Table 1.
list of strains, plasmids, and primers used in this study.
| Strains | Description | Source |
|---|---|---|
| E.coli DH5α | F− φ80dlacZΔM15 Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17(rK−, m +) phoA supE44 λ− thi-1 gyrA96 relA1 | Invitrogen |
| Z. mobilis | Ethanologenic strain | PTCC 1718 |
| S. cerevisiae | Ethanologenic strain | PTCC 5080 |
| B. subtilis WB600 | trpC2 nprE aprE epr bpr mpr nprB; Emr | 11 |
| B. subtilis WBN | Bacillus subtilis WB600, ldh::pDHtldh | This study |
| B. subtilis NC | WBN harboring pHY300PLK | This study |
| B. subtilis NZ | WBN harboring pHYpdcZ-adhZ | This study |
| B. subtilis NZS | WBN harboring pHYpdcZ-adhS | This study |
| B. subtilis NZS2 | WBN harboring pHYpdcZ-(adhS)2 | This study |
| B. subtilis N(ZS)2 | WBN harboring pHY(pdcZ-adhS)2 | This study |
| B. subtilis NZ:S | WBN harboring pHYpdcZ:adhS | This study |
| B. subtilis NS:Z | WBN harboring pHYadhS:pdcZ | This study |
| Plasmids | Description | Source |
|---|---|---|
| pHY300PLK | Shuttle vector, Tet and Amp resistant | 12 |
| pHYpdcZ-adhZ | pHY300PLK with pdc and adhII of Z. mobilis | This study |
| pHYpdcZ-adhS | pHY300PLK with Z. mobilis pdc and S. cerevisiae adhI | This study |
| pHYpdcZ-(adhS)2 | pHY300PLK with Z. mobilis pdc and two copies of S. cerevisiae adhI | This study |
| pHY(pdcZ-adhS)2 | pHY300PLK with two copies of both Z. mobilis pdc and S. cerevisiae adhI | This study |
| pHYadhS:pdcZ | pHY300PLK with adhS:pdcZ fusion gene | This study |
| pHYpdcZ:adhS | pHY300PLK with pdcZ:adhS fusion gene | This study |
| pDH88 | Integration vector, chloramphenicol resistant | 13 |
| pDHtldh | pHD88 with a 325 bp fragment from within B. subtilis ldh | This study |
| Primers | Sequence | Restriction enzymes |
|---|---|---|
| pdcZ-F | ATTGAATTCCATATGAAGGAGGAGTAAGCAATGAGTTATACTGTCGG | EcoRI |
| pdcZ-R | ATAGGATCCCTAGAGGAGCTTGTTAACAGGCTTACG | BamHI |
| adhZ-F | ATAGGATCCAAGGAGGGTATAGCTATGGCTTCTTCAACTTTTTATATTC | BamHI |
| adhZ-R | ATATCTAGACATATGTCAGAAAGCGCTCAGGAAGAGTTCTTCAAC | XbaI |
| adhS-F | ATGGATCCAAAGGAGGCGATTTGATGTCTATCCCAGAAACTC | BamHI |
| adhS-R | ATTCTAGATTAATGATGATGATGATGATGTTTAGAAGTGTCAACAAC | XbaI |
| tldh-F | ATAAAGCTTTCTTACGGAACATATGAAGACTGC | HindIII |
| tldh-R | ATATCTAGACGTGTACGTTTTGAGGCGC | XbaI |
| dldh-F | GTAGCTTTAATCGGAGCGGG | – |
| dldh-R | GCGACATCGTATAACGTTACTGG | – |