Table 1.
Bacterial strains, plasmids, and primers
| Strain, plasmid, or primer | Relevant characteristics | Reference or source |
|---|---|---|
| Strains | ||
| LY180 | ΔfrdBC::(Zm frg celYEc) ΔldhA::(Zmfrg casA BKo) adhE::(ZmfrgestZPp FRT) ΔackA::FRT rrlE::(pdc adhA adhB FRT) ΔmgsA::FRT | 30 |
| EM322 | LY180 ΔyqhD::FRT | 30 |
| BL21(λDE3) | F−ompT gal dcm lon hsdSB(rB−mB−) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) | Promega, Madison, WI |
| MM160 | Hydrolysate-resistant derivative of LY180, yqhD frameshift | 13 |
| XW042 | MM160 ldhA::ldhL Δ(rrlE::[pdc adhA adhB FRT]), Δ(Zm frg estZPp FRT) l-lactate production | This study |
| XW043 | MM160 ldhA restored, Δ(rrlE::[pdc adhA adhB FRT]), Δ(Zm frg estZ Pp FRT) d-lactate production | This study |
| XW059 | XW042 after serial transfer with xylose l-lactate production | This study |
| XW068 | XW043 after serial transfer with xylose d-lactate production | This study |
| Plasmids | ||
| pTrc99A | pTrc bla oriR rrnB lacIq | 4 |
| pCR2.1 | TOPO cloning vector | Invitrogen |
| pLOI4162 | PacI-flanked cat-sacB cassette | 17 |
| pET15b | T7 expression vector | Novagen |
| fucO cloning and expression | ||
| pLOI4319 | fucO in pTrc99A | This study |
| pLOI4322 | fucO in pET15b | This study |
| Deletion of (rrlE::[pdc adhA adhB FRT]) | ||
| pLOI4780 | pdc-adhA-adhB from LY180 cloned into pCR2.1-TOPO vector | This study |
| pLOI4781 | cat-sacB cassette cloned into pdc-adhA-adhB in pLOI4780 | This study |
| pLOI4782 | PacI digestion of pLOI4781; self-ligated to delete pdc-adhA-adhB | This study |
| Deletion of (adhE::[Zm frg estZPp FRT]) | ||
| pLOI4811 | adhE::(Zm frg estZPp FRT) region cloned into pCR2.1-TOPO vector | This study |
| pLOI4824 | cat-sacB cassette cloned into the adhE::(Zm frg estZPpFRT) region of pLOI4811 | This study |
| pLOI5167 | E. coli adhE ORF and its adjacent regions cloned into pCR2.1 TOPO vector | This study |
| pLOI5168 | cat-sacB cassette cloned into adhE in pLOI5167 | This study |
| pLOI5169 | PacI digestion of pLOI5168; self-ligated to delete adhE | This study |
| ldhA restoring | ||
| pLOI4652 | ldhA (PCR) from E. coli cloned into the pCR2.1-TOPO vector | 41 |
| pLOI4653 | cat-sacB cassette cloned into ldhA of pLOI4652 | 41 |
| ldhL integration | ||
| pLOI5161 | ldhA ORF and its adjacent regions cloned into pCR2.1 TOPO vector | This study |
| pLOI5174 | ldhL ORF from TG108 was cloned and used to replace the ldhA ORF in pLOI5161 | This study |
| Primers | ||
| fucO cloning | ||
| fucO for EcoRI | CGCGCGGAATTCGATTGCCGTAGTGCTGGAGA | This study |
| fucO rev BamHI | CGCGCGGGATCCTGCGGTTGGTACGGTAACGG | This study |
| ldhA and ldhL integration | ||
| ldhA for | GATAACGGAGATCGGGAATG (for construction of pLOI4652) | 41 |
| ldhA rev | CTTTGGCTGTCAGTTCACCA (for construction of pLOI4652) | 41 |
| ldhA-1 | TCTGGAAAAAGGCGAAACCT (for construction of pLOI4653) | 41 |
| ldhA-2 | TTTGTGCTATAAACGGCGAGT (for construction of pLOI4653) | 41 |
| ldhL ORF up | ATGTCTAATATTCAAAATCATCAAAAAGTTGTCCTCGTCG (for construction of pLOI5174) | This study |
| ldhL ORF down | TTATTTGTCTTGTTTTTCAGCAAGAGCGTTTAGAC (for construction of pLOI5174) | This study |
| ldhA rev1 | AAGACTTTCTCCAGTGATGTTGAATCAC (for construction of pLOI 5174) | This study |
| ldhA for1 | TCTTGCCGCTCCCCT (for construction of pLOI5174) | This study |
| Deletion of (rrlE::[pdc adhA adhB FRT]) | ||
| pdc for | TGGTCTCAAGCATCACTTCG | This study |
| adhB rev | TTGGTCAGAGCACAAGCATC | This study |
| adhB-1 | CCCACGCATTTGAAGCTTAT | This study |
| pdc-2 | ATCGATTTTAGCCGGAGCTT | This study |
| Deletion of (Zm frg estZPp FRT) | ||
| estZ for | ACTGGCATCTGAGTTCTCTG | This study |
| estZ rev | TTCCATGGCGTGAGTTACTG | This study |
| estZ-1 | CAGACCGTGCGGAATATGGA | This study |
| estZ-2 | CAGCCTCGATTCGCATGACA | This study |
| adhE for | CAATACGCCTTTTGACAGCA | This study |
| adhE rev | GCCATCAATGGCAAAAAGTT | This study |
| adhE-1 | TCAGTAGCGCTGTCTGGCA | This study |
| adhE-2 | AATGCTCTCCTGATAATGTTAAACTTTTTTAGTA | This study |
| Amplification and sequencing of yqhD region | ||
| yqhD for | TATGATGCCAGGCTCGTACA | This study |
| yqhD rev | GATCATGCCTTTCCATGCTT | This study |