhE2 | E. coli atoB gene and C. acetobutylicum adhE2 gene under the control of Ptrc (Ampr, oriR pBR322) | This study |
| pTH.atoDAB.adc | E. coli atoD, atoA, and atoB genes and C. acetobutylicum adc gene under the control of Ptrc (Ampr, oriR pBR322) | This study |
| pZS.sadh | C. acetobutylicum sadH gene under the control of PLtetO-1 (Tetr, oriR SC101*, cat) | This study |
| pZS.ackA.pta | E. coli ackA-pta operon under the control of PLtetO-1 (Tetr, oriR SC101*, cat) | This study |
| pZS.ackA.pta.sadh | E. coli ackA-pta operon and C. acetobutylicum sadH gene under the control of PLtetO-1 (Tetr, oriR SC101*, cat) | This study |
| pTrc.scpABC | E. coli scpA, scpB, and scpC genes under the control of PLtetO-1 (Ampr, oriR pBR322) | This study |
| Primersa | ||
| v-adhE | GTTTAACATTATCAGGAG; GTCAACTAATCCTTAAC | This study |
| v-sucA | CACATCACTGTGCGTGGTAGTATCC; CAGGTCAGGGACCAGAATATCTACG | This study |
| v-sdhB | CTTCCGTACCGAAAGCCGTG; ACCACGCACAGTGATGTGCG | This study |
| v-sucD | GACAGCGGCCTGAATATTATTGCAG; CATCGCGATAAGCACAAAAAAGGCC | This study |
| m-adhE | CATCCGGAAACTCACTTCGAAAAGCTGGCGCTG; CAGCGCCAGCTTTTCGAAGTGAGTTTCCGGA | This study |
| c-adhE | CATTAAAGAGGAGAAAGGTACCATGGCTGTTACTAATG; GATGCCTCTAGCACGCGTTTAAGCGGATTTTTTCG | This study |
| c-ackA.pta | CATTAAAGAGGAGAAAGGTACCATGTCGAGTAAGTTAG; GATGCCTCTAGCACGCGTTTACTGCTGCTGTGC | This study |
| c-ackA.pta.sadh | CTGCACAGCAGCAGTAAACGCGTGAGGAATGAAAGGCTTTGCG; GATGCCTCTAGCACGCGTTTACAGAATCACCACCGC | This study |
| c-sadh | CATTAAAGAGGAGAAAGGTACCATGAAAGGCTTTGCGATGCTG; GATGCCTCTAGCACGCGTTTACAGAATCACCACCGC | This study |
| c-crt.bcd.etfAB.hbd | GATGGTACCATGGAACTAAACAATGTCATCCTTG; GATCACGCGTTTATTTTGAATAATCGTAGAAACC | This study |
| c-atoB | GAGATCTGCAGCTGGTACCATGAAAAATTGTGTCATC; CTTTTTGATTTGTAACTTTCATTTAATTCAACCGTTCAATCACC | This study |
| c-adhE2 | GGTGATTGAACGGTTGAATTAAATGAAAGTTACAAATCAAAAAG; CGGGCCCAAGCTTCGAATTCTTAAAATGATTTTATATAGATATCC | This study |
| c-atoB.adhE2 | GAGATCTGCAGCTGGTACCATGAAAAATTGTGTCATC; CGGGCCCAAGCTTCGAATTCTTAAAATGATTTTATATAGATATCC | This study |
| c-scpABC | GCTCTAGAATGTCTTATCAGTATGTTAAGG; GCTCTAGATTAATCATGATGCTGGC | This study |
| c-scpAargKscpBC | CGGAATTCATGTCTAACGTGCAGGAGTGG; GACAAGCTTTTAACCCAGCATCGAGCCG | This study |
| c-atoDA | CGGGATCCATGAAAACAAAATTGATGAC; GAGGTACCTCATAAATCACCCCGTTG | This study |
| c-adc | GCGGTACCATGTTAAAGGATGAAG; CGGAATTCTTAAGATAATCATATATAAC | This study |
| c-atoB | GACGGTACCAGGAGGAAATGAAAAATTGTGTCATCGTCAGTGC; GACGGTACCTTCCTCCTTTAATTCAACCGTTCAATCACCATCGC | This study |
a
v, primer sequences that were used for verification purposes during the creation of disruption mutants by phage transduction. m, primers used for site-directed mutagenesis of adhE. c, primers used for cloning purposes. Sequences are in the 5′-to-3′ direction, and the forward follows the reverse sequence in each case, separated by a semicolon. Genes or operons deleted or cloned are apparent from the primer names.