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 |